Engineered terminal nucleotidyl transferase variants

Abstract

The present invention provides engineered terminal nucleotidyl transferase (TnT) polypeptides useful in template-independent polynucleotide synthesis, as well as compositions, methods of utilizing these engineered polypeptides, and polynucleotides encoding the engineered terminal nucleotidyl transferases.

Description

Docket No. CX10-267WO3 ENGINEERED TERMINAL NUCLEOTIDYL TRANSFERASE VARIANTS REFERENCE TO SEQUENCE LISTING, TABLE OR COMPUTER PROGRAM

[0001] This application claims the benefit of U.S. Provisional Application No.63 / 634,824, filed April 16, 2024, and U.S. Provisional Application 63 / 706,470, filed October 11, 2024, all of which are incorporated by reference herein. TECHNICAL FIELD

[0002] The present invention provides engineered terminal nucleotidyl transferase (TnT) polypeptides useful in template-independent polynucleotide synthesis, as well as compositions and methods of utilizing these engineered polypeptides. REFERENCE TO SEQUENCE LISTING, TABLE OR COMPUTER PROGRAM

[0003] The official copy of the Sequence Listing is submitted concurrently with the specification as an XML file, with a file name of “CX10-267WO3_ST26.xml,” a creation date of April 16, 2025, and a size of 7,615,530 bytes. The Sequence Listing filed is part of the specification and is incorporated in its entirety by reference herein. BACKGROUND

[0004] Synthetic biology is becoming established in a diverse range of high value, high growth markets. From food and agriculture to therapeutics, diagnostics, and vaccines; tools such as gene editing, DNA sequencing and gene synthesis are being used to build value-added products with advanced functionality (e.g., cell bioreactors, etc.) and desired end products (e.g., drugs, chemicals, etc.). The barrier to widespread implementation of these technologies is the ability to efficiently synthesize RNA, DNA, and other polynucleotides.

[0005] In particular, silencing RNA (siRNA) therapeutics are a promising class of drugs that have the potential to treat numerous difficult to treat conditions in a highly targeted manner by binding to known mRNA targets (Hu et al. (2020). Sig Transduct Target Ther 5, 101; Zhang et al. (2021). Bioch. Pharmac., 189, 114432.) As these therapies become more common and are targeted at larger patient populations, the ability to produce large amounts of the oligonucleotide active pharmaceutical ingredient (API) becomes critical.

[0006] To date, short RNA oligonucleotides have been synthesized almost exclusively by iterative addition of nucleotides in the form of activated phosphoramidites, plus additional processing steps, to a growing immobilized nucleotide chain (Brown, T. Nucleic Acids Book. See at: www.atdbio.com / nucleic-acids-book (accessed 2022-10-10).)

[0007] Phosphoramidite chemistry has been developed extensively over the years to synthesize small amounts of DNA and for more complex therapeutic RNA syntheses but suffers from several cost and sustainability issues that are potentially limiting as API demand grows to triple-quadruple digit kilograms per year (Andrews et al. (2021). J. Org. Chem.86, 49−61). Additionally, RNA synthesis using phosphoramiditeDocket No. CX10-267WO3 synthesis chemistry is limited to producing short oligonucleotides of approximately 200 basepairs (Beaucage & Caruthers. (1981). Tetrahedron Lett.22 (20): 1859.)

[0008] The phosphoramidite iterative methodology is multi-step and based on phosphorous (III) coupling chemistry that requires (i) coupling (ii) capping (iii) oxidation to P(V) forming phosphodiester or phosphorothioate diester (iv) deblocking of 5’-O group. After chain synthesis is complete, the final oligo is cleaved from the support where deblocking of phosphate cyanoethyl group and nucleobases can also occur (Brown, T. Nucleic Acids Book. See at: www.atdbio.com / nucleic-acids-book (accessed 2022-10-10).) Washes with organic solvents at each step are also required. The phosphate cyanoethyl blocking group and nucleobase protecting groups can be removed in parallel to oligonucleotide cleavage from the solid support to generate the oligonucleotide product, or the cyanoethyl group can be removed under milder condition before chain cleavage, if required.

[0009] Many aspects of the environmental impact of the current phosphoramidite methodology and potential advances have been reviewed (Andrews et al. (2021). J. Org. Chem.86, 49−61). Even at an aspirational high oligonucleotide loading of 20% mass final oligonucleotide to mass solid support, at least five-fold mass of support is required over the final product mass.

[0010] In addition to the high cost of mass support required to immobilize the oligonucleotide, the use of organic solvents and 5’-O-blocking groups entail additional waste and process inefficiencies. Organic solvents such as acetonitrile are required for solubilization of the phosphoramidite coupling partners, or dichloromethane or toluene for deprotection steps. These solvents need to be anhydrous to reduce undesired hydrolysis of the phosphoramidite partners and can come from non-sustainable sources, adding cost, sustainability questions, and potential supply issues to the process.

[0011] The phosphoramidite coupling partners themselves carry a required blocking group at the 5’O- position, the nucleobase nitrogen atom (in A, C and G), and the nascent phosphate. The most common 5’O- blocking group, dimethoxytrityl, has a molecular mass of ~ 303 Da that approaches that of the heaviest native ribonucleotide fragment Gp with a mass of ~ 345 Da. This protecting group requires energy, resources, and effort to produce and append, and then requires disposal when separated from the desired materials.

[0012] In conclusion, a paradigm shift in oligonucleotide synthesis is necessary to enable siRNA therapeutics by lowering environmental impact, improving economic efficiency, and increasing scalability. New methods of oligonucleotide synthesis are, therefore, of great interest to the pharmaceutical industry. Template-Independent Enzymatic Synthesis

[0013] Enzymatic synthesis may facilitate production of high volumes of complex or long polynucleotides (>200 base pairs) while minimizing toxic waste. A variety of prokaryotic and eukaryotic DNA and RNA polymerases are known to naturally synthesize polynucleotides of thousands of base pairs or more. Most of these polymerases function during DNA replication associated with cell division or transcription of RNA from DNA associated with gene or protein expression. Both of these processes involve template-dependent polynucleotide synthesis, wherein the polymerase uses an existing template polynucleotide strand to synthesize a complementary polynucleotide strand.Docket No. CX10-267WO3

[0014] The potential of template-independent enzymatic polynucleotide synthesis to produce defined sequences has long been recognized. One early report suggested using NTPs with blocked 3’ groups to allow stepwise addition of specific nucleotide residues (Bollum, J Biol Chem, 1962, 237, 1945–1949).

[0015] However, few polymerases are known to catalyze template-independent polynucleotide synthesis. These include polymerase lambda, polymerase mu, and terminal deoxynucleotidyl transferase (TdT), all members of the X family of DNA polymerases, many of which participate in DNA repair processes (Domínguez et al., EMBO, 2000, 19(7), 1731–1742.) Of these, TdT is known to generate diversity in antigen receptors by indiscriminately adding nucleosides to the 3’ end of a single-stranded polynucleotide in a template-independent process (Bentolila et al., EMBO, 1995, 14(17), 4221–4229.)

[0016] Others have published a method of polynucleotide synthesis using a nucleoside 5'-triphosphate with a 3'-OH position protected with a removable blocking moiety and, specifically, a template-independent polynucleotide polymerase, including a terminal deoxynucleotidyl transferase (U.S. Pat.5,763,594). The blocking group, also known to those skilled in the art as an inhibitor or reversible terminating group, may include a variety of groups that prevent the TdT from adding additional NTPs to the nascent polynucleotide chain. This may include charged molecules, large molecules and moieties, or other blocking groups known to those skilled in the art. Appropriate removable blocking groups may include carbonitriles, phosphates, carbonates, carbamates, esters, ethers, borates, nitrates, sugars, phosphoramidates, phenylsulfenates, and sulfates. Other 3’ blocking groups are also known in the art, including 3’-O-amines and methylamines (U.S. Pat.7,544,794) and 3’-O-azides (U.S. Pat.10,407,721).

[0017] Although initially promising, use of 3’-blocked NTPs in template-independent synthesis catalyzed by TdT has proven difficult in practice, as TdT struggles to accept 3'-O-blocked NTPs as substrates. Further, wild-type TdTs have low tolerance for oligo acceptor substrates containing one or more modified nucleotides (e.g., 2’ modifications).

[0018] Additionally, synthesis of RNA strands presents unique challenges due to the additional, reactive 2’- OH on the ribose. While protection of the 2’ position facilitates RNA synthesis, this approach reduces efficiency because of steric hindrance by the 2’ protecting groups and requires maintenance and removal of the protecting group (CB Reese, Org Biomol Chem., 2005, 3, 3851–3868.)

[0019] Recently several reports have described template-independent synthesis methods that use modified NTPs with blocking groups attached to the purine or pyrimidine base, leaving the 3’-OH unmodified and available for additional rounds of synthesis. These base blocking groups may include a cleavable linker that allows removal of the blocking group after each NTP addition step. The cleavable linker may also be attached to a detectable label (U.S. Pat.7,057,026, among others). A variety of cleavable linkers are known to those skilled in the art. These include linkers attached via reducible disulfide bonds, photocleavable, electrophilic or nucleophilic, pH sensitive, temperature sensitive, and linkers cleaved by enzymes. One drawback to using cleavable linkers is that, typically, some atoms of the linker moiety remain attached to the NTP following cleavage, leaving a “scar” that may interfere with synthesis of a complementary strand after initial template- independent synthesis of the primary polynucleotide strand.Docket No. CX10-267WO3

[0020] Recently, modified NTPs with bases attached to blocking groups with cleavable linkers that are “scarless” and leave the nascent DNA ready for the next round of synthesis have been developed. In one example, the blocking group and cleavable linker are attached to the base via a disulfide bond. Upon addition of a reducing agent, the blocking group is removed, and the remaining atoms of the linker self-cyclize to leave the nascent DNA free of any linker atoms (U.S. Pat.8,808,989, U.S. Pat.9,695,470, U.S. Pat.10,041,110). Methods of using NTPs attached to cleavable blocking groups to synthesize polynucleotides are known, including using a microfluidic device or ink jet printing technology (U.S. Pat.9,279,149). An exonuclease may also be used in a method to synthesize polynucleotides to shorten or completely degrade polynucleotide strands that have not successfully added an NTP after the polynucleotide extension step and prior to removing the blocking group (U.S. Pat.9,771,613).

[0021] However, NTP bases with bulky blocking groups attached via cleavable linkers are not optimal for efficient synthesis of complex or long oligonucleotides. The large labels may negatively impact enzyme kinetics, and linker scars may lead to an unacceptable rate of misincorporation when synthesizing the oligonucleotide strand. Additionally, larger linkers and necessary deblocking steps may increase the cost, time, and inefficiency of the process as a whole, rendering these methods economically infeasible.

[0022] Recently, several groups have explored modifying the structure or amino acid sequence of TdT or other polymerases to allow template-independent synthesis using 3’-O-blocked groups. Efcavitch et al. describes incorporation of 3’ modified dNTPs by TdT in template-independent synthesis using a murine or bacterial TdT with substituted amino acid residues (U.S. Pat.10,059,929). Other reports describe engineered bovine and gar (Lepisosteus oculatus) TdTs that displayed improved activity over wild-type TdT (U.S. Pat. 10,745,727, PCT / GB2020 / 050247). Similarly, a variety of mutations have been described to improve the activity of Pol X family enzymes (WO 2017216472 A2). Finally, an N-terminal truncation of the BRCT domain (or alternatively mutation of the BRCT domain) of TdT has also been described as enhancing activity in the addition of reversibly blocked NTPs to the 3’-OH of a nucleic acid (US20210164008A1).

[0023] Feasible methods of template-independent enzymatic synthesis of complex or long polynucleotides have recognized commercial value. Recently, research efforts devoted to resolving challenges in this field have yielded improved engineered enzymes with activity in the template independent synthesis of polynucleotides, including DNA (PCT / US2022 / 078071) and RNA (PCT / US2023 / 076667). However, engineered terminal nucleotidyl transferase (TnT) enzymes with further improvements are necessary to enable template-independent enzymatic synthesis of a variety of complex or long polynucleotides. In particular, TnT enzymes with improved activity under industrial process conditions that are able to accept and incorporate NTPs with an extensive range of modifications to highly modified polynucleotide oligo acceptor substrates are necessary to enable economically feasible synthesis processes. SUMMARY

[0024] The present invention provides engineered terminal nucleotidyl transferase (TnT) polypeptides useful in template-independent polynucleotide synthesis, as well as compositions and methods of utilizing these engineered polypeptides. The TnTs of the present invention are variants of a previously engineered TnTDocket No. CX10-267WO3 enzyme (PCT / US2023 / 076667), which is an engineered variant of a predicted splice variant of the wild-type gene from Monodelphis domestica.

[0025] As used herein, the term terminal nucleotidyl transferase (TnT) is used to distinguish an engineered enzyme with activity on a variety of nucleoside triphosphates, including ribonucleoside triphosphates with 3' modifications or with 2' modifications or with 2' and 3' modifications, from a wild-type TdT enzyme having wild-type TdT activity.

[0026] These engineered TnTs are capable of adding nucleoside triphosphates with a 3’-O-removable blocking group and other natural or modified NTPs to the 3’-OH end of a growing oligonucleotide or polynucleotide chain in a template-independent manner. After removal of the blocking group, additional rounds of NTP addition can be used to synthesize a polynucleotide with a defined sequence of bases without using a complementary template strand as a guide for NTP incorporation (template-independent synthesis).

[0027] In some embodiments, the present invention provides an engineered TnT polypeptide comprising an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, sequence identity to a reference sequence corresponding to amino acid residues 12 to the carboxy terminus of an even-numbered SEQ ID NO. of SEQ ID NOs: 2, 4-1056, 1130-1880, and 1928-3008, or to a reference sequence corresponding to an even- numbered SEQ ID NO. of SEQ ID NOs: 2, 4-1056, 1130-1880, and comprising an amino acid residue difference (e.g., at least one or one substitution set) at one or more amino acid positions, wherein the positions are numbered with reference to SEQ ID NOs: 2, 8, 70, 148, 184, 234, 256, 478, 668, 682, 730, 734, 742, 932, 992, 1144, 1212, 1236, 1264, 1414, 1504, 1522, 1702, 1742, 1928, 1944, 1946, 1984, 2064, 2104, 2210, 2256, 2280, 2342, 2438, 2452, 2562, 2576, 2562, 2714, 2876, 2884, 2910, 2974, 2998, or 3002, and an improved property described herein.

[0028] In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, sequence identity to a reference sequence of SEQ ID NOs: 2, 8, 70, 148, 184, 234, 256, 478, 668, 682, 730, 734, 742, 932, 992, 1144, 1212, 1236, 1264, 1414, 1504, 1522, 1702, 1742, 1928, 1944, 1946, 1984, 2064, 2104, 2210, 2256, 2280, 2342, 2438, 2452, 2562, 2576, 2562, 2714, 2876, 2884, 2910, 2974, 2998, or 3002 and comprising an amino acid residue difference (e.g., one substitution or one substitution set) at one or more amino acid positions, wherein the positions are numbered with reference to SEQ ID NOs: 2, 8, 70, 148, 184, 234, 256, 478, 668, 682, 730, 734, 742, 932, 992, 1144, 1212, 1236, 1264, 1414, 1504, 1522, 1702, 1742, 1928, 1944, 1946, 1984, 2064, 2104, 2210, 2256, 2280, 2342, 2438, 2452, 2562, 2576, 2562, 2714, 2876, 2884, 2910, 2974, 2998, or 3002, and an improved property described herein.

[0029] In some embodiments, the engineered terminal nucleotidyl transferase comprises an amino acid sequence comprising at least an amino acid residue difference at amino acid position 22, 27, 34, 41, 42, 43, 45, 52, 60, 92, 105, 133, 152, 160, 161, 162, 164, 169, 173, 174, 175, 176, 177, 179, 182, 183, 184, 186, 187, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 208, 212, 213, 218, 219, 221, 222, 223, 224, 227, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 241, 242, 244, 246, 249, 250, 251, 252, 254, 259, 260, 261, 262, 263, 264, 267, 268, 272, 273, 274, 275, 277, 278, 280, 281, 282, 288, 290, 291,Docket No. CX10-267WO3 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 307, 308, 309, 310, 312, 313, 314, 315, 316, 317, 318, 319, 321, 322, 323, 324, 325, 328, 329, 333, 334, 335, 336, 337, 339, 340, 341, 342, 343, 347, 352, 353, 354, 357, 359, 360, 361, 362, 363, 364, 366, 367, 368, 370, 371, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 387, 388, 389, 390, 392, 393, 394, 395, 396, 397, 398, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 446, 448, 450, 451, 456, 457, 458, 459, 460, 461, 462, 463, 465, 466, 467, 468, 474, 477, 479, 480, 481, 484, 485, 487, 490, 492, 493, 494, 495, 497, 499, 500, 501, 502, 503, 504, 506, 507, 508, 513, 515, 517, 518, 522, 523, 525, or 526, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12 to the carboxy terminus of SEQ ID NO: 2, 8, 70, 148, 184, 234, 256, 478, 668, 682, 730, 734, 742, 932, 992, 1144, 1212, 1236, 1264, 1414, 1504, 1522, 1702, 1742, 1928, 1944, 1946, 1984, 2064, 2104, 2210, 2256, 2280, 2342, 2438, 2452, 2562, 2576, 2562, 2714, 2876, 2884, 2910, 2974, 2998, or 3002, or to a reference sequence corresponding to SEQ ID NO: 2, 8, 70, 148, 184, 234, 256, 478, 668, 682, 730, 734, 742, 932, 992, 1144, 1212, 1236, 1264, 1414, 1504, 1522, 1702, 1742, 1928, 1944, 1946, 1984, 2064, 2104, 2210, 2256, 2280, 2342, 2438, 2452, 2562, 2576, 2562, 2714, 2876, 2884, 2910, 2974, 2998, or 3002.

[0030] In some embodiments, the engineered TnT polypeptide has improved thermostability, increased activity at elevated temperatures, increased soluble expression or isolated protein yield, decreased by-product formation, increased specific activity on NTP-3’-O-RBG and other natural or modified NTP substrates, and / or increased activity on various oligo acceptor substrates as compared to a wild-type TnT or other TnTs or template-independent polymerases known to those of skill in the art. These engineered TnT polypeptides with one or more amino acid substitutions or substitution sets are described, below, in the detailed description of the invention.

[0031] In some additional embodiments, the engineered terminal nucleotidyl transferase polypeptide comprises an amino acid sequence with at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to the carboxy terminus of an even-numbered sequence set forth in SEQ ID NOs: 2-1056, 1130-1880, and 1928-3008, or to a reference sequence corresponding to an even-numbered sequence set forth in SEQ ID NOs: 2-1056, 1130-1880, and 1928-3008.

[0032] In some embodiments, the engineered terminal nucleotidyl transferase polypeptide comprises an amino acid sequence comprising residues 12 to carboxy terminal of an even-numbered sequence of an even- numbered sequence set forth in SEQ ID NOs: 2-1056, 1130-1880, and 1928-3008, or an amino acid sequence comprising an even-numbered sequence set forth in SEQ ID NOs: 2-1056, 1130-1880, and 1928-3008.

[0033] In some additional embodiments, the engineered terminal nucleotidyl transferase polypeptide comprises an amino acid sequence with at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to the carboxy terminus of SEQ ID NO: 2, 8, 70, 148, 184, 234, 256, 478, 668, 682, 730, 734, 742, 932, 992, 1144, 1212, 1236, 1264, 1414, 1504, 1522, 1702, 1742, 1928, 1944, 1946, 1984, 2064, 2104, 2210, 2256, 2280, 2342, 2438, 2452, 2562, 2576, 2562,Docket No. CX10-267WO3 2714, 2876, 2884, 2910, 2974, 2998, or 3002, or to a reference sequence corresponding to SEQ ID NO: 2, 8, 70, 148, 184, 234, 256, 478, 668, 682, 730, 734, 742, 932, 992, 1144, 1212, 1236, 1264, 1414, 1504, 1522, 1702, 1742, 1928, 1944, 1946, 1984, 2064, 2104, 2210, 2256, 2280, 2342, 2438, 2452, 2562, 2576, 2562, 2714, 2876, 2884, 2910, 2974, 2998, or 3002.

[0034] In some embodiments, the engineered terminal nucleotidyl transferase polypeptide comprises an amino acid sequence comprising amino acid residues 12 to the carboxy terminus of SEQ ID NO: 2, 8, 70, 148, 184, 234, 256, 478, 668, 682, 730, 734, 742, 932, 992, 1144, 1212, 1236, 1264, 1414, 1504, 1522, 1702, 1742, 1928, 1944, 1946, 1984, 2064, 2104, 2210, 2256, 2280, 2342, 2438, 2452, 2562, 2576, 2562, 2714, 2876, 2884, 2910, 2974, 2998, or 3002, or an amino acid sequence comprising SEQ ID NO: 2, 8, 70, 148, 184, 234, 256, 478, 668, 682, 730, 734, 742, 932, 992, 1144, 1212, 1236, 1264, 1414, 1504, 1522, 1702, 1742, 1928, 1944, 1946, 1984, 2064, 2104, 2210, 2256, 2280, 2342, 2438, 2452, 2562, 2576, 2562, 2714, 2876, 2884, 2910, 2974, 2998, or 3002.

[0035] In an additional embodiment, the engineered polypeptide of the present invention further comprises an N-terminal truncation of 1-156 amino acids of the polypeptide sequence relative to any even-numbered sequence set forth in SEQ ID NOs: 2-1056, 1130-1880, and 1928-3008.

[0036] In an additional embodiment, the engineered polypeptide of the present invention is immobilized.

[0037] The present invention also provides an engineered polynucleotide encoding at least one engineered polypeptide described in the above paragraphs and as disclosed herein.

[0038] In some embodiments, the engineered polynucleotide comprises a polynucleotide sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference polynucleotide sequence corresponding to nucleotide residues 34 to 3’-terminus of SEQ ID NO: 1, 7, 69, 147, 183, 233, 255, 477, 667, 681, 729, 733, 741, 931, 991, 1143, 1211, 1235, 1263, 1413, 1503, 1521, 1701, 1741, 1927, 1943, 1945, 1983, 2063, 2103, 2209, 2255, 2279, 2341, 2437, 2451, 2561, 2575, 2753, 2943, 2952, 2977, 3041, 3065, 3125, 3201, or 3227, or to a reference polynucleotide sequence corresponding to SEQ ID NO: 1, 7, 69, 147, 183, 233, 255, 477, 667, 681, 729, 733, 741, 931, 991, 1143, 1211, 1235, 1263, 1413, 1503, 1521, 1701, 1741, 1927, 1943, 1945, 1983, 2063, 2103, 2209, 2255, 2279, 2341, 2437, 2451, 2561, 2575, 2753, 2943, 2952, 2977, 3041, 3065, 3125, 3201, or 3227, wherein the polynucleotide sequence encodes a terminal nucleotidyl transferase.

[0039] In some embodiments, the engineered polynucleotide comprises a polynucleotide sequence polynucleotide sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference polynucleotide sequence corresponding to nucleotide residues 34 to 3’-terminus of an odd numbered SEQ ID NO. of SEQ ID NOs: 3-1055, 1129-1879, and 1927-3007, or to a reference polynucleotide sequence corresponding to an odd numbered SEQ ID NO. of SEQ ID NOs: 3-1055, 1129-1879, and 1927-3007, wherein the polynucleotide sequence encodes a terminal nucleotidyl transferase.Docket No. CX10-267WO3

[0040] In some embodiments, the engineered polynucleotide comprises polynucleotide sequence comprising nucleotide residues 34 to 3’-terminus of an odd-numbered sequence set forth in SEQ ID NOs: 1-1055, 1129- 1879, and 1927-3007, or a polynucleotide sequence comprising an odd-numbered sequence set forth in SEQ ID NOs: 1-1055, 1129-1879, and 1927-3007.

[0041] In another aspect, the present invention further provides vectors comprising at least one engineered polynucleotide described above. In some embodiments, the vectors further comprise at least one control sequence.

[0042] In a further aspect, the present invention also provides host cells comprising the vectors provided herein. In some embodiments, the host cell produces at least one engineered polypeptide provided herein.

[0043] In a further aspect, the present invention provides methods of producing an engineered TnT polypeptide, comprising the steps of culturing the host cell provided herein under conditions such that the engineered polynucleotide is expressed, and the engineered polypeptide is produced. In some embodiments, the methods further comprise the step of recovering the engineered polypeptide.

[0044] In another aspect, the present disclosure provides a method of template independent oligonucleotide synthesis, the method comprising contacting an oligonucleotide acceptor substrate with an engineered terminal nucleotidyl transferase described herein in presence of at least a nucleotide triphosphate under reaction conditions sufficient for addition of a nucleotide to the 3’-end of the oligonucleotide acceptor substrate.

[0045] In some embodiments, the nucleotide triphosphate comprises a modified nucleotide trisphosphate, where the modification is at the sugar moiety, nucleobase, and / or phosphate group. In some embodiments of the method the modified nucleotide triphosphate comprises a 3’-reversible blocking group (RGB). DESCRIPTION OF THE INVENTION

[0046] Unless defined otherwise, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Generally, the nomenclature used herein, and the laboratory procedures of cell culture, molecular genetics, microbiology, organic chemistry, analytical chemistry and nucleic acid chemistry described below are those well-known and commonly employed in the art. Such techniques are well-known and described in numerous texts and reference works well known to those of skill in the art. Standard techniques, or modifications thereof, are used for chemical syntheses and chemical analyses. All patents, patent applications, articles and publications mentioned herein, both supra and infra, are hereby expressly incorporated herein by reference.

[0047] Although any suitable methods and materials similar or equivalent to those described herein find use in the practice of the present invention, some methods and materials are described herein. It is to be understood that this invention is not limited to the particular methodology, protocols, and reagents described, as these may vary, depending upon the context they are used by those of skill in the art. Accordingly, the terms defined immediately below are more fully described by reference to the invention as a whole.

[0048] It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the present invention. The section headings used herein are for organizational purposes only and not to be construed as limiting the subjectDocket No. CX10-267WO3 matter described. Numeric ranges are inclusive of the numbers defining the range. Thus, every numerical range disclosed herein is intended to encompass every narrower numerical range that falls within such broader numerical range, as if such narrower numerical ranges were all expressly written herein. It is also intended that every maximum (or minimum) numerical limitation disclosed herein includes every lower (or higher) numerical limitation, as if such lower (or higher) numerical limitations were expressly written herein.

[0049] As used in this specification and the appended claims, the singular forms “a”, “an” and “the” include plural referents unless the context clearly indicates otherwise. Thus, for example, reference to “a polypeptide” includes more than one polypeptide. Similarly, “comprise,” “comprises,” “comprising” “include,” “includes,” and “including” are interchangeable and not intended to be limiting.

[0050] It is to be understood that where descriptions of various embodiments use the term “comprising,” those skilled in the art would understand that in some specific instances, an embodiment can be alternatively described using language “consisting essentially of” or “consisting of.” It is to be further understood that where descriptions of various embodiments use the term “optional” or “optionally” the subsequently described event or circumstance may or may not occur, and that the description includes instances where the event or circumstance occurs and instances in which it does not. It is to be understood that both the foregoing general description, and the following detailed description are exemplary and explanatory only and are not restrictive of this disclosure. The section headings used herein are for organizational purposes only and not to be construed as limiting the subject matter described. Abbreviations

[0051] The abbreviations used for the genetically encoded amino acids are conventional and are as follows: Amino Acid Three-Letter Abbreviation One-Letter Abbreviation Al i Al ADocket No. CX10-267WO3 Amino Acid Three-Letter Abbreviation One-Letter Abbreviation Threonine Thr T“L” or a “D” or clear from the context in which the abbreviation is used, the amino acid may be in either the L- or D-configuration about α-carbon (Cα). For example, whereas “Ala” designates alanine without specifying the configuration about the α-carbon, “D-Ala” and “L-Ala” designate D-alanine and L-alanine, respectively.

[0053] When the one-letter abbreviations are used, upper case letters designate amino acids in the L- configuration about the α-carbon and lower-case letters designate amino acids in the D-configuration about the α-carbon. For example, “A” designates L-alanine and “a” designates D-alanine. When polypeptide sequences are presented as a string of one-letter or three-letter abbreviations (or mixtures thereof), the sequences are presented in the amino (N) to carboxy (C) direction in accordance with common convention.

[0054] The abbreviations used for the genetically encoding nucleosides are conventional and are as follows: adenosine (A); guanosine (G); cytidine (C); thymidine (T); and uridine (U). These abbreviations are also used interchangeably for nucleosides and nucleotides (nucleosides with one or more phosphate groups). Unless specifically delineated, the abbreviated nucleosides or nucleotides may be either ribonucleosides (or ribonucleotides) or 2’-deoxyribonucleosides (or 2’-deoxyribonucleotides). The nucleosides or nucleotides may also be modified at the 3’ position. The nucleosides or nucleotides may be specified as being either ribonucleosides (or ribonucleotides) or 2’-deoxyribonucleosides (or 2’-deoxyribonucleotides) on an individual basis or on an aggregate basis. When nucleic acid sequences are presented as a string of one-letter abbreviations, the sequences are presented in the 5’ to 3’ direction in accordance with common convention, and the phosphates are not indicated. Definitions

[0055] In reference to the present invention, the technical and scientific terms used in the descriptions herein will have the meanings commonly understood by one of ordinary skill in the art, unless specifically defined otherwise. Accordingly, the following terms are intended to have the following meanings.

[0056] “EC” number refers to the Enzyme Nomenclature of the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (NC-IUBMB). The IUBMB biochemical classification is a numerical classification system for enzymes based on the chemical reactions they catalyze.

[0057] “ATCC” refers to the American Type Culture Collection whose biorepository collection includes genes and strains.

[0058] “NCBI” refers to National Center for Biological Information and the sequence databases provided therein.Docket No. CX10-267WO3

[0059] “Protein,” “polypeptide,” and “peptide” are used interchangeably herein to denote a polymer of at least two amino acids covalently linked by an amide bond, regardless of length or post-translational modification (e.g., glycosylation, phosphorylation, lipidation, myristilation, ubiquitination, etc.). Included within this definition are D- and L-amino acids, and mixtures of D- and L-amino acids, as well as polymers comprising D- and L-amino acids, and mixtures of D- and L-amino acids.

[0060] “Amino acids” are referred to herein by either their commonly known three-letter symbols or by the one-letter symbols recommended by IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single letter codes.

[0061] As used herein, “polynucleotide,” “oligonucleotide,” and “nucleic acid’’ are used interchangeably herein and refer to two or more nucleosides or nucleotides that are covalently linked together. The polynucleotide may be wholly comprised of ribonucleotides (i.e., RNA), wholly comprised of 2’ deoxyribonucleotides (i.e., DNA), wholly comprised of other synthetic nucleotides or comprised of mixtures of synthetic, ribo- and / or 2’ deoxyribonucleotides. The polynucleotides may also include modified nucleotides with substitutions, including 2’ substitutions (e.g., 2’-fluoro, 2’-O-methyl, 2’-O-methoxyethyl, locked or constrained ethyl modifications, and others known to those skilled in the art). Nucleosides will be linked together via standard phosphodiester linkages or via one or more non-standard linkages, including but not limited to phosphorothioate linkages. The polynucleotide may be single-stranded or double-stranded or may include both single-stranded regions and double-stranded regions. Moreover, while a polynucleotide will typically be composed of the naturally occurring encoding nucleobases (i.e., adenine, guanine, uracil, thymine, and cytosine), it may include one or more modified and / or synthetic nucleobases, such as, for example, inosine, xanthine, hypoxanthine, etc. In some embodiments, such modified or synthetic nucleobases are nucleobases encoding amino-acid sequences. Nucleobases that are modified or synthetic may comprise any known or hypothetical or future discovered modification or structure that would be recognized by one of skill in the art as a modified or synthetic nucleobase. Similarly, the terms “polynucleotide,” “oligonucleotide,” and “nucleic acid’’ are intended to comprise any modified or synthetic structure that is now known or discovered in the future that would be recognized by one of skill in the art as being or having the function of a “polynucleotide,” “oligonucleotide,” or “nucleic acid.’’ An example of a modified or synthetic structure having the function of a “polynucleotide,” “oligonucleotide,” or “nucleic acid’’ is PNA or peptide nucleic acid.

[0062] As used herein, “oligo acceptor substrate” and “acceptor substrate” and “growing oligo acceptor substrate strand” and “growing oligonucleotide chain” and “growing polynucleotide strand” are used interchangeably herein and refer to any oligo or nucleotide chain or similar moiety with an exposed 3’-OH or equivalent thereof that may be recognized by a wild-type TnT or polymerase or an engineered TnT or template-independent polymerase of the current disclosure as a substrate for nucleoside addition or synthesis. In some embodiments, the acceptor substrate may be single stranded. In yet other embodiments, the acceptor substrate may be double stranded or partially doubled stranded. In some embodiments, the acceptor substrate may comprise a nucleotide chain consisting of 1-10 nucleotides, 5-20 nucleotides, 15-50 nucleotides, 30-100 nucleotides, or greater than 100 nucleotides. In some embodiments, the acceptor substrate may comprise aDocket No. CX10-267WO3 chemical moiety that is not a nucleotide chain but contains a free -OH capable of being recognized as a substrate by a wild-type or engineered TnT, referred to herein as a “3’-OH equivalent”. Exemplary oligo acceptor substrates are provided in the Examples.

[0063] As used herein, “nucleoside triphosphate-3’-O-removable blocking group” and “nucleotide triphosphate-3’-O-removable blocking group” and “reversible terminator” and “NTP-3’-O-RBG” are used interchangeably herein and refer to a ribonucleoside triphosphate or a deoxyribonucleoside triphosphate or a synthetic or nucleoside triphosphate composed of an alternate or modified sugar with a removable blocking group attached at the 3’ position of the sugar moiety. An NTP-3’-O-RBG may also include other modifications as described herein, including but not limited to modifications at the 2’ position, modifications to the nucleobase, and modifications to the phosphates. A polynucleotide may also have a 3’-O-RBG, as is expected after reaction of an NTP-3’-O-RBG with an engineered TnT of the present disclosure and an oligo acceptor substrate.

[0064] As used herein, “oligo acceptor product” and “growing oligonucleotide chain” and “oligo acceptor extension product” are used interchangeably herein and refer to the product of a NTP-3’-O-RBG or other natural or modified NTP substrate and an oligo acceptor substrate, wherein a TnT or related polymerase has catalyzed the extension or addition of a nucleotide-3’-O-RBG or other natural or modified nucleotide substrate to an oligo acceptor substrate via reaction with one or more NTP-3'-O-RBGs or other natural or modified NTP substrates.

[0065] As used herein, “removable blocking group” and “blocking group” and “terminator group” and “reversible terminating group" and “inhibitor group” and related variations of these terms are used interchangeably herein and refer to a chemical group that would hinder addition of a second NTP-3’-O-RBG or other natural or modified NTP substrate to the 3’ end of the growing oligo acceptor substrate strand prior to removal of the removable blocking from the first round of addition. In some embodiments, the NTP-3’-O- RBG or other natural or modified NTP substrate may comprise a removable blocking group selected from the group consisting of NTP-3’-O-NH2, or NTP-3’-O-PO3. In some embodiments, the NTP- 3’-O-RBG or other natural or modified NTP substrate may have a natural purine or pyrimidine base, such as adenine, guanine, cytosine, thymine, or uridine. In some embodiments, NTP- 3’-O-RBG or other natural or modified NTP substrates may have an unnatural base analog such as inosine, xanthine, hypoxanthine, or another base analog, as is known in the art. In some embodiments the blocking group may comprise or may additionally comprise a modification at the 2’ position.

[0066] As used herein, “template-independent synthesis” refers to synthesis of an oligonucleotide or a polynucleotide without the use of template strand as a guide for synthesis of a complementary oligo or polynucleotide strand. Thus, template-independent synthesis refers to an iterative process, whereby, successive nucleotides are added to a growing oligo or nucleotide chain or acceptor substrate. Template- independent synthesis may be in a sequence defined manner or may be random, as is the case with the wild- type TnT in creating antigen receptor diversity. Processes for template-independent synthesis are further described herein.Docket No. CX10-267WO3

[0067] “Coding sequence” refers to that portion of a nucleic acid (e.g., a gene) that encodes an amino acid sequence of a protein.

[0068] “Naturally-occurring” or “wild-type” refers to the form found in nature. For example, a naturally occurring or wild-type polypeptide or polynucleotide sequence is a sequence present in an organism that can be isolated from a source in nature and which has not been intentionally modified by human manipulation.

[0069] As used herein, “recombinant,” “engineered,” and “non-naturally occurring” when used with reference to a cell, nucleic acid, or polypeptide, refer to a material, or a material corresponding to the natural or native form of the material, that has been modified in a manner that would not otherwise exist in nature. In some embodiments, the cell, nucleic acid, or polypeptide is identical to a naturally occurring cell, nucleic acid, or polypeptide, but is produced or derived from synthetic materials and / or by manipulation using recombinant techniques. Non-limiting examples include, among others, recombinant cells expressing genes that are not found within the native (non-recombinant) form of the cell or expressed native genes that are otherwise expressed at a different level.

[0070] “Percentage of sequence identity” and “percentage homology” are used interchangeably herein to refer to comparisons among polynucleotides or polypeptides and are determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide or polypeptide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence for optimal alignment of the two sequences. The percentage may be calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity. Alternatively, the percentage may be calculated by determining the number of positions at which either the identical nucleic acid base or amino acid residue occurs in both sequences or a nucleic acid base or amino acid residue is aligned with a gap to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity. Those of skill in the art appreciate that there are many established algorithms available to align two sequences. Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith and Waterman (Smith and Waterman, Adv. Appl. Math., 1981, 2:482), by the homology alignment algorithm of Needleman and Wunsch (Needleman and Wunsch, J. Mol. Biol., 1970, 48:443), by the search for similarity method of Pearson and Lipman (Pearson and Lipman, Proc. Natl. Acad. Sci. USA, 1988, 85:2444), by computerized implementations of these algorithms (e.g., GAP, BESTFIT, FASTA, and TFASTA in the GCG Wisconsin Software Package), or by visual inspection, as known in the art. Examples of algorithms that are suitable for determining percent sequence identity and sequence similarity include, but are not limited to the BLAST and BLAST 2.0 algorithms, which are described by Altschul et al. (See, Altschul et al., J. Mol. Biol., 1990, 215:403-410; and Altschul et al., Nucl. Acids Res., 1977, 3389-3402, respectively). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information website. This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words ofDocket No. CX10-267WO3 length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as, the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11, an expectation (E) of 10, M=5, N=-4, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a wordlength (W) of 3, an expectation (E) of 10, and the BLOSum62 scoring matrix (See, Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA, 1989, 89:10915). Exemplary determination of sequence alignment and % sequence identity can employ the BESTFIT or GAP programs in the GCG Wisconsin Software package (Accelrys, Madison WI), using default parameters provided.

[0071] “Reference sequence” refers to a defined sequence used as a basis for a sequence comparison. A reference sequence may be a subset of a larger sequence, for example, a segment of a full-length gene or polypeptide sequence. Generally, a reference sequence is at least 20 nucleotide or amino acid residues in length, at least 25 residues in length, at least 50 residues in length, or the full length of the nucleic acid or polypeptide. Since two polynucleotides or polypeptides may each (1) comprise a sequence (i.e., a portion of the complete sequence) that is similar between the two sequences, and (2) may further comprise a sequence that is divergent between the two sequences, sequence comparisons between two (or more) polynucleotides or polypeptide are typically performed by comparing sequences of the two polynucleotides or polypeptides over a “comparison window” to identify and compare local regions of sequence similarity. In some embodiments, a “reference sequence” can be based on a primary amino acid sequence, where the reference sequence is a sequence that can have one or more changes in the primary sequence. For instance, a “reference sequence based on SEQ ID NO: 4 having at the residue corresponding to X14 a valine” or X14V refers to a reference sequence in which the corresponding residue at X14 in SEQ ID NO: 4, which is a tyrosine, has been changed to valine.

[0072] “Comparison window” refers to a conceptual segment of at least about 20 contiguous nucleotide positions or amino acids residues wherein a sequence may be compared to a reference sequence of at least 20 contiguous nucleotides or amino acids and wherein the portion of the sequence in the comparison window may comprise additions or deletions (i.e., gaps) of 20 percent or less as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The comparison window can be longer than 20 contiguous residues, and includes, optionally 30, 40, 50, 100, or longer windows.Docket No. CX10-267WO3

[0073] As used herein, “substantial identity” refers to a polynucleotide or polypeptide sequence that has at least 80 percent sequence identity, at least 85 percent identity, at least between 89 to 95 percent sequence identity, or more usually, at least 99 percent sequence identity as compared to a reference sequence over a comparison window of at least 20 residue positions, frequently over a window of at least 30-50 residues, wherein the percentage of sequence identity is calculated by comparing the reference sequence to a sequence that includes deletions or additions which total 20 percent or less of the reference sequence over the window of comparison. In some specific embodiments applied to polypeptides, the term “substantial identity” means that two polypeptide sequences, when optimally aligned, such as by the programs GAP or BESTFIT using default gap weights, share at least 80 percent sequence identity, preferably at least 89 percent sequence identity, at least 95 percent sequence identity or more (e.g., 99 percent sequence identity). In some embodiments, residue positions that are not identical in sequences being compared differ by conservative amino acid substitutions.

[0074] “Corresponding to,” “reference to,” and “relative to” when used in the context of the numbering of a given amino acid or polynucleotide sequence refer to the numbering of the residues of a specified reference sequence when the given amino acid or polynucleotide sequence is compared to the reference sequence. In other words, the residue number or residue position of a given polymer is designated with respect to the reference sequence rather than by the actual numerical position of the residue within the given amino acid or polynucleotide sequence. For example, a given amino acid sequence, such as that of an engineered TnT, can be aligned to a reference sequence by introducing gaps to optimize residue matches between the two sequences. In these cases, although the gaps are present, the numbering of the residue in the given amino acid or polynucleotide sequence is made with respect to the reference sequence to which it has been aligned.

[0075] “Amino acid difference” or “residue difference” refers to a change in the amino acid residue at a position of a polypeptide sequence relative to the amino acid residue at a corresponding position in a reference sequence. The positions of amino acid differences generally are referred to herein as “Xn,” where n refers to the corresponding position in the reference sequence upon which the residue difference is based. For example, a “residue difference at position X25 as compared to SEQ ID NO: 2” refers to a change of the amino acid residue at the polypeptide position corresponding to position 25 of SEQ ID NO:2. Thus, if the reference polypeptide of SEQ ID NO: 2 has a valine at position 25, then a “residue difference at position X25 as compared to SEQ ID NO:2” an amino acid substitution of any residue other than valine at the position of the polypeptide corresponding to position 25 of SEQ ID NO: 2. In most instances herein, the specific amino acid residue difference at a position is indicated as “XnY” where “Xn” specified the corresponding position as described above, and “Y” is the single letter identifier of the amino acid found in the engineered polypeptide (i.e., the different residue than in the reference polypeptide). In some embodiments, more than one amino acid can appear in a specified residue position (i.e., the alternative amino acids can be listed in the form XnY / Z, where Y and Z represent alternate amino acid residues). In some instances (e.g., in Tables 5.2, 6.2, 7.2, 8.2, 9.2, 10.2, 11.2, 12.2, 13.2, 14.2, 15.2, 16.2, 17.2, 18.2, 19.2, 20.2, 21.2, 22.2, 23.2, 24.2, 25.2, 26.2, 27.2, 28.2, 29.2, 30.2, 35.2, 36.2, 37.2, 38.2, 39.2, 40.2, 41.2, 42.2, 43.2, 44.2, 45.2, 46.2, 47.2, 48.2, 49.2, 50.2, 51.2, 52.2, 53.2, 54.2, 56.2, 57.2, 58.2, 59.2, 60.2, 61.2, 62.2, 63.2, 64.2, 65.2, 66.2, 67.2, 68.2, 69.2, 70.2, 71.2, 72.2, 73.2, 74.2, 75.2, 76.2, 77.2, 78.2, 79.2, 80.2, 81.2, 82.2, 83.2, 84.2, 85.2, 86.2, 87.2, and 88.2.) theDocket No. CX10-267WO3 present invention also provides specific amino acid differences denoted by the conventional notation “AnB”, where A is the single letter identifier of the residue in the reference sequence, “n” is the number of the residue position in the reference sequence, and B is the single letter identifier of the residue substitution in the sequence of the engineered polypeptide. Furthermore, in some instances, a polypeptide of the present invention can include one or more amino acid residue differences relative to a reference sequence, which is indicated by a list of the specified positions where changes are made relative to the reference sequence. In some additional embodiments, the present invention provides engineered polypeptide sequences comprising both conservative and non-conservative amino acid substitutions.

[0076] As used herein, “conservative amino acid substitution” refers to a substitution of a residue with a different residue having a similar side chain, and thus typically involves substitution of the amino acid in the polypeptide with amino acids within the same or similar defined class of amino acids. By way of example and not limitation, an amino acid with an aliphatic side chain is substituted with another aliphatic amino acid (e.g., alanine, valine, leucine, and isoleucine); an amino acid with an hydroxyl side chain is substituted with another amino acid with a hydroxyl side chain (e.g., serine and threonine); an amino acid having an aromatic side chain is substituted with another amino acid having an aromatic side chain (e.g., phenylalanine, tyrosine, tryptophan, and histidine); an amino acid with a basic side chain is substituted with another amino acid with a basic side chain (e.g., lysine and arginine); an amino acid with an acidic side chain is substituted with another amino acid with an acidic side chain (e.g., aspartic acid or glutamic acid); and / or a hydrophobic or hydrophilic amino acid is replaced with another hydrophobic or hydrophilic amino acid, respectively. Exemplary conservative substitutions are provided in Table 1 below. Table 1. Conservative Amino Acid Substitution Examples

[0077] Non-conservative substitution refers to substitution of an amino acid in the polypeptide with an amino acid with significantly differing side chain properties. Non-conservative substitutions may use amino acids between, rather than within, the defined groups and affects (a) the structure of the peptide backbone in the area of the substitution (e.g., proline for glycine), (b) the charge or hydrophobicity, or (c) the bulk of the side chain. By way of example and not limitation, an exemplary non-conservative substitution can be an acidic amino acid substituted with a basic or aliphatic amino acid; an aromatic amino acid substituted with a small amino acid; and a hydrophilic amino acid substituted with a hydrophobic amino acid.

[0078] “Deletion” refers to modification to the polypeptide by removal of one or more amino acids from the reference polypeptide. Deletions can comprise removal of 1 or more amino acids, 2 or more amino acids, 5 orDocket No. CX10-267WO3 more amino acids, 10 or more amino acids, 15 or more amino acids, or 20 or more amino acids, up to 10% of the total number of amino acids, or up to 20% of the total number of amino acids making up the reference enzyme while retaining enzymatic activity and / or retaining the improved properties of an engineered TnT enzyme. Deletions can be directed to the internal portions and / or terminal portions of the polypeptide. In various embodiments, the deletion can comprise a continuous segment or can be discontinuous.

[0079] “Insertion” refers to modification to the polypeptide by addition of one or more amino acids from the reference polypeptide. In some embodiments, the improved engineered TnT enzymes comprise insertions of one or more amino acids to the naturally occurring polypeptide as well as insertions of one or more amino acids to other improved TnT polypeptides. Insertions can be in the internal portions of the polypeptide, or to the carboxy or amino terminus. Insertions as used herein include fusion proteins as is known in the art. The insertion can be a contiguous segment of amino acids or separated by one or more of the amino acids in the naturally occurring polypeptide.

[0080] “Fragment” as used herein refers to a polypeptide that has an amino-terminal and / or carboxy-terminal deletion, but where the remaining amino acid sequence is identical to the corresponding positions in the sequence. Fragments can be at least 14 amino acids long, at least 20 amino acids long, at least 50 amino acids long or longer, and up to 70%, 80%, 90%, 95%, 98%, and 99% of the full-length TnT polypeptide, for example the polypeptide of SEQ ID NO: 2 or an TnT provided in the even-numbered sequences of SEQ ID NO: 2-1056, 1130-1880, and 1928-3008.

[0081] “Functional fragment” and “biologically active fragment” are used interchangeably herein to refer to a polypeptide that has an amino-terminal and / or carboxy-terminal deletion(s) and / or internal deletions, but where the remaining amino acid sequence is identical to the corresponding positions in the sequence to which it is being compared (e.g., a full-length engineered pyruvate oxidase of the present invention) and that retains substantially all of the activity of the full-length polypeptide.

[0082] “Isolated polypeptide” refers to a polypeptide which is substantially separated from other contaminants that naturally accompany it, e.g., protein, lipids, and polynucleotides. The term embraces polypeptides which have been removed or purified from their naturally-occurring environment or expression system (e.g., host cell or in vitro synthesis). The engineered TnT enzymes may be present within a cell, present in the cellular medium, or prepared in various forms, such as lysates or isolated preparations. As such, in some embodiments, the engineered TnT enzyme can be an isolated polypeptide.

[0083] “Substantially pure polypeptide” refers to a composition in which the polypeptide species is the predominant species present (i.e., on a molar or weight basis it is more abundant than any other individual macromolecular species inand is generally a substantially purified composition when the object species comprises at least about 50 percent of the macromolecular species present by mole or % weight. Generally, a substantially pure TnT composition will comprise about 60 % or more, about 70% or more, about 80% or more, about 90% or more, about 95% or more, and about 98% or more of all macromolecular species by mole or % weight present in the composition. In some embodiments, the object species is purified to essential homogeneity (i.e., contaminant species cannot be detected in the composition by conventional detection methods) wherein the composition consists essentially of a single macromolecular species. SolventDocket No. CX10-267WO3 species, small molecules (<500 Daltons), and elemental ion species are not considered macromolecular species. In some embodiments, the isolated engineered TnT polypeptide is a substantially pure polypeptide composition.

[0084] As used herein, “improved enzyme property” refers to at least one improved property of an enzyme. In some embodiments, the present invention provides engineered TnT polypeptides that exhibit an improvement in any enzyme property as compared to a reference TnT polypeptide and / or a wild-type TnT polypeptide, and / or another engineered TnT polypeptide. For the engineered TnT polypeptides described herein, the comparison is generally made to the wild-type enzyme from which the TnT is derived, although in some embodiments, the reference enzyme can be another improved engineered TnT. Thus, the level of “improvement” can be determined and compared between various TnT polypeptides, including wild-type, as well as engineered TnTs. Improved properties include, but are not limited, to such properties as enzymatic activity (which can be expressed in terms of percent conversion of the substrate), thermostability, solvent stability, pH activity profile, cofactor requirements, refractoriness to inhibitors (e.g., substrate or product inhibition), activity at elevated temperatures, increased soluble expression, decreased by-product formation, increased specific activity on NTP-3’-O-RBG substrates, increased incorporation efficiency in extension of oligo acceptor substrates, and / or increased activity on various oligo acceptor substrates (including enantioselectivity).

[0085] “Increased enzymatic activity” refers to an improved property of the TnT polypeptides, which can be represented by an increase in specific activity (e.g., product produced / time / weight protein) or an increase in percent conversion of the substrate to the product (e.g., percent conversion of starting amount of substrate to product in a specified time period using a specified amount of TnT) as compared to the reference TnT enzyme. Exemplary methods to determine enzyme activity are provided in the Examples. Any property relating to enzyme activity may be affected, including the classical enzyme properties of Km, Vmax or kcat, changes of which can lead to increased enzymatic activity. Improvements in enzyme be fromabout 1.2 times the enzymatic activity of the corresponding wild-type enzyme, to as much as 2 times, 5 times, 10 times, 20 times, 25 times, 50 times or more enzymatic activity than the naturally occurring or another engineered TnT from which the TnT polypeptides were derived. TnT activity can be measured by any one of standard assays, such as by monitoring changes in properties of substrates, cofactors, or products. In some embodiments, the amount of products generated can be measured by Liquid Chromatography-Mass Spectrometry (LC-MS), HPLC, or other methods, as known in the art. Comparisons of enzyme activities are made using a defined preparation of enzyme, a defined assay under a set condition, and one or more defined substrates, as further described in detail herein. Generally, when lysates are compared, the numbers of cells and the amount of protein assayed are determined as well as use of identical expression systems and identical host cells to minimize variations in amount of enzyme produced by the host cells and present in the lysates.

[0086] “Conversion” refers to the enzymatic conversion of the substrate(s) to the corresponding product(s). “Percent conversion” refers to the percent of the substrate that is converted to the product within a period of time under specified conditions. Thus, the “enzymatic activity” or “activity” of a TnT polypeptide can be expressed as “percent conversion” of the substrate to the product.Docket No. CX10-267WO3

[0087] “Thermostable” refers to a polypeptide that maintains similar activity (more than 60% to 80% for example) after exposure to elevated temperatures (e.g., 40-80 °C) for a period of time (e.g., 0.5-24 hrs) compared to the wild-type enzyme exposed to the same elevated temperature.

[0088] “Solvent stable” refers to a polypeptide that maintains similar activity (more than e.g., 60% to 80%) after exposure to varying concentrations (e.g., 5-99%) of solvent (ethanol, isopropyl alcohol, dimethylsulfoxide (DMSO), tetrahydrofuran, 2-methyltetrahydrofuran, acetone, toluene, butyl acetate, methyl tert-butyl ether, etc.) for a period of time (e.g., 0.5-24 hrs) compared to the wild-type enzyme exposed to the same concentration of the same solvent.

[0089] “Thermo- and solvent stable” refers to a polypeptide that is both thermostable and solvent stable.

[0090] The term “stringent hybridization conditions” is used herein to refer to conditions under which nucleic acid hybrids are stable. As known to those of skill in the art, the stability of hybrids is reflected in the melting temperature (Tm) of the hybrids. In general, the stability of a hybrid is a function of ion strength, temperature, G / C content, and the presence of chaotropic agents. The Tmvalues for polynucleotides can be calculated using known methods for predicting melting temperatures (See e.g., Baldino et al., Meth. Enzymol., 1989, 168:761-777; Bolton et al., Proc. Natl. Acad. Sci. USA, 1962, 48:1390; Bresslauer et al., Proc. Natl. Acad. Sci. USA, 1986, 83:8893-8897; Freier et al., Proc. Natl. Acad. Sci. USA, 1986, 83:9373-9377; Kierzek et al., Biochem., 1986, 25:7840-7846; Rychlik et al., Nucl. Acids Res., 1990, 18:6409-6412 (erratum, Nucl. Acids Res., 1991, 19:698); Sambrook et al., supra); Suggs et al., 1981, in Developmental Biology Using Purified Genes, Brown et al., eds., pp.683-693, Academic Press, Cambridge, MA (1981); and Wetmur, Crit. Rev. Biochem. Mol. Biol., 1991, 26:227-259). In some embodiments, the polynucleotide encodes the polypeptide disclosed herein and hybridizes under defined conditions, such as moderately stringent or highly stringent conditions, to the complement of a sequence encoding an engineered TnT enzyme of the present invention.

[0091] “Hybridization stringency” relates to hybridization conditions, such as washing conditions, in the hybridization of nucleic acids. Generally, hybridization reactions are performed under conditions of lower stringency, followed by washes of varying but higher stringency. The term “moderately stringent hybridization” refers to conditions that permit target-DNA to bind a complementary nucleic acid that has about 60% identity, preferably about 75% identity, about 85% identity to the target DNA, with greater than about 90% identity to target-polynucleotide. Exemplary moderately stringent conditions are conditions equivalent to hybridization in 50% formamide, 5× Denhart's solution, 5×SSPE, 0.2% SDS at 42 °C, followed by washing in 0.2×SSPE, 0.2% SDS, at 42 °C. “High stringency hybridization” refers generally to conditions that are about 10 °C or less from the thermal melting temperature Tmas determined under the solution condition for a defined polynucleotide sequence. In some embodiments, a high stringency condition refers to conditions that permit hybridization of only those nucleic acid sequences that form stable hybrids in 0.018M NaCl at 65 °C (i.e., if a hybrid is not stable in 0.018M NaCl at 65 °C, it will not be stable under high stringency conditions, as contemplated herein). High stringency conditions can be provided, for example, by hybridization in conditions equivalent to 50% formamide, 5× Denhart's solution, 5×SSPE, 0.2% SDS at 42 °C, followed by washing in 0.1×SSPE, and 0.1% SDS at 65 °C. Another high stringency condition is hybridizingDocket No. CX10-267WO3 in conditions equivalent to hybridizing in 5X SSC containing 0.1% (w:v) SDS at 65 °C and washing in 0.1x SSC containing 0.1% SDS at 65 °C. Other high stringency hybridization conditions, as well as moderately stringent conditions, are described in the references cited above.

[0092] “Heterologous” polynucleotide refers to any polynucleotide that is introduced into a host cell by laboratory techniques and includes polynucleotides that are removed from a host cell, subjected to laboratory manipulation, and then reintroduced into a host cell.

[0093] “Codon optimized” refers to changes in the codons of the polynucleotide encoding a protein to those preferentially used in a particular organism such that the encoded protein is efficiently expressed in the organism of interest. Although the genetic code is degenerate in that most amino acids are represented by several codons, called “synonyms” or “synonymous” codons, it is well known that codon usage by particular organisms is nonrandom and biased towards particular codon triplets. This codon usage bias may be higher in reference to a given gene, genes of common function or ancestral origin, highly expressed proteins versus low copy number proteins, and the aggregate protein coding regions of an organism's genome. In some embodiments, the polynucleotides encoding the TnT enzymes may be codon optimized for optimal production from the host organism selected for expression.

[0094] As used herein, “preferred, optimal, high codon usage bias codons” refers interchangeably to codons that are used at higher frequency in the protein coding regions than other codons that code for the same amino acid. The preferred codons may be determined in relation to codon usage in a single gene, a set of genes of common function or origin, highly expressed genes, the codon frequency in the aggregate protein coding regions of the whole organism, codon frequency in the aggregate protein coding regions of related organisms, or combinations thereof. Codons whose frequency increases with the level of gene expression are typically optimal codons for expression. A variety of methods are known for determining the codon frequency (e.g., codon usage, relative synonymous codon usage) and codon preference in specific organisms, including multivariate analysis, for example, using cluster analysis or correspondence analysis, and the effective number of codons used in a gene (See e.g., GCG CodonPreference, Genetics Computer Group Wisconsin Package; CodonW, Peden, University of Nottingham; McInerney, Bioinform., 1998, 14:372-73; Stenico et al., Nucl. Acids Res., 1994, 222437-46; Wright, Gene, 1999, 87:23-29). Codon usage tables are available for many different organisms (See e.g., Wada et al., Nucl. Acids Res., 1992, 20:2111-2118; Nakamura et al., Nucl. Acids Res., 2000, 28:292; Duret, et al., supra; Henaut and Danchin, in Escherichia coli and Salmonella, Neidhardt, et al. (eds.), ASM Press, Washington D.C., p.2047-2066 (1996)). The data source for obtaining codon usage may rely on any available nucleotide sequence capable of coding for a protein. These data sets include nucleic acid sequences actually known to encode expressed proteins (e.g., complete protein coding sequences-CDS), expressed sequence tags (ESTS), or predicted coding regions of genomic sequences (See e.g., Mount, Bioinformatics: Sequence and Genome Analysis, Chapter 8, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2001); Uberbacher, Meth. Enzymol., 1996, 266:259-281; and Tiwari et al., Comput. Appl. Biosci., 1997, 13:263-270).

[0095] “Control sequence” is defined herein to include all components, which are necessary or advantageous for the expression of a polynucleotide and / or polypeptide of the present invention. Each control sequence mayDocket No. CX10-267WO3 be native or foreign to the nucleic acid sequence encoding the polypeptide. Such control sequences include, but are not limited to, a leader, polyadenylation sequence, propeptide sequence, promoter, signal peptide sequence, and transcription terminator. At a minimum, the control sequences include a promoter, and transcriptional and translational stop signals. The control sequences may be provided with linkers for the purpose of introducing specific restriction sites facilitating ligation of the control sequences with the coding region of the nucleic acid sequence encoding a polypeptide.

[0096] “Operably linked” is defined herein as a configuration in which a control sequence is appropriately placed (i.e., in a functional relationship) at a position relative to a polynucleotide of interest such that the control sequence directs or regulates the expression of the polynucleotide and / or polypeptide of interest.

[0097] “Promoter sequence” refers to a nucleic acid sequence that is recognized by a host cell for expression of a polynucleotide of interest, such as a coding sequence. The promoter sequence contains transcriptional control sequences, which mediate the expression of a polynucleotide of interest. The promoter may be any nucleic acid sequence which shows transcriptional activity in the host cell of choice including mutant, truncated, and hybrid promoters, and may be obtained from genes encoding extracellular or intracellular polypeptides either homologous or heterologous to the host cell.

[0098] “Suitable reaction conditions” refer to those conditions in the biocatalytic reaction solution (e.g., ranges of enzyme loading, substrate loading, cofactor loading, temperature, pH, buffers, co-solvents, etc.) under which a TnT polypeptide of the present invention is capable of converting one or more substrate compounds to a product compound (e.g., addition of a nucleotide-3’-O-RBG or other natural or modified nucleotide substrate to an oligo acceptor substrate via reaction with NTP-3'-O-RBG or other natural or modified NTP substrate). Exemplary “suitable reaction conditions” are provided in the present invention and illustrated by the Examples.

[0099] “Composition” refers to a mixture or combination of one or more substances, wherein each substance or component of the composition retains its individual properties. As used herein, a biocatalytic composition refers to a combination of one or more substances useful for biocatalysis.

[0100] “Loading”, such as in “compound loading” or “enzyme loading” or “cofactor loading” refers to the concentration or amount of a component in a reaction mixture at the start of the reaction.

[0101] “Substrate” in the context of a biocatalyst mediated process refers to the compound or molecule acted on by the biocatalyst. For example, a TnT biocatalyst used in the synthesis processes disclosed herein acts on an NTP-3’-O-RBG substrate or other natural or modified NTP substrate and an oligo acceptor substrate.

[0102] “Product” in the context of a biocatalyst mediated process refers to the compound or molecule resulting from the action of the biocatalyst. For example, an exemplary product for a TnT biocatalyst used in a process disclosed herein is an oligo acceptor extension product, as depicted in Schemes 1 and 2.

[0103] “Alkyl” refers to saturated hydrocarbon groups of from 1 to 18 carbon atoms inclusively, either straight chained or branched, more preferably from 1 to 8 carbon atoms inclusively, and most preferably 1 to 6 carbon atoms inclusively. An alkyl with a specified number of carbon atoms is denoted in parenthesis (e.g., (C1-C6)alkyl refers to an alkyl of 1 to 6 carbon atoms).Docket No. CX10-267WO3

[0104] “Alkenyl” refers to hydrocarbon groups of from 2 to 12 carbon atoms inclusively, either straight or branched containing at least one double bond but optionally containing more than one double bond.

[0105] “Alkynyl” refers to hydrocarbon groups of from 2 to 12 carbon atoms inclusively, either straight or branched containing at least one triple bond but optionally containing more than one triple bond, and additionally optionally containing one or more double bonded moieties.

[0106] “Heteroalkyl, “heteroalkenyl,” and heteroalkynyl,” refer respectively, to alkyl, alkenyl and alkynyl as defined herein in which one or more of the carbon atoms are each independently replaced with the same or different heteroatoms or heteroatomic groups. Heteroatoms and / or heteroatomic groups which can replace the carbon atoms include, but are not limited to -O-, -S-, -S-O-, -NRg-, -PH-, -S(O)-, -S(O)2-, -S(O) NRg-, - S(O)2NRg, and the like, including combinations thereof, where each Rgis independently selected from hydrogen, alkyl, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl.

[0107] “Amino” refers to the group -NH2. Substituted amino refers to the group –NHRh, NRhRh, and NRhRhRh, where each Rhis independently selected from substituted or unsubstituted alkyl, cycloalkyl, cycloheteroalkyl, alkoxy, aryl, heteroaryl, heteroarylalkyl, acyl, alkoxycarbonyl, sulfanyl, sulfinyl, sulfonyl, and the like. Typical amino groups include, but are limited to, dimethylamino, diethylamino, trimethylammonium, triethylammonium, methylysulfonylamino, furanyl-oxy-sulfamino, and the like.

[0108] “Aminoalkyl” refers to an alkyl group in which one or more of the hydrogen atoms are replaced with one or more amino groups, including substituted amino groups.

[0109] “Aminocarbonyl” refers to -C(O)NH2. Substituted aminocarbonyl refers to –C(O)NRhRh, where the amino group NRhRhis as defined herein.

[0110] “Oxy” refers to a divalent group -O-, which may have various substituents to form different oxy groups, including ethers and esters.

[0111] “Alkoxy” or “alkyloxy” are used interchangeably herein to refer to the group –ORz, wherein Rzis an alkyl group, including optionally substituted alkyl groups.

[0112] “Carboxy” refers to -COOH.

[0113] “Carbonyl” refers to -C(O)-, which may have a variety of substituents to form different carbonyl groups including acids, acid halides, aldehydes, amides, esters, and ketones.

[0114] “Carboxyalkyl” refers to an alkyl in which one or more of the hydrogen atoms are replaced with one or more carboxy groups.

[0115] “Aminocarbonylalkyl” refers to an alkyl substituted with an aminocarbonyl group, as defined herein.

[0116] “Halogen” or “halo” refers to fluoro, chloro, bromo and iodo.

[0117] “Haloalkyl” refers to an alkyl group in which one or more of the hydrogen atoms are replaced with a halogen. Thus, the term “haloalkyl” is meant to include monohaloalkyls, dihaloalkyls, trihaloalkyls, etc. up to perhaloalkyls. For example, the expression “(C1- C2) haloalkyl” includes 1-fluoromethyl, difluoromethyl, trifluoromethyl, 1-fluoroethyl, 1,1-difluoroethyl, 1,2-difluoroethyl, 1,1,1 trifluoroethyl, perfluoroethyl, etc.Docket No. CX10-267WO3

[0118] “Hydroxy” refers to -OH.

[0119] “Hydroxyalkyl” refers to an alkyl group in which in which one or more of the hydrogen atoms are replaced with one or more hydroxy groups.

[0120] “Thiol” or “sulfanyl” refers to –SH. Substituted thiol or sulfanyl refers to –S-Rh, where Rhis an alkyl, aryl, or other suitable substituent.

[0121] “Sulfonyl” refers to –SO2-. Substituted sulfonyl refers to –SO2-Rh, where Rhis an alkyl, aryl, or other suitable substituent.

[0122] “Alkylsulfonyl" refers to –SO2-Rz, where Rzis an alkyl, which can be optionally substituted. Typical alkylsulfonyl groups include, but are not limited to, methylsulfonyl, ethylsulfonyl, n-propylsulfonyl, and the like.

[0123] “Phosphate” as used herein refers to a functional group comprised of an orthophosphate ion (phosphorous atom covalently linked to four oxygen atoms). The orthophosphate ion is commonly found with one or more hydrogen atoms or organic groups.

[0124] “Phosphorylated” as used herein refers to the addition or presence of one of more phosphoryl groups (phosphorous atom covalently linked to the three oxygen atoms).

[0125] “Optionally substituted” as used herein with respect to the foregoing chemical groups means that positions of the chemical group occupied by hydrogen can be substituted with another atom (unless otherwise specified) exemplified by, but not limited to carbon, oxygen, nitrogen, or sulfur, or a chemical group, exemplified by, but not limited to, hydroxy, oxo, nitro, methoxy, ethoxy, alkoxy, substituted alkoxy, trifluoromethoxy, haloalkoxy, fluoro, chloro, bromo, iodo, halo, methyl, ethyl, propyl, butyl, alkyl, alkenyl, alkynyl, substituted alkyl, trifluoromethyl, haloalkyl, hydroxyalkyl, alkoxyalkyl, thio, alkylthio, acyl, carboxy, alkoxycarbonyl, carboxamido, substituted carboxamido, alkylsulfonyl, alkylsulfinyl, alkylsulfonylamino, sulfonamido, substituted sulfonamido, cyano, amino, substituted amino, alkylamino, dialkylamino, aminoalkyl, acylamino, amidino, amidoximo, hydroxamoyl, phenyl, aryl, substituted aryl, aryloxy, arylalkyl, arylalkenyl, arylalkynyl, pyridyl, imidazolyl, heteroaryl, substituted heteroaryl, heteroaryloxy, heteroarylalkyl, heteroarylalkenyl, heteroarylalkynyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloalkyl, cycloalkenyl, cycloalkylalkyl, substituted cycloalkyl, cycloalkyloxy, pyrrolidinyl, piperidinyl, morpholino, heterocycle, (heterocycle)oxy, and (heterocycle)alkyl; where preferred heteroatoms are oxygen, nitrogen, and sulfur. Additionally, where open valences exist on these substitute chemical groups they can be further substituted with alkyl, cycloalkyl, aryl, heteroaryl, and / or heterocycle groups, that where these open valences exist on carbon they can be further substituted by halogen and by oxygen-, nitrogen-, or sulfur-bonded substituents, and where multiple such open valences exist, these groups can be joined to form a ring, either by direct formation of a bond or by formation of bonds to a new heteroatom, preferably oxygen, nitrogen, or sulfur. It is further contemplated that the above substitutions can be made provided that replacing the hydrogen with the substituent does not introduce unacceptable instability to the molecules of the present invention and is otherwise chemically reasonable. One of ordinary skill in the art would understand that with respect to any chemical group described as optionally substituted, only sterically practical and / or syntheticallyDocket No. CX10-267WO3 feasible chemical groups are meant to be included. “Optionally substituted” as used herein refers to all subsequent modifiers in a term or series of chemical groups. For example, in the term "optionally substituted arylalkyl,” the “alkyl” portion and the “aryl” portion of the molecule may or may not be substituted, and for the series “optionally substituted alkyl, cycloalkyl, aryl and heteroaryl,” the alkyl, cycloalkyl, aryl, and heteroaryl groups, independently of the others, may or may not be substituted.

[0126] “Reaction” as used herein refers to a process in which one or more substances or compounds or substrates is converted into one or more different substances, compounds, or processes. Template-Independent Synthesis by Engineered TnTs

[0127] New methods of efficiently synthesizing high purity strands of DNA, RNA, and other polynucleotides are necessary to overcome the limitations of existing phosphoramidite chemical synthesis methods in order to enable a range of emerging and existing synthetic biology applications.

[0128] The present invention provides novel terminal nucleotidyl transferases that have improved activity in the template-independent synthesis of polynucleotides using 5’-nucleoside triphosphates (“NTPs”) modified with a 3’-O-removable blocking group (NTP-3’-O-RBG) or other natural or modified NTP substrates. The TnTs of the present disclosure have improved thermostability, activity at elevated temperatures, increased soluble expression or isolated protein yield, decreased by-product formation, increased affinity for NTP-3’-O- RBG and other natural or modified NTP substrates, increased affinity for oligo acceptor substrates, increased activity or specific activity on NTP-3’-O-RBG and other natural or modified NTP substrates, and / or increased activity or specific activity on various oligo acceptor substrates as compared to a wild-type TnT or other TnTs or template-independent polymerases known to those of skill in the art. The engineered polypeptides of the present disclosure are variants of the engineered TnT enzyme of SEQ ID NO: 2 (PCT / US2023 / 076667), which is an engineered variant of a predicted splice variant of the wild-type gene from Monodelphis domestica. These engineered TnTs are capable of template-independent synthesis of oligonucleotides and polynucleotides.

[0129] Template-independent synthesis of a defined polynucleotide sequence using an engineered TnT is a multistep process. In one embodiment, an oligo acceptor substrate with a 3’-OH allows addition of a defined modified NTP substrate (in this example, an NTP-3’-O-RBG) by an engineered TnT, as depicted in Scheme 1, below.Docket No. CX10-267WO3

[0130] After reaction of the NTP-3 -O-RBG with the 3 -OH of oligo acceptor substrate or the growing polynucleotide chain, the TnT is blocked from further reaction by the 3’-O-RBG. The RBG is then removed, exposing the 3’-OH and allowing another round of addition. After each round of addition, the blocking group of the nucleotide-3’-O-RBG or natural or modified nucleotide from the previous round is removed and a new NTP-3’-O-RBG or natural or modified NTP substrate is added to sequentially and efficiently create a defined polynucleotide sequence by addition at the 3’-OH end of the polynucleotide or oligo acceptor substrate without a complimentary strand or templating primer sequence. After synthesis of the defined polynucleotide is complete, the oligonucleotide chain may be cleaved or released from the oligo acceptor substrate.

[0131] A variety of oligo acceptor substrates and NTP-3’-O-RBG or natural or modified NTP substrates may be used in this process, as may be envisioned by one of skill in the art. An example of one reaction is detailed in Scheme 2, below. Scheme 2 depicts the TnT catalyzed reaction of 5’-6-FAM-[N]15AT*mC and 3’-phos- mATP, while other examples of suitable oligo acceptor substrate and NTP-3’-O-RBG or natural or modified NTP pairs are described in the Examples. These examples are non-limiting.Docket No. CX10-267WO3

[0132] Occasionally, undesired synthesis products are created by the TnT during the addition step. This includes incorporation of NTPs that have lost their blocking group, addition of more than one NTP, or the excision or pyrophosphorolysis of the TnT on the growing polynucleotide chain.

[0133] In some embodiments, one or more additional quality control steps are used, such as adding an exonuclease prior to removing the blocking group and initiating a new round of synthesis. In some embodiments, a phosphatase, such as a pyrophosphatase, is used to breakdown inorganic phosphate and push the reversible TnT reaction toward synthesis.

[0134] As described further herein, the engineered TnT polypeptides of the current disclosure exhibit one of more improved properties in the template-independent polynucleotide synthesis process depicted in Schemes 1 and 2.

[0135] In some embodiments, the present invention provides an engineered TnT polypeptide comprising an amino acid sequence having at least 60% sequence identity to an amino acid reference sequence of SEQ ID NO: 2 and further comprising one or more amino acid residue differences as compared to the reference amino acid sequence, wherein the engineered TnT polypeptide has improved thermostability, increased activity at elevated temperatures, increased soluble expression or isolated protein yield, decreased by-product formation, increased specific activity on NTP-3’-O-RBG or natural or modified NTP substrates, and / or increased activity on various oligo acceptor substrates as compared to a wild-type TnT or other TnTs or template-independent polymerases known to those of skill in the art.

[0136] In particular, the engineered TnTs polypeptides of the present disclosure have been engineered for efficient synthesis of polynucleotides having a defined sequence using NTP-3’-O-RBG or natural or modified NTP substrates in the process described above.Docket No. CX10-267WO3

[0137] A variety of suitable reaction conditions are known to those skilled in the art, as detailed below and in the Examples. Engineered Terminal Nucleotidyl Transferase (TnT) Polypeptides

[0138] The present invention provides engineered terminal nucleotidyl transferase (TnT) polypeptides useful in template-independent polynucleotide synthesis using an NTP-3’-O-RBG or natural or modified NTP substrate, as well as compositions and methods of utilizing these engineered polypeptides in template- independent oligonucleotide synthesis.

[0139] The present invention provides TnT polypeptides, polynucleotides encoding the polypeptides, methods of preparing the polypeptides, and methods for using the polypeptides. Where the description relates to polypeptides, it is to be understood that it can describe the polynucleotides encoding the polypeptides.

[0140] Suitable reaction conditions under which the above-described improved properties of the engineered polypeptides carry out the desired reaction can be determined with respect to concentrations or amounts of polypeptide, substrate, co-substrate, buffer, solvent, pH, conditions including temperature and reaction time, and / or conditions with the polypeptide immobilized on a solid support, as further described below and in the Examples.

[0141] In some embodiments, exemplary engineered TnTs comprise an amino acid sequence that has one or more residue differences as compared to SEQ ID NO: 2 at the residue positions indicated in Tables 5.2, 6.2, 7.2, 8.2, 9.2, 10.2, 11.2, 12.2, 13.2, 14.2, 15.2, 16.2, 17.2, 18.2, 19.2, 20.2, 21.2, 22.2, 23.2, 24.2, 25.2, 26.2, 27.2, 28.2, 29.2, 30.2, 35.2, 36.2, 37.2, 38.2, 39.2, 40.2, 41.2, 42.2, 43.2, 44.2, 45.2, 46.2, 47.2, 48.2, 49.2, 50.2, 51.2, 52.2, 53.2, 54.2, 56.2, 57.2, 58.2, 59.2, 60.2, 61.2, 62.2, 63.2, 64.2, 65.2, 66.2, 67.2, 68.2, 69.2, 70.2, 71.2, 72.2, 73.2, 74.2, 75.2, 76.2, 77.2, 78.2, 79.2, 80.2, 81.2, 82.2, 83.2, 84.2, 85.2, 86.2, 87.2, and 88.2.

[0142] The structure and function information for the exemplary engineered polypeptides of the present invention are based on the conversion of an oligo acceptor substrate and a NTP -3’-O-RBG or a dideoxy NTP (e.g., a 2',3'-dideoxy NTP), the results of which are shown below in Tables 5.2, 6.2, 7.2, 8.2, 9.2, 10.2, 11.2, 12.2, 13.2, 14.2, 15.2, 16.2, 17.2, 18.2, 19.2, 20.2, 21.2, 22.2, 23.2, 24.2, 25.2, 26.2, 27.2, 28.2, 29.2, 30.2, 35.2, 36.2, 37.2, 38.2, 39.2, 40.2, 41.2, 42.2, 43.2, 44.2, 45.2, 46.2, 47.2, 48.2, 49.2, 50.2, 51.2, 52.2, 53.2, 54.2, 56.2, 57.2, 58.2, 59.2, 60.2, 61.2, 62.2, 63.2, 64.2, 65.2, 66.2, 67.2, 68.2, 69.2, 70.2, 71.2, 72.2, 73.2, 74.2, 75.2, 76.2, 77.2, 78.2, 79.2, 80.2, 81.2, 82.2, 83.2, 84.2, 85.2, 86.2, 87.2, and 88.2, as further described in the Examples. The odd numbered sequence identifiers (i.e., SEQ ID NOs) in these Tables refer to the nucleotide sequence encoding the amino acid sequence provided by the even numbered SEQ ID NOs in these Tables. Exemplary sequences are provided in the electronic Sequence Listing file accompanying this disclosure, which is hereby incorporated by reference herein. The amino acid residue differences are based on comparison to the reference sequence of SEQ ID NO: 2, 8, 70, 148, 184, 234, 256, 478, 668, 682, 730, 734, 742, 932, 992, 1144, 1212, 1236, 1264, 1414, 1504, 1522, 1702, 1742, 1928, 1944, 1946, 1984, 2064, 2104, 2210, 2256, 2280, 2342, 2438, 2452, 2562, 2576, 2714, 2876, 2884, 2910, 2974, 2998, or 3002, as indicated. It is to be understood that the amino acid residue differences can also be made in comparison to the reference sequence without the histidine-tag, e.g., a reference sequence corresponding to amino acid residues 12 to theDocket No. CX10-267WO3 carboxy terminus of an even-numbered SEQ ID NO: 2, 8, 70, 148, 184, 234, 256, 478, 668, 682, 730, 734, 742, 932, 992, 1144, 1212, 1236, 1264, 1414, 1504, 1522, 1702, 1742, 1928, 1944, 1946, 1984, 2064, 2104, 2210, 2256, 2280, 2342, 2438, 2452, 2562, 2576, 2714, 2876, 2884, 2910, 2974, 2998, or 3002.

[0143] Terminal deoxynucleotidyl transferase, a member of the Pol X family, has been identified in many species. Members of the diverse Pol X family are known to share certain residues, which are conserved across family members. TdT also has a high level of conservation across species for residues thought to be involved in binding divalent metal ions, ternary complex formation, and binding dNTP and DNA ligands (Domínguez et al. (2000). EMBO, 19(7), 1731–1742.) Additionally, TdTs are known to have splice variants which are N- terminal truncations, lacking a BRCT domain.

[0144] Other template-independent polymerases (including, but not limited to Poly(N) polymerases, Polμ polymerases, Polβ polymerases, Polλ polymerases, terminal urildylytransferases, and Polθ polymerases, and others) are also known in the art and may be used to practice the invention (see for example, U.S. Pat. 10059929, U.S. Pat.10,760,063, U.S. Pat.10,774,316, U.S. Pat.11,390,858, U.S. Pat.10,745,727, PCT / GB2020 / 050247, US20210164008A1, US20210407509, WO2016GB50301, WO17216472, WO18215803, WO20072715, WO20077227, WO21122539, WO2022029427, and WO21116270, from Molecular Assemblies, Nuclera, DNA Script, and others, each of which is specifically incorporated herein).

[0145] Similarly, other polymerases are known to be capable of template-independent synthesis (including but not limited to reverse transcriptases) and may be used to practice the invention.

[0146] As used herein, the term terminal nucleotidyl transferase (TnT) is used to distinguish an engineered enzyme with activity on a variety of nucleoside triphosphates, including ribonucleoside triphosphates with 3' modifications or with 2' modifications or with 2' and 3' modifications, from a wild-type TdT enzyme having wild-type TdT activity.

[0147] The engineered TnT enzyme of SEQ ID NO: 2 (PCT / US2023 / 076667, published as WO2024081770), which is an engineered variant of a predicted splice variant of the wild-type gene from Monodelphis domestica, was selected for evolution. The TnT polypeptides of the present disclosure are engineered variants of SEQ ID NO: 2 with a N-terminal 6-histidine tag.

[0148] The polypeptides of the present disclosure have residue differences that result in improved properties necessary to develop an efficient TnT enzyme, capable of template-independent synthesis of polynucleotides, particularly polynucleotides having a defined sequence. Various residue differences, at both conserved and non-conserved positions, have been discovered to be related to improvements in various enzymes properties, including improved thermostability, increased activity at elevated temperatures, increased soluble expression or isolated protein yield, decreased by-product formation, increased specific activity on NTP-3’-O-RBG or natural or modified NTP substrates, increased incorporation efficiency in extension of oligo acceptor substrates, and / or increased activity on various oligo acceptor substrates as compared to a wild-type TnT or other TnTs or template-independent polymerases known to those of skill in the art. In some embodiments, the engineered TnT polypeptides exhibit increased incorporation efficiency in extension of an oligo acceptor substrate by addition of an NTP or NQP of greater than 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%,Docket No. CX10-267WO3 93%, 94%, 95%, 96%, 97%, 98%, or 99%. Exemplary incorporation efficiency of engineered TnTs is described in the Examples.

[0149] The polypeptides of the present disclosure are capable of incorporation of various NTP-3’-O-RBG or natural or modified NTP substrates using an optionally modified oligo acceptor substrate with a length of three to seven nucleotides, as provided in Examples 31 and 32. In some embodiments, the incorporation efficiency of the TnT polypeptides described herein is enhanced by addition of a phosphate to the 5’ end of an optionally modified oligo acceptor substrate with a length of three to seven nucleotides.

[0150] The activity of each engineered TnT relative to the reference polypeptide of SEQ ID NOs: 2, 8, 70, 148, 184, 234, 256, 478, 668, 682, 730, 734, 742, 932, 992, 1144, 1212, 1236, 1264, 1414, 1504, 1522, 1702, 1742, 1928, 1944, 1946, 1984, 2064, 2104, 2210, 2256, 2280, 2342, 2438, 2452, 2562, 2576, 2714, 2876, 2884, 2910, 2974, 2998, or 3002, was determined as conversion of the substrates described in the Examples herein. In some embodiments, a shake flask purified enzyme (SFP) is used to assess the properties of the engineered TnTs, the results of which are provided in the Examples.

[0151] In some embodiments, the specific enzyme properties are associated with the residues differences as compared to SEQ ID NOs: 2, 8, 70, 148, 184, 234, 256, 478, 668, 682, 730, 734, 742, 932, 992, 1144, 1212, 1236, 1264, 1414, 1504, 1522, 1702, 1742, 1928, 1944, 1946, 1984, 2064, 2104, 2210, 2256, 2280, 2342, 2438, 2452, 2562, 2576, 2714, 2876, 2884, 2910, 2974, 2998, or 3002 at the residue positions indicated herein. In some embodiments, residue differences affecting polypeptide expression can be used to increase expression of the engineered TnTs.

[0152] In light of the guidance provided herein, it is further contemplated that any of the exemplary engineered polypeptides comprising the sequences of SEQ ID NOs: 2, 8, 70, 148, 184, 234, 256, 478, 668, 682, 730, 734, 742, 932, 992, 1144, 1212, 1236, 1264, 1414, 1504, 1522, 1702, 1742, 1928, 1944, 1946, 1984, 2064, 2104, 2210, 2256, 2280, 2342, 2438, 2452, 2562, 2576, 2714, 2876, 2884, 2910, 2974, 2998, or 3002 find use as the starting amino acid sequence for synthesizing other TnT polypeptides, for example by subsequent rounds of evolution that incorporate new combinations of various amino acid differences from other polypeptides in Tables 5.2, 6.2, 7.2, 8.2, 9.2, 10.2, 11.2, 12.2, 13.2, 14.2, 15.2, 16.2, 17.2, 18.2, 19.2, 20.2, 21.2, 22.2, 23.2, 24.2, 25.2, 26.2, 27.2, 28.2, 29.2, 30.2, 35.2, 36.2, 37.2, 38.2, 39.2, 40.2, 41.2, 42.2, 43.2, 44.2, 45.2, 46.2, 47.2, 48.2, 49.2, 50.2, 51.2, 52.2, 53.2, 54.2, 56.2, 57.2, 58.2, 59.2, 60.2, 61.2, 62.2, 63.2, 64.2, 65.2, 66.2, 67.2, 68.2, 69.2, 70.2, 71.2, 72.2, 73.2, 74.2, 75.2, 76.2, 77.2, 78.2, 79.2, 80.2, 81.2, 82.2, 83.2, 84.2, 85.2, 86.2, 87.2, and 88.2, and other residue positions described herein. Further improvements may be generated by including amino acid differences at residue positions that had been maintained as unchanged throughout earlier rounds of evolution.

[0153] In one aspect, the engineered terminal nucleotidyl transferase, or a functional fragment thereof, comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to the carboxy terminus of an even-numbered SEQ ID NO. of SEQ ID NOs: 2, 4-1056, 1130-1880, and 1928-3008, or to a reference sequence corresponding to SEQ ID NO: 2, 4-1056, 1130-1880, and 1928-3008, wherein the amino acid sequenceDocket No. CX10-267WO3 comprises one or more amino differences relative to the reference sequence corresponding to amino acid residues 12 to the carboxy terminus of SEQ ID NO: 2, 8, 70, 148, 184, 234, 256, 478, 668, 682, 730, 734, 742, 932, 992, 1144, 1212, 1236, 1264, 1414, 1504, 1522, 1702, 1742, 1928, 1944, 1946, 1984, 2064, 2104, 2210, 2256, 2280, 2342, 2438, 2452, 2562, 2576, 2714, 2876, 2884, 2910, 2974, 2998, or 3002, or to the reference sequence corresponding to SEQ ID NO: 2, 8, 70, 148, 184, 234, 256, 478, 668, 682, 730, 734, 742, 932, 992, 1144, 1212, 1236, 1264, 1414, 1504, 1522, 1702, 1742, 1928, 1944, 1946, 1984, 2064, 2104, 2210, 2256, 2280, 2342, 2438, 2452, 2562, 2576, 2714, 2876, 2884, 2910, 2974, 2998, or 3002.

[0154] In some embodiments, the engineered terminal nucleotidyl transferase comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to the carboxy terminus of SEQ ID NO: 2, 8, 70, 148, 184, 234, 256, 478, 668, 682, 730, 734, 742, 932, 992, 1144, 1212, 1236, 1264, 1414, 1504, 1522, 1702, 1742, 1928, 1944, 1946, 1984, 2064, 2104, 2210, 2256, 2280, 2342, 2438, 2452, 2562, 2576, 2714, 2876, 2884, 2910, 2974, 2998, or 3002, or to a reference sequence corresponding to SEQ ID NO: 2, 8, 70, 148, 184, 234, 256, 478, 668, 682, 730, 734, 742, 932, 992, 1144, 1212, 1236, 1264, 1414, 1504, 1522, 1702, 1742, 1928, 1944, 1946, 1984, 2064, 2104, 2210, 2256, 2280, 2342, 2438, 2452, 2562, 2576, 2714, 2876, 2884, 2910, 2974, 2998, or 3002, wherein the amino acid sequence comprises one or more amino differences relative to the reference sequence corresponding to amino acid residues 12 to the carboxy terminus of SEQ ID NO: 2, 8, 70, 148, 184, 234, 256, 478, 668, 682, 730, 734, 742, 932, 992, 1144, 1212, 1236, 1264, 1414, 1504, 1522, 1702, 1742, 1928, 1944, 1946, 1984, 2064, 2104, 2210, 2256, 2280, 2342, 2438, 2452, 2562, 2576, 2714, 2876, 2884, 2910, 2974, 2998, or 3002, or to the reference sequence corresponding to SEQ ID NO: 2, 8, 70, 148, 184, 234, 256, 478, 668, 682, 730, 734, 742, 932, 992, 1144, 1212, 1236, 1264, 1414, 1504, 1522, 1702, 1742, 1928, 1944, 1946, 1984, 2064, 2104, 2210, 2256, 2280, 2342, 2438, 2452, 2562, 2576, 2714, 2876, 2884, 2910, 2974, 2998, or 3002.

[0155] In some embodiments, the engineered terminal nucleotidyl transferase comprises an amino acid sequence comprising at least an amino acid residue difference at amino acid position 22, 27, 34, 41, 42, 43, 45, 52, 60, 92, 105, 133, 152, 160, 161, 162, 164, 169, 173, 174, 175, 176, 177, 179, 182, 183, 184, 186, 187, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 208, 212, 213, 218, 219, 221, 222, 223, 224, 227, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 241, 242, 244, 246, 249, 250, 251, 252, 254, 259, 260, 261, 262, 263, 264, 267, 268, 272, 273, 274, 275, 277, 278, 280, 281, 282, 288, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 307, 308, 309, 310, 312, 313, 314, 315, 316, 317, 318, 319, 321, 322, 323, 324, 325, 328, 329, 333, 334, 335, 336, 337, 339, 340, 341, 342, 343, 347, 352, 353, 354, 357, 359, 360, 361, 362, 363, 364, 366, 367, 368, 370, 371, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 387, 388, 389, 390, 392, 393, 394, 395, 396, 397, 398, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 446, 448, 450, 451, 456, 457, 458, 459, 460, 461, 462, 463, 465, 466, 467, 468, 474, 477, 479, 480, 481, 484, 485, 487, 490, 492, 493, 494, 495, 497, 499, 500, 501, 502, 503, 504, 506, 507, 508, 513, 515, 517, 518, 522, 523, 525, or 526, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12 to the carboxyDocket No. CX10-267WO3 terminus of SEQ ID NO: 2, 8, 70, 148, 184, 234, 256, 478, 668, 682, 730, 734, 742, 932, 992, 1144, 1212, 1236, 1264, 1414, 1504, 1522, 1702, 1742, 1928, 1944, 1946, 1984, 2064, 2104, 2210, 2256, 2280, 2342, 2438, 2452, 2562, 2576, 2714, 2876, 2884, 2910, 2974, 2998, or 3002, or to a reference sequence corresponding to SEQ ID NO: 2, 8, 70, 148, 184, 234, 256, 478, 668, 682, 730, 734, 742, 932, 992, 1144, 1212, 1236, 1264, 1414, 1504, 1522, 1702, 1742, 1928, 1944, 1946, 1984, 2064, 2104, 2210, 2256, 2280, 2342, 2438, 2452, 2562, 2576, 2714, 2876, 2884, 2910, 2974, 2998, or 3002.

[0156] In some embodiments, the engineered terminal nucleotidyl transferase comprises an amino acid sequence comprising at least an amino acid residue difference or amino acid residue 22P, 27P, 34Y, 41T, 42T, 43- / T, 45T, 52G, 60H, R92C, 105G, 133E, 152H, 160A / D / T / W, 161A / G / L / Q / T, 162P, 164Q, 169L, 173G / H / P, 174L / V / W, 175E / H / L / P / R / S / T / V, 176H, 177C / K, 179C / K / L / R / V, 182I / V, 183G / R / S / T / V, 184K / R, 186G / L / M / R / T / V, 187L / T / V / Y, 190A / F / G / H / L / M / N / Q / R / T / V, 191F / W / Y, 192A / H / S, 193A / G / I / Q / R / S / T / W, 194L / M, 195F / Q / R / T / W, 196D / H / K / L / Q / R / S / V, 197I / L / M / Q / R, 198E / H / P / Q / R / S / T, 199E / F / V, 200Q, 201D / E / P / Q / R / S, 202H / I / M / S / V, 203F / L / M / Q, 204M / V, 205L 208A / D / E / G / M / N / Q / T, 212R, 213G, 218I, 219D / Q / S, 221I / Q / V, 222A / N, 223H / L / T, 224L / V, 227G / S, 229F / G / L / S, 230C / L / V, 231C / M / N / T, 232E / G, 233I / P, 234T, 235N / R, 236R / S, 237L, 238F / I / L / V, 239G / L / R / S, 241L, 242L, 244G / I / R / S, 246L / V, 249L / M / Q, 250G, 251A / E / G / M / R / S / W, 252E / H / L / M / R / S, 254I / N / Q / R, 259F, 260K / R / S / T, 261G / R / T / V, 262L / M, 263A / D / K / R / S / T / W, 264L / R / S, 267G / S, 268L, 272R, 273E / G / I / K / L / M / / N / Q / R, 274L / N / P / R / S / T / V, 275I / L / R / S, 277E / S / T, 278A / D / L / R / S, 280W, 281A / C / G / H / L / S, 282N, 288D, 290L, 291K / L, 292A, 293R, 294G / T, 295A / D / N / T, 296M / V, 297A / G / L / M / P / Q / S / T / V / W / Y, 298I, 299A / G / H / N / P / R / S, 300A / E / F / G / I / K / L / M / N / Q / R / S / V / Y, 301A / F / V, 302S, 303E / R / T, 304L, 307D / E / K / M / N / S, 308H / L, 309H, 310D / S, 312M, 313H / Q / R / S / T, 314A / E / I / S / T, 315K / R, 316L, 317R, 318D / I / K / L / P / Q / S / T, 319G / L / M / P / Q / T / V / W, 321R / T, 322A / E, 323T, 324A / T, 325G / S / V / Y, 328A / I, 329H / L, 333N / S, 334S, 335V, 336C / T, 337S, 339I / S / T / V, 340L / M / T / V, 341L / T, 342S / T, 343L / M / R / T / V, 347A / S, 352A / E / L / M / N / S / T / V, 353E / K / L / R / T / V, 354G, 357I, 359A / C / F / V, 360V, 361L / V, 362A / M, 363G / P / T, 364S, 366D / F / H / P, 367N / Q / R / S / V, 368L / N / R / S, 370E / F / L / R, 371G / R, 376A / E / G / H / R / S / T / V, 377R, 378M, 379L / T, 380A / K / L / S / V, 381S / Y, 382L / M, 383L / Q, 384A / G / S / T, 385V, 387T / V, 388V, 389A / V, 390I / L / M / S / T, 392E / H / N / Q / R / S, 393A / G / I / L / M / S / T / Y, 394K / N / S, 395E / F / I / L / M / Q / S / T, 396T, 397E / F / G / H / I / K / L / M / R / S / T / V / W / Y, 398L / M, 402A / D / K / M / P / S / T, 403E / F / I / P, 404A / E / G / I / L / M / Q / R / S / T / V, 405A / G / L / P / Q / T / V / Y, 406A / D / E / H / M / N / P / R / T, 407A / D / G / L / N / P / Q / S / V / Y, 408D / G / L / N / S / V, 409E / G / K / N / P / Q / R / S / V / Y, 410V, 411E / H / K / R / T / V, 412M / N / P / Q / S / V, 413S / T, 414M / Q / R / V, 415G / P / S, 416A / G / H, 417A / G / S, 418C / S / T, 419A / I / M, 420C / L / V, 424P, 425R, 426G, 427A / M, 428A / L / T, 429A / S, 430G / Q / S / W, 431G, 432I / V, 433A / P / T / V, 434G / L / P, 435F / L / N / T / V, 436C / G / H / L / S / T, 437A / H / P / S, 438P / V, 439C / L / P, 440D / M, 441E / T, 442E / L / M / R / V, 443A / D / P / V, 444G, 446D / G / H / S / T, 448R, 450V, 451R, 456M, 457T, 458K, 459L, 460A / D / H / L / Q / R / S / V, 461A / V, 462H / L / M / Y, 463P / S, 465G, 466I / L, 467M, 468H / I / M, 474H / I / L / V, 477A / E / G / I / L / Q / S / T / W, 479M / T / V, 480K / M / W, 481H, 484A / L / W / Y, 485I / L / V, 487R, 490A / I / L / V, 492A / C / G / H / M / R / S / Y, 493R, 494L / M / Q / R / T / V / Y, 495A / G / L / T, 497L, 499L / N / R / S, 500A / N, 501K / L, 502M, 503L / R / V, 504P / S / T, 506A / K / L, 507G, 508C / D / L / Q, 513V, 515G, 517I, 518S, 522A / F / M / V, 523A / D / H, 525G / H / I / R / T / V / W, or 526E / G, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding toDocket No. CX10-267WO3 amino acid residues 12 to the carboxy terminus of SEQ ID NO: 2, or to a reference sequence corresponding to SEQ ID NO: 2.

[0157] In some embodiments, the engineered terminal nucleotidyl transferase comprises an amino acid sequence comprising at least an amino acid residue difference or amino acid residue F22P, Q27P, D34Y, I41T, K42T, F43- / T, E45T, E52G, R60H, R92C, E105G, K133E, R152H, M160A / D / T / W, K161A / G / L / Q / T, T162P, S164Q, Q169L, T173G / H / P, L174L / V / W, N175E / H / L / P / R / S / T / V, N176H, R177C / K, Q179C / K / L / R / V, T182I / V, D183G / R / S / T / V, S184K / R, E186G / L / M / R / T / V, I187L / T / V / Y, E190A / F / G / H / L / M / N / Q / R / T / V, N191F / W / Y, Y192A / H / S, V193A / G / I / Q / R / S / T / W, F194L / M, K195F / Q / R / T / W, G196D / H / K / L / Q / R / S / V, N197I / L / M / Q / R, D198E / H / P / Q / R / S / T, D199E / F / V, K200Q, A201D / E / P / Q / R / S, L202H / I / M / S / V, E203F / L / M / Q, F204M / V, R205L I208A / D / E / G / M / N / Q / T, K212R, S213G, V218I, R219D / Q / S, L221I / Q / V, K222A / N, D223H / L / T, T224L / V, L227G / S, W229F / G / L / S, I230C / L / V, S231C / M / N / T, D232E / G, E233I / P, V234T, K235N / R, G236R / S, I237L, M238F / I / L / V, E239G / L / R / S, I241L, I242L, V244G / I / R / S, E246L / V, E249L / M / Q, V250G, Q251A / E / G / M / R / S / W, A252E / H / L / M / R / S, L254I / N / Q / R, Y259F, Q260K / R / S / T, S261G / R / T / V, F262L / M, H263A / D / K / R / S / T / W, A264L / R / S, R267G / S, V268L, G272R, V273E / G / I / K / L / M / / N / Q / R, G274L / N / P / R / S / T / V, T275I / L / R / S, D277E / S / T, K278A / D / L / R / S, Y280W, Q281A / C / G / H / L / S, M282N, E288D, V290L, R291K / L, S292A, S293R, K294G / T, T295A / D / N / T, L296M / V, K297A / G / L / M / P / Q / S / T / V / W / Y, L298I, T299A / G / H / N / P / R / S, T300A / E / F / G / I / K / L / M / N / Q / R / S / V / Y, W301A / F / V, A302S, K303E / R / T, V304L, T307D / E / K / M / N / S, Y308H / L, Y309H, E310D / S, L312M, A313H / Q / R / S / T, R314A / E / I / S / T, G315K / R, V316L, T317R, R318D / I / K / L / P / Q / S / T, , A319G / L / M / P / Q / T / V / W, A321R / T, D322A / E, A323T, I324A / T, W325G / S / V / Y, V328A / I, Q329H / L, A333N / S, T334S, F335V, D336C / T, P337S, A339I / S / T / V, I340L / M / T / V, V341L / T, E342S / T, I343L / M / R / T / V, Q347A / S, P352A / E / L / M / N / S / T / V, Q353E / K / L / R / T / V, S354G, V357I, L359A / C / F / V, L360V, I361L / V, S362A / M, S363G / P / T, P364S, A366D / F / H / P, E367N / Q / R / S / V, K368L / N / R / S, Q370E / F / L / R, E371G / R, Q376A / E / G / H / R / S / T / V, K377R, V378M, M379L / T, R380A / K / L / S / V, L381S / Y, W382L / M, K383L / Q, K384A / G / S / T, Q385V, L387T / V, L388V, L389A / V, C390I / L / M / S / T, K392E / H / N / Q / R / S, V393A / G / I / L / M / S / T / Y, Y394K / N / S, W395E / F / I / L / M / Q / S / T, S396T, Q397E / F / G / H / I / K / L / M / R / S / T / V / W / Y, W398L / M, G402A / D / K / M / P / S / T, L403E / F / I / P, P404A / E / G / I / L / M / Q / R / S / T / V, S405A / G / L / P / Q / T / V / Y, G406A / D / E / H / M / N / P / R / T, K407A / D / G / L / N / P / Q / S / V / Y, A408D / G / L / N / S / V, D409E / G / K / N / P / Q / R / S / V / Y, E410V, G411E / H / K / R / T / V, D412M / N / P / Q / S / V, G413S / T, E414M / Q / R / V, A415G / P / S, S416A / G / H, C417A / G / S, F418C / S / T, L419A / I / M, I420C / L / V, Y424P, D425R, H426G, L427A / M, V428A / L / T, R429A / S, K430G / Q / S / W, S431G, K432I / V, H433A / P / T / V, N434G / L / P, M435F / L / N / T / V, P436C / G / H / L / S / T, T437A / H / P / S, G438P / V, S439C / L / P, N440D / M, M441E / T, G442E / L / M / R / V, E443A / D / P / V, S444G, P446D / G / H / S / T, K448R, I450V, K451R, R456M, C457T, P458K, R459L, G460A / D / H / L / Q / R / S / V, S461A / V, F462H / L / M / Y / , A463P / S, A465G, M466I / L, L467M, Q468H / I / M, M474H / I / L / V, R477A / E / G / I / L / Q / S / T / W, L479M / T / V, R480K / M / W, D481H, R484A / L / W / Y, E485I / L / V, K487R, M490A / I / L / V, D492A / C / G / H / M / R / S / Y, N493R, H494L / M / Q / R / T / V / Y, S495A / G / L / T, Y497L, M499L / N / R / S, T500A / N, R501K / L, G502M, I503L / R / V, Q504P / S / T, P506A / K / L, A507G, K508C / D / L / Q, I513V, E515G, L517I, G518S, L522A / F / M / V, E523A / D / H, F525G / H / I / R / T / V / W, or Y526E / G, or combinations thereof, wherein the amino acid positions are relative to the reference sequenceDocket No. CX10-267WO3 corresponding to amino acid residues 12 to the carboxy terminus of SEQ ID NO: 2, or to a reference sequence corresponding to SEQ ID NO: 2.

[0158] In some embodiments, the engineered terminal nucleotidyl transferase comprises an amino acid sequence comprising at least an amino acid residue difference at amino acid position 22, 34, 60, 160, 162, 173, 174, 176, 177, 179, 187, 190, 191, 193, 194, 197, 203, 204, 205, 208, 222, 224, 230, 231, 236, 246, 251, 254, 260, 261, 262, 263, 264, 267, 273, 274, 277, 278, 295, 296, 297, 299, 300, 308, 310, 313, 318, 319, 325, 329, 334, 335, 339, 342, 343, 352, 353, 357, 361, 363, 367, 368, 376, 379, 381, 390, 396, 397, 402, 404, 405, 406, 407, 408, 409, 412, 416, 417, 420, 433, 437, 444, 451, 460, 462, 465, 466, 477, 480, 485, 487, 490, 492, 495, 503, 504, 508, or 525, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12 to the carboxy terminus of SEQ ID NO: 2, or to a reference sequence corresponding to SEQ ID NO: 2.

[0159] In some embodiments, the engineered terminal nucleotidyl transferase comprises an amino acid sequence comprising at least an amino acid residue difference or amino acid residue 22P, 34Y, 60H, 160T, 162P, 173P, 174I / V / W, 176H, 177K, 179K, 187T / Y, 190R / T, 191F, 193S / T, 194L, 197M, 203M, 204M, 205L, 208E / G / Q, 222A, 224V, 230V, 231M / N, 236R, 246V, 251A, 254I / R, 260R, 261V, 262M, 263R / S, 264L, 267S, 273I / K / R / M, 274P, 277S, 278S, 295D, 296V, 297S, 299S, 300K / S, 308H, 310D, 313R, 318K / P / Q, 319T / V, 325V, 329L, 334S, 335V, 339S / T / V, 342T, 343L, 352T, 353L / R / V, 357I, 361V, 363P, 367Q, 368S, 376E, 379L, 381Y, 390M / S, 396T, 397K, 402S, 404S, 405Q, 406P, 407A / P, 408V, 409E, 412P, 416A, 417A, 420V, 433P, 437H, 444G, 451R, 460Q, 462L / M, 465G, 466I, 477A / L / Q, 480K, 485I, 487R, 490V, 492H / M, 495G, 503L, 504S, 508Q, or 525V, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12 to the carboxy terminus of SEQ ID NO: 2, or to a reference sequence corresponding to SEQ ID NO: 2.

[0160] In some embodiments, the engineered terminal nucleotidyl transferase comprises an amino acid sequence comprising at least an amino acid residue difference or amino acid residue F22P, D34Y, R60H, M160T, T162P, T173P, L174I / V / W, N176H, R177K, Q179K, I187T / Y, E / R190R / T, N191F, V193S / T, H194L, N197M, E203M, F204M, R205L, I208E / G / Q, K222A, T224V, I230V, S231M / N, G236R, E246V, Q251A, L / R254I / R, Q260R, S261V, F262M, H263R / S, A264L, R267S, V273I / K / R / M, G274P, D277S, K278S, T295D, L296V, K297S, T299S, T300K / S, Y308H, E310D, A313R, R318K / P / Q, A / T319T / V, W325V, Q329L, T334S, F335V, A339S / T / V, E342T, I343L, P352T, Q353L / R / V, V357I, I361V, S363P, E367Q, K368S, Q376E, M379L, L381Y, C390M / S, S396T, Q397K, G402S, P404S, S405Q, G406P, K / P407A / P, A408V, D409E, D412P, S416A, C417A, I420V, H433P, T437H, S444G, K451R, G460Q, F / M462L / M, A465G, M466I, R477A / L / Q, R480K, E485I, K487R, M490V, D / M492H / M, S495G, I503L, Q504S, K508Q, F525V, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12 to the carboxy terminus of SEQ ID NO: 2, or to a reference sequence corresponding to SEQ ID NO: 2.

[0161] In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to aminoDocket No. CX10-267WO3 acid residues 12 to carboxy terminal of SEQ ID NO: 2, or to a reference sequence corresponding to SEQ ID NO: 2, and one or more residue differences as compared to SEQ ID NO: 2 at amino acid position(s) 233 / 402, 296 / 384 / 412 / 415 / 451 / 477 / 480, 296 / 415 / 451 / 477 / 480, 296 / 451 / 477, 296 / 451 / 480, 384 / 412 / 451 / 480, 384 / 451 / 477, 402 / 404 / 460, 404, 404 / 431, 415 / 451 / 477 / 480 / 503, 451, 451 / 477, or 477. In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to carboxy terminal of SEQ ID NO: 2, or to a reference sequence corresponding to SEQ ID NO: 2, and at least an amino acid residue difference(s), or amino acid residue 233P / 402K, 296V / 384S / 412M / 415P / 451R / 477Q / 480W, 296V / 415P / 451R / 477Q / 480W, 296V / 451R / 477Q, 296V / 451R / 480W, 384A / 412M / 451R / 480W, 384S / 451R / 477S, 402K / 404A / 460A, 402K / 404A / 460S, 404A, 404A / 431G, 415P / 451R / 477Q / 480W / 503R, 451R, 451R / 477Q, or 477Q. In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to carboxy terminal of SEQ ID NO: 2, or to a reference sequence corresponding to SEQ ID NO: 2, and at least an amino acid residue difference(s), or amino acid residue E233P / G402K, L296V / K384S / D412M / A415P / K451R / R477Q / R480W, L296V / A415P / K451R / R477Q / R480W, L296V / K451R / R477Q, L296V / K451R / R480W, K384A / D412M / K451R / R480W, K384S / K451R / R477S, G402K / P404A / G460A, G402K / P404A / G460S, P404A, P404A / S431G, A415P / K451R / R477Q / R480W / I503R, K451R, K451R / R477Q, and R477Q.

[0162] In some embodiments, the engineered terminal nucleotidyl transferase comprises an amino acid sequence comprising at least an amino acid residue difference at amino acid position 22, 27, 34, 41, 42, 43, 45, 52, 60, 92, 105, 133, 152, 160, 161, 162, 164, 169, 173, 174, 175, 176, 177, 179, 182, 183, 184, 186, 187, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 208, 212, 213, 218, 219, 221, 222, 223, 224, 227, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 241, 242, 244, 246, 249, 250, 251, 252, 254, 259, 260, 261, 262, 263, 264, 267, 268, 272, 273, 274, 275, 277, 278, 280, 281, 282, 288, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 307, 308, 309, 310, 312, 313, 314, 315, 316, 317, 318, 319, 321, 322, 323, 324, 325, 328, 329, 333, 334, 335, 336, 337, 339, 340, 341, 342, 343, 347, 352, 353, 354, 357, 359, 360, 361, 362, 363, 364, 366, 367, 368, 370, 371, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 387, 388, 389, 390, 392, 393, 394, 395, 396, 397, 398, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 446, 448, 450, 451, 456, 457, 458, 459, 460, 461, 462, 463, 465, 466, 467, 468, 474, 477, 479, 480, 481, 484, 485, 487, 490, 492, 493, 494, 495, 497, 499, 500, 501, 502, 503, 504, 506, 507, 508, 513, 515, 517, 518, 522, 523, 525, or 526, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12 to the carboxy terminus of SEQ ID NO: 8, 70, 148, 184, 234, 256, 478, 668, 682, 730, 734, 742, 932, 992, 1144, 1212, 1236, 1264, 1414, 1504, 1522, 1702, 1742, 1928, 1944, 1946, 1984, 2064, 2104, 2210, 2256, 2280, 2342, 2438, 2452, 2562, 2576, 2714, 2876, 2884, 2910, 2974, 2998, or 3002, or to the reference sequence corresponding to SEQ ID NO: 8, 70, 148, 184, 234, 256, 478, 668, 682, 730, 734, 742, 932, 992, 1144, 1212, 1236, 1264, 1414,Docket No. CX10-267WO3 1504, 1522, 1702, 1742, 1928, 1944, 1946, 1984, 2064, 2104, 2210, 2256, 2280, 2342, 2438, 2452, 2562, 2576, 2714, 2876, 2884, 2910, 2974, 2998, or 3002.

[0163] In some embodiments, the engineered terminal nucleotidyl transferase comprises an amino acid sequence comprising at least an amino acid residue difference or amino acid residue 22P, 27P, 34D / Y, 41T, 42T, 43- / T, 45T, 52G, 60H, 92C, 105G, 133E, 152H, 160A / D / T / W, 161A / G / L / Q / T, 162P, 164Q, 169L, 173G / H / P, 174L / V / W, 175E / H / L / P / R / S / T / V, 176H / N, 177C / K, 179C / K / L / R / V, 182I / V, 183G / R / S / T / V, 184K / R, 186G / L / M / R / T / V, 187L / T / V / Y, 190A / F / G / H / L / M / N / Q / R / T / V, 191F / W / Y, 192A / H / S, 193A / G / I / Q / R / S / T / V / W, 194L / M, 195F / Q / R / T / W, 196D / H / K / L / Q / R / S / V, 197I / L / M / Q / R, 198E / H / P / Q / R / S / T, 199E / F / V, 200Q, 201D / E / P / Q / R / S, 202H / I / M / S / V, 203E / F / L / M / Q, 204M / V, 205L / R, 208A / D / E / G / M / N / Q / T, 212R, 213G, 218I, 219D / Q / S, 221I / Q / V, 222A / N, 223H / L / T, 224L / V, 227G / S, 229F / G / L / S, 230C / I / L / V, 231C / M / N / T, 232E / G, 233I / P, 234T, 235N / R, 236R / S, 237L, 238F / I / L / V, 239G / L / R / S, 241L, 242L, 244G / I / R / S, 246L / V, 249L / M / Q, 250G, 251A / E / G / M / R / S / W, 252E / H / L / M / R / S, 254I / N / Q / R, 259F, 260K / Q / R / S / T, 261G / R / S / T / V, 262L / M, 263A / D / H / K / R / S / T / W, 264A / L / R / S, 267G / S, 268L, 272R, 273E / G / I / K / L / M / / N / Q / R / V, 274L / N / P / R / S / T / V, 275I / L / R / S, 277D / E / S / T, 278A / D / L / R / S, 280W, 281A / C / G / H / L / S, 282N, 288D, 290L, 291K / L, 292A, 293R, 294G / T, 295A / D / N / T, 296L / M / V, 297A / G / L / M / P / Q / S / T / V / W / Y, 298I, 299A / G / H / N / P / R / S, 300A / E / F / G / I / K / L / M / N / Q / R / S / V / Y, 301A / F / V, 302S, 303E / R / T, 304L, 307D / E / K / M / N / S, 308H / L / Y, 309H, 310D / S, 312M, 313A / H / Q / R / S / T, 314A / E / I / S / T, 315K / R, 316L, 317R, 318D / I / K / L / P / Q / R / S / T, , 319A / G / L / M / P / Q / T / V / W, 321R / T, 322A / E, 323T, 324A / T, 325G / S / V / Y, 328A / I, 329H / L / Q, 333N / S, 334S, 335V, 336C / T, 337S, 339A / I / S / T / V, 340L / M / T / V, 341L / T, 342S / T, 343I / L / M / R / T / V, 347A / S, 352A / E / L / M / N / S / T / V, 353E / K / L / R / T / V, 354G, 357I, 359A / C / F / V, 360V, 361I / L / V, 362A / M, 363G / P / T, 364S, 366D / F / H / P, 367E / N / Q / R / S / V, 368L / N / R / S, 370E / F / L / R, 371G / R, 376A / E / G / H / R / S / T / V, 377R, 378M, 379L / M / T, 380A / K / L / S / V, 381S / Y, 382L / M, 383L / Q, 384A / G / S / T, 385V, 387T / V, 388V, 389A / V, 390I / L / M / S / T, 392E / H / N / Q / R / S, 393A / G / I / L / M / S / T / Y, 394K / N / S, 395E / F / I / L / M / Q / S / T, S396T, 397E / F / G / H / I / K / L / M / Q / R / S / T / V / W / Y, 398L / M, 402A / D / K / M / P / S / T, 403E / F / I / P, 404A / E / G / I / L / M / Q / R / S / T / V, 405A / G / L / P / Q / S / T / V / Y, 406A / D / E / G / H / M / N / P / R / T, K407A / D / G / L / N / P / Q / S / V / Y, 408A / D / G / L / N / S / V, 409D / E / G / K / N / P / Q / R / S / V / Y, 410V, 411E / H / K / R / T / V, 412M / N / P / Q / S / V, 413S / T, 414M / Q / R / V, 415G / P / S, 416A / G / H / S, 417A / G / S, 418C / S / T, 419A / I / M, 420C / L / V, 424P, 425R, 426G, 427A / M, 428A / L / T, 429A / S, 430G / Q / S / W, 431G, 432I / V, 433A / P / T / V, 434G / L / P, 435F / L / N / T / V, 436C / G / H / L / S / T, 437A / H / P / S / T, 438P / V, 439C / L / P, 440D / M, 441E / T, 442E / L / M / R / V, 443A / D / P / V, 444G, 446D / G / H / S / T, 448R, 450V, 451R, 456M, 457T, 458K, 459L, 460A / D / H / L / Q / R / S / V, 461A / V, 462F / H / L / M / Y, 463P / S, 465G, 466I / L, 467M, 468H / I / M, 474H / I / L / V, 477A / E / G / I / L / Q / R / S / T / W, 479M / T / V, 480K / M / W, 481H, 484A / L / W / Y, 485E / I / L / V, 487K / R, 490A / I / L / V, 492A / C / G / H / M / R / S / Y, 493R, 494L / M / Q / R / T / V / Y, 495A / G / L / S / T, 497L, 499L / N / R / S, 500A / N, 501K / L, 502M, 503L / R / V, 504P / S / T, 506A / K / L, 507G, 508C / D / K / L / Q, 513V, 515G, 517I, 518S, 522A / F / M / V, 523A / D / H, 525G / H / I / R / T / V / W, or 526E / G, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12 to the carboxy terminus of SEQ ID NO: 8, 70, 148, 184, 234, 256, 478, 668, 682, 730, 734, 742, 932, 992, 1144, 1212, 1236, 1264, 1414, 1504, 1522, 1702, 1742, 1928, 1944, 1946, 1984, 2064, 2104, 2210, 2256, 2280, 2342, 2438, 2452, 2562, 2576, 2714, 2876, 2884, 2910, 2974, 2998, or 3002, or to the reference sequence corresponding to SEQ ID NO: 8,Docket No. CX10-267WO3 70, 148, 184, 234, 256, 478, 668, 682, 730, 734, 742, 932, 992, 1144, 1212, 1236, 1264, 1414, 1504, 1522, 1702, 1742, 1928, 1944, 1946, 1984, 2064, 2104, 2210, 2256, 2280, 2342, 2438, 2452, 2562, 2576, 2714, 2876, 2884, 2910, 2974, 2998, or 3002.

[0164] In some embodiments, the engineered terminal nucleotidyl transferase comprises an amino acid sequence comprising at least an amino acid residue difference or amino acid residue F22P, Q27P, D34D / Y, I41T, K42T, F43- / T, E45T, E52G, R60H, R92C, E105G, K133E, R152H, M160A / D / T / W, K161A / G / L / Q / T, T162P, S164Q, Q169L, T173G / H / P, L174L / V / W, N175E / H / L / P / R / S / T / V, N176H / N, R177C / K,H263A / D / H / K / R / S / T / W, A264A / L / R / S, R267G / S, V268L, G272R, V273E / G / I / K / L / M / / N / Q / R / V, G274L / N / P / R / S / T / V, T275I / L / R / S, D277D / E / S / T, K278A / D / L / R / S, Y280W, Q281A / C / G / H / L / S, M282N, E288D, V290L, R291K / L, S292A, S293R, K294G / T, T295A / D / N / T, L296L / M / V, K297A / G / L / M / P / Q / S / T / V / W / Y, L298I, T299A / G / H / N / P / R / S, T300A / E / F / G / I / K / L / M / N / Q / R / S / V / Y, W301A / F / V, A302S, K303E / R / T, V304L, T307D / E / K / M / N / S, Y308H / L / Y, Y309H, E310D / S, L312M, A313A / H / Q / R / S / T, R314A / E / I / S / T, G315K / R, V316L, T317R, R318D / I / K / L / P / Q / R / S / T, , A319A / G / L / M / P / Q / T / V / W, A321R / T, D322A / E, A323T, I324A / T, W325G / S / V / Y, V328A / I, Q329H / L / Q, A333N / S, T334S, F335V, D336C / T, P337S, A339A / I / S / T / V, I340L / M / T / V, V341L / T, E342S / T, I343I / L / M / R / T / V, Q347A / S, P352A / E / L / M / N / S / T / V, Q353E / K / L / R / T / V, S354G, V357I, L359A / C / F / V, L360V, I361I / L / V, S362A / M, S363G / P / T, P364S, A366D / F / H / P, E367E / N / Q / R / S / V, K368L / N / R / S, Q370E / F / L / R, E371G / R, Q376A / E / G / H / R / S / T / V, K377R, V378M, M379L / M / T, R380A / K / L / S / V, L381S / Y, W382L / M, K383L / Q, K384A / G / S / T, Q385V, L387T / V, L388V, L389A / V, C390I / L / M / S / T, K392E / H / N / Q / R / S, V393A / G / I / L / M / S / T / Y, Y394K / N / S, W395E / F / I / L / M / Q / S / T, S396T, Q397E / F / G / H / I / K / L / M / Q / R / S / T / V / W / Y, W398L / M, G402A / D / K / M / P / S / T, L403E / F / I / P, P404A / E / G / I / L / M / Q / R / S / T / V, S405A / G / L / P / Q / S / T / V / Y, G406A / D / E / G / H / M / N / P / R / T, K407A / D / G / L / N / P / Q / S / V / Y, A408A / D / G / L / N / S / V, D409D / E / G / K / N / P / Q / R / S / V / Y, E410V, G411E / H / K / R / T / V, D412M / N / P / Q / S / V, G413S / T, E414M / Q / R / V, A415G / P / S, S416A / G / H / S, C417A / G / S, F418C / S / T, L419A / I / M, I420C / L / V, Y424P, D425R, H426G, L427A / M, V428A / L / T, R429A / S, K430G / Q / S / W, S431G, K432I / V, H433A / P / T / V, N434G / L / P, M435F / L / N / T / V, P436C / G / H / L / S / T, T437A / H / P / S / T, G438P / V, S439C / L / P, N440D / M, M441E / T, G442E / L / M / R / V, E443A / D / P / V, S444G, P446D / G / H / S / T, K448R, I450V, K451R, R456M, C457T, P458K, R459L, G460A / D / H / L / Q / R / S / V, S461A / V, F462F / H / L / M / Y / , A463P / S, A465G, M466I / L, L467M, Q468H / I / M, M474H / I / L / V, R477A / E / G / I / L / Q / R / S / T / W, L479M / T / V, R480K / M / W, D481H, R484A / L / W / Y, E485E / I / L / V, K487K / R, M490A / I / L / V, D492A / C / G / H / M / R / S / Y, N493R, H494L / M / Q / R / T / V / Y, S495A / G / L / S / T, Y497L,Docket No. CX10-267WO3 M499L / N / R / S, T500A / N, R501K / L, G502M, I503L / R / V, Q504P / S / T, P506A / K / L, A507G, K508C / D / K / L / Q, I513V, E515G, L517I, G518S, L522A / F / M / V, E523A / D / H, F525G / H / I / R / T / V / W, or Y526E / G, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12 to the carboxy terminus of SEQ ID NO: 8, 70, 148, 184, 234, 256, 478, 668, 682, 730, 734, 742, 932, 992, 1144, 1212, 1236, 1264, 1414, 1504, 1522, 1702, 1742, 1928, 1944, 1946, 1984, 2064, 2104, 2210, 2256, 2280, 2342, 2438, 2452, 2562, 2576, 2714, 2876, 2884, 2910, 2974, 2998, or 3002, or to the reference sequence corresponding to SEQ ID NO: 8, 70, 148, 184, 234, 256, 478, 668, 682, 730, 734, 742, 932, 992, 1144, 1212, 1236, 1264, 1414, 1504, 1522, 1702, 1742, 1928, 1944, 1946, 1984, 2064, 2104, 2210, 2256, 2280, 2342, 2438, 2452, 2562, 2576, 2714, 2876, 2884, 2910, 2974, 2998, or 3002.

[0165] In some embodiments, the engineered terminal nucleotidyl transferase comprises an amino acid sequence comprising at least an amino acid residue difference at amino acid position 22, 34, 60, 160, 162, 173, 174, 176, 177, 179, 187, 190, 191, 193, 194, 197, 203, 204, 205, 208, 222, 224, 230, 231, 236, 246, 251, 254, 260, 261, 262, 263, 264, 267, 273, 274, 277, 278, 295, 296, 297, 299, 300, 308, 310, 313, 318, 319, 325, 329, 334, 335, 339, 342, 343, 352, 353, 357, 361, 363, 367, 368, 376, 379, 381, 390, 396, 397, 402, 404, 405, 406, 407, 408, 409, 412, 416, 417, 420, 433, 437, 444, 451, 460, 462, 465, 466, 477, 480, 485, 487, 490, 492, 495, 503, 504, 508, or 525, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12 to the carboxy terminus of SEQ ID NO: 8, 70, 148, 184, 234, 256, 478, 668, 682, 730, 734, 742, 932, 992, 1144, 1212, 1236, 1264, 1414, 1504, 1522, 1702, 1742, 1928, 1944, 1946, 1984, 2064, 2104, 2210, 2256, 2280, 2342, 2438, 2452, 2562, 2576, 2714, 2876, 2884, 2910, 2974, 2998, or 3002, or to the reference sequence corresponding to SEQ ID NO: 8, 70, 148, 184, 234, 256, 478, 668, 682, 730, 734, 742, 932, 992, 1144, 1212, 1236, 1264, 1414, 1504, 1522, 1702, 1742, 1928, 1944, 1946, 1984, 2064, 2104, 2210, 2256, 2280, 2342, 2438, 2452, 2562, 2576, 2714, 2876, 2884, 2910, 2974, 2998, or 3002.

[0166] In some embodiments, the engineered terminal nucleotidyl transferase comprises an amino acid sequence comprising at least an amino acid residue difference or amino acid residue 22P, 34Y, 60H, 160T, 162P, 173P, 174I / V / W, 176H, 177K, 179K, 187T / Y, 190R / T, 191F, 193S / T, 194L, 197M, 203M, 204M, 205L / R, 208E / G / Q, 222A, 224V, 230I / V, 231M / N, 236R, 246V, 251A, 254I / R, 260R, 261V, 262M, 263R / S, 264L, 267S, 273I / K / R / M, 274P, 277D / S, 278S, 295D, 296L / V, 297S, 299S, 300K / S, 308H, 310D, 313R, 318K / P / Q, 319T / V, 325V, 329L, 334S, 335V, 339S / T / V, 342T, 343L, 352T, 353L / R / V, 357I, 361V, 363P, 367Q, 368S, 376E, 379L, 381Y, 390M / S, 396T, 397K, 402S, 404S, 405Q, 406P, 407A / P, 408A / V, 409D / E, 412P, 416A, 417A, 420V, 433P, 437H, 444G, 451R, 460Q, 462L / M, 465G, 466I, 477A / L / Q, 480K, 485I, 487R, 490V, 492H / M, 495G, 503L, 504S, 508Q, or 525V, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12 to the carboxy terminus of SEQ ID NO: 8, 70, 148, 184, 234, 256, 478, 668, 682, 730, 734, 742, 932, 992, 1144, 1212, 1236, 1264, 1414, 1504, 1522, 1702, 1742, 1928, 1944, 1946, 1984, 2064, 2104, 2210, 2256, 2280, 2342, 2438, 2452, 2562, 2576, 2714, 2876, 2884, 2910, 2974, 2998, or 3002, or to the reference sequence corresponding to SEQ ID NO: 8, 70, 148, 184, 234, 256, 478, 668, 682, 730, 734, 742, 932, 992, 1144, 1212, 1236, 1264, 1414, 1504, 1522, 1702, 1742, 1928, 1944, 1946, 1984, 2064, 2104, 2210, 2256, 2280, 2342, 2438, 2452, 2562, 2576, 2714, 2876, 2884, 2910, 2974, 2998, or 3002.Docket No. CX10-267WO3

[0167] In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to carboxy terminal of SEQ ID NO: 8, or to a reference sequence corresponding to SEQ ID NO: 8, and one or more residue differences as compared to SEQ ID NO: 8 at amino acid positions(s) 92 / 273 / 277 / 462, 267 / 313 / 314, 267 / 313 / 314 / 408 / 485, 267 / 313 / 367 / 485, 267 / 313 / 406, 267 / 314 / 408 / 441, 267 / 367 / 485, 267 / 406 / 408 / 485, 273 / 277, 273 / 277 / 381, 273 / 277 / 381 / 404 / 462 / 492, 273 / 277 / 381 / 429, 273 / 277 / 381 / 462, 273 / 277 / 381 / 462 / 492, 273 / 277 / 381 / 492, 273 / 277 / 429, 273 / 277 / 429 / 492, 273 / 277 / 462, 273 / 381, 273 / 381 / 492, 273 / 462, 273 / 462 / 492, 277 / 381, 277 / 381 / 429 / 462 / 492, 277 / 381 / 429 / 492, 277 / 381 / 462 / 492, 277 / 381 / 492, 277 / 429 / 462 / 492, 277 / 429 / 492, 277 / 492, 309 / 313 / 314 / 412 / 485, 381 / 462 / 492, and 381 / 492, or combinations thereof. In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to carboxy terminal of SEQ ID NO: 8, or to a reference sequence corresponding to SEQ ID NO: 8, and one or more residue differences as compared to SEQ ID NO: 8, or amino acid residues selected from 92C / 273R / 277T / 462M, 267S / 313S / 314A / 408V / 485L, 267S / 313S / 367S / 485L, 267S / 313S / 406H, 267S / 313T / 314A, 267S / 314A / 408V / 441E, 267S / 367S / 485L, 267S / 406H / 408V / 485L, 273R / 277S, 273R / 277S / 381Y, 273R / 277S / 381Y / 404S / 462M / 492M, 273R / 277S / 381Y / 429A, 273R / 277S / 381Y / 462M, 273R / 277S / 381Y / 462M / 492M, 273R / 277S / 381Y / 462M / 492Y, 273R / 277S / 429A, 273R / 277S / 429A / 492S, 273R / 277S / 462M, 273R / 277T, 273R / 277T / 381Y, 273R / 277T / 381Y / 429A, 273R / 277T / 381Y / 492M, 273R / 277T / 429A, 273R / 277T / 462M, 273R / 381Y, 273R / 381Y / 492M, 273R / 462M, 273R / 462M / 492S, 273R / 462M / 492Y, 277S / 381Y, 277S / 381Y / 429A / 462M / 492M, 277S / 381Y / 429A / 492M, 277S / 381Y / 462M / 492M, 277S / 381Y / 462M / 492S, 277S / 429A / 462M / 492S, 277S / 429A / 492Y, 277S / 492S, 277T / 381Y, 277T / 381Y / 429A / 462M / 492M, 277T / 381Y / 492M, 277T / 381Y / 492S, 309H / 313S / 314A / 412N / 485L, 381Y / 462M / 492Y, and 381Y / 492S. In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to carboxy terminal of SEQ ID NO: 8, or to a reference sequence corresponding to SEQ ID NO: 8, and one or more residue differences as compared to SEQ ID NO: 8, or amino acid residues selected from R92C / V273R / D277T / F462M, R267S / A313S / R314A / A408V / E485L, R267S / A313S / E367S / E485L, R267S / A313S / G406H, R267S / A313T / R314A, R267S / R314A / A408V / M441E, R267S / E367S / E485L, R267S / G406H / A408V / E485L, V273R / D277S, V273R / D277S / L381Y, V273R / D277S / L381Y / P404S / F462M / D492M, V273R / D277S / L381Y / R429A, V273R / D277S / L381Y / F462M, V273R / D277S / L381Y / F462M / D492M, V273R / D277S / L381Y / F462M / D492Y, V273R / D277S / R429A, V273R / D277S / R429A / D492S, V273R / D277S / F462M, V273R / D277T, V273R / D277T / L381Y, V273R / D277T / L381Y / R429A, V273R / D277T / L381Y / D492M, V273R / D277T / R429A, V273R / D277T / F462M, V273R / L381Y, V273R / L381Y / D492M, V273R / F462M, V273R / F462M / D492S, V273R / F462M / D492Y, D277S / L381Y, D277S / L381Y / R429A / F462M / D492M,Docket No. CX10-267WO3 D277S / L381Y / R429A / D492M, D277S / L381Y / F462M / D492M, D277S / L381Y / F462M / D492S, D277S / R429A / F462M / D492S, D277S / R429A / D492Y, D277S / D492S, D277T / L381Y, D277T / L381Y / R429A / F462M / D492M, D277T / L381Y / D492M, D277T / L381Y / D492S, Y309H / A313S / R314A / D412N / E485L, L381Y / F462M / D492Y, and L381Y / D492S.

[0168] In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to carboxy terminal of SEQ ID NO: 8, or to a reference sequence corresponding to SEQ ID NO: 8, and one or more residue differences as compared to SEQ ID NO: 8 at amino acid positions(s) 186, 193, 196, 198, 198 / 515, 274, 394, and 409, or combinations thereof. In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to carboxy terminal of SEQ ID NO: 8, or to a reference sequence corresponding to SEQ ID NO: 8, and one or more residue differences as compared to SEQ ID NO: 8, or amino acid residues selected from 186R, 193Q, 193T, 193W, 196K, 198H, 198Q, 198R, 198R / 515G, 198S, 274R, 394S, and 409R, or combinations thereof. In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to carboxy terminal of SEQ ID NO: 8, or to a reference sequence corresponding to SEQ ID NO: 8, and one or more residue differences as compared to SEQ ID NO: 8, or amino acid residues selected from E186R, V193Q, V193T, V193W, G196K, D198H, D198Q, D198R, D198R / E515G, D198S, G274R, Y394S, and D409R, or combinations thereof.

[0169] In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to carboxy terminal of SEQ ID NO: 70, or to a reference sequence corresponding to SEQ ID NO: 70, and one or more residue differences as compared to SEQ ID NO: 70 at amino acid positions(s) 190, 190 / 208 / 267 / 274, 190 / 208 / 267 / 274 / 309 / 417, 190 / 208 / 267 / 274 / 417 / 462, 190 / 208 / 274 / 309 / 313 / 417 / 462, 190 / 208 / 309 / 462, 190 / 267 / 274, 190 / 274 / 417, 190 / 274 / 417 / 477, 208 / 274 / 309 / 313 / 430, 267 / 274 / 477, 273, 274, 274 / 309, 274 / 309 / 313 / 417 / 462, 274 / 309 / 417 / 477, 274 / 313, and 274 / 462. In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to carboxy terminal of SEQ ID NO: 8, or to a reference sequence corresponding to SEQ ID NO: 70, and one or more residue differences as compared to SEQ ID NO: 8, or amino acid residues selected from 190A, 190A / 208E / 267S / 274P, 190A / 208E / 309H / 462H, 190A / 208G / 267S / 274P / 417A / 462H, 190A / 274P / 417A, 190R / 208G / 267S / 274P / 309H / 417A, 190R / 208G / 274P / 309H / 313S / 417A / 462H, 190R / 267S / 274P, 190R / 274P / 417A / 477R, 208E / 274P / 309H / 313S / 430Q, 267S / 274P / 477R, 273V, 274P, 274P / 309H,Docket No. CX10-267WO3 274P / 309H / 313S / 417A / 462H, 274P / 309H / 417A / 477R, 274P / 313S, and 274P / 462H. In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to carboxy terminal of SEQ ID NO: 70, or to a reference sequence corresponding to SEQ ID NO: 70, and one or more residue differences as compared to SEQ ID NO: 70, or amino acid residues selected from E190A, E190A / I208E / R267S / G274P, E190A / I208E / Y309H / M462H, E190A / I208G / R267S / G274P / C417A / M462H, E190A / G274P / C417A, E190R / I208G / R267S / G274P / Y309H / C417A, E190R / I208G / G274P / Y309H / A313S / C417A / M462H, E190R / R267S / G274P, E190R / G274P / C417A / Q477R, I208E / G274P / Y309H / A313S / K430Q, R267S / G274P / Q477R, R273V, G274P, G274P / Y309H, G274P / Y309H / A313S / C417A / M462H, G274P / Y309H / C417A / Q477R, G274P / A313S, and G274P / M462H.

[0170] In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to carboxy terminal of SEQ ID NO: 148, or to a reference sequence corresponding to SEQ ID NO: 148, and one or more residue differences as compared to SEQ ID NO: 148 at amino acid positions(s) 183 / 208 / 309 / 313 / 407 / 462, 208 / 343 / 407, 208 / 407, and 309. In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to carboxy terminal of SEQ ID NO: 148, or to a reference sequence corresponding to SEQ ID NO: 148, and one or more residue differences as compared to SEQ ID NO: 148, or amino acid residues selected from 183R / 208E / 309H / 313S / 407P / 462H, 208D / 343L / 407P, 208E / 343L / 407P, 208E / 407P, or 309H. In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to carboxy terminal of SEQ ID NO: 148, or to a reference sequence corresponding to SEQ ID NO: 148, and one or more residue differences as compared to SEQ ID NO: 148, or amino acid residues selected from D183R / I208E / Y309H / A313S / K407P / M462H, I208D / I343L / K407P, I208E / I343L / K407P, I208E / K407P, and Y309H.

[0171] In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to carboxy terminal of SEQ ID NO: 148, or to a reference sequence corresponding to SEQ ID NO: 148, and one or more residue differences as compared to SEQ ID NO: 148 at amino acid positions(s) 190, 268, 275, 303, 397, 409, 415, 417, and 477. In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to carboxy terminal of SEQ ID NO: 148, or to aDocket No. CX10-267WO3 reference sequence corresponding to SEQ ID NO: 148, and one or more residue differences as compared to SEQ ID NO: 148, or amino acid residues selected from 190H, 268L, 275L, 275S, 303T, 397V, 409P, 415G, 415P, 417S, and 477R. In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to carboxy terminal of SEQ ID NO: 148, or to a reference sequence corresponding to SEQ ID NO: 148, and one or more residue differences as compared to SEQ ID NO: 148, or amino acid residues selected from R190H, V268L, T275L, T275S, K303T, Q397V, D409P, A415G, A415P, C417S, and Q477R.

[0172] In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to carboxy terminal of SEQ ID NO: 148, or to a reference sequence corresponding to SEQ ID NO: 148, and one or more residue differences as compared to SEQ ID NO: 148 at amino acid positions 186, 304, 318, 347, 353, 409, 416, 417, and 420. In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to carboxy terminal of SEQ ID NO: 148, or to a reference sequence corresponding to SEQ ID NO: 148, and one or more residue differences as compared to SEQ ID NO: 148, or amino acid residues selected from 186L, 186T, 186V, 304L, 318L, 347A, 353L, 409P, 416H, 417S, and 420L. In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to carboxy terminal of SEQ ID NO: 148, or to a reference sequence corresponding to SEQ ID NO: 148, and one or more residue differences as compared to SEQ ID NO: 148, or amino acid residues selected from E186L, E186T, E186V, V304L, R318L, Q347A, Q353L, D409P, S416H, C417S, and I420L.

[0173] In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to carboxy terminal of SEQ ID NO: 184, or to a reference sequence corresponding to SEQ ID NO: 184, and one or more residue differences as compared to SEQ ID NO: 184 at amino acid positions 193 / 274 / 411 / 465 / 525, 193 / 408 / 465, 193 / 465, 274 / 352 / 353 / 465, 281 / 411 / 465, 343 / 408 / 465, 343 / 411, 411, 411 / 525, and 525. In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to carboxy terminal of SEQ ID NO: 184, or to a reference sequence corresponding to SEQ ID NO: 184, and one or more residue differences as compared to SEQ ID NO: 184, or amino acid residues selected from 193S / 274N / 411T / 465G / 525H, 193S / 408L / 465G, 193S / 465G,Docket No. CX10-267WO3 274N / 352M / 353T / 465G, 281S / 411T / 465G, 343I / 408L / 465G, 343I / 411T, 411T, 411T / 525H, and 525H. In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to carboxy terminal of SEQ ID NO: 184, or to a reference sequence corresponding to SEQ ID NO: 184, and one or more residue differences as compared to SEQ ID NO: 184, or amino acid residues selected from V193S / P274N / G411T / A465G / F525H, V193S / A408L / A465G, V193S / A465G, P274N / P352M / Q353T / A465G, Q281S / G411T / A465G, L343I / A408L / A465G, L343I / G411T, G411T, G411T / F525H, and F525H.

[0174] In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to carboxy terminal of SEQ ID NO: 234, or to a reference sequence corresponding to SEQ ID NO: 234, and one or more residue differences as compared to SEQ ID NO: 234 at amino acid positions 275, 299, 300, 392, 393, and 397. In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to carboxy terminal of SEQ ID NO: 234, or to a reference sequence corresponding to SEQ ID NO: 234, and one or more residue differences as compared to SEQ ID NO: 234, or amino acid residues selected from 275I, 299G, 299P, 299R, 299S, 300F, 300G, 300I, 300K, 300L, 300M, 300N, 300Q, 300R, 300Y, 392H, 392N, 393G, 393Y, 397F, 397H, 397I, 397K, 397M, 397R, 397V, 397W, and 397Y. In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to carboxy terminal of SEQ ID NO: 234, or to a reference sequence corresponding to SEQ ID NO: 234, and one or more residue differences as compared to SEQ ID NO: 234, or amino acid residues selected from T275I, T299G, T299P, T299R, T299S, T300F, T300G, T300I, T300K, T300L, T300M, T300N, T300Q, T300R, T300Y, K392H, K392N, V393G, V393Y, Q397F, Q397H, Q397I, Q397K, Q397M, Q397R, Q397V, Q397W, and Q397Y.

[0175] In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to carboxy terminal of SEQ ID NO: 234, or to a reference sequence corresponding to SEQ ID NO: 234, and one or more residue differences as compared to SEQ ID NO: 234 at amino acid positions 299, 300, 392, 393, 395, and 397. In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to carboxy terminal of SEQ ID NO: 234, or to a reference sequence corresponding to SEQ ID NO: 234, and one or more residue differences as compared to SEQ ID NO: 234, orDocket No. CX10-267WO3 amino acid residues selected from 299A, 299G, 299N, 299R, 299S, 300A, 300E, 300F, 300L, 300M, 300N, 300S, 300V, 300Y, 392E, 392N, 392Q, 392S, 393A, 393I, 393L, 393M, 393S, 393T, 395F, 395I, 395L, 395M, 395Q, 395T, 397E, 397L, and 397M. In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to carboxy terminal of SEQ ID NO: 234, or to a reference sequence corresponding to SEQ ID NO: 234, and one or more residue differences as compared to SEQ ID NO: 234, or amino acid residues selected from T299A, T299G, T299N, T299R, T299S, T300A, T300E, T300F, T300L, T300M, T300N, T300S, T300V, T300Y, K392E, K392N, K392Q, K392S, V393A, V393I, V393L, V393M, V393S, V393T, W395F, W395I, W395L, W395M, W395Q, W395T, Q397E, Q397L, and Q397M.

[0176] In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to carboxy terminal of SEQ ID NO: 234, or to a reference sequence corresponding to SEQ ID NO: 234, and one or more residue differences as compared to SEQ ID NO: 234 at amino acid positions 307, 359, 405, 420, 492, and 525. In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to carboxy terminal of SEQ ID NO: 234, or to a reference sequence corresponding to SEQ ID NO: 234, and one or more residue differences as compared to SEQ ID NO: 234, or amino acid residues selected from 307S, 359F, 405T, 420V, 492H, and 525T. In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to carboxy terminal of SEQ ID NO: 234, or to a reference sequence corresponding to SEQ ID NO: 234, and one or more residue differences as compared to SEQ ID NO: 234, or amino acid residues selected from T307S, L359F, S405T, I420V, M492H, and F525T.

[0177] In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to carboxy terminal of SEQ ID NO: 234, or to a reference sequence corresponding to SEQ ID NO: 234, and one or more residue differences as compared to SEQ ID NO: 234 at amino acid positions 298, 308, 312, 318, 319, 322, 328, 359, 361, 379, 382, 389, 390, 392, 394, 398, 404, 405, 409, 410, 411, 412, 414, 415, 417, 419, 420, 457, 458, 466, 468, 492, 493, 494, 495, 513, 517, and 525. In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to carboxy terminal of SEQ ID NO: 234, or to a reference sequence corresponding to SEQ ID NO: 234, and one or moreDocket No. CX10-267WO3 residue differences as compared to SEQ ID NO: 234, or amino acid residues selected from 298I, 308L, 312M, 318I, 319M, 319T, 322A, 328I, 359V, 361L, 379T, 382M, 389A, 390I, 392R, 394K, 398M, 404I, 404L, 404R, 405Y, 409G, 409R, 409S, 409Y, 410V, 411H, 411R, 411V, 412V, 414R, 415S, 417G, 419I, 419M, 420C, 457T, 458K, 466L, 468H, 468I, 468M, 492C, 492G, 492H, 492R, 492S, 493R, 494Q, 495G, 495L, 495T, 513V, 517I, and 525I. In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to carboxy terminal of SEQ ID NO: 234, or to a reference sequence corresponding to SEQ ID NO: 234, and one or more residue differences as compared to SEQ ID NO: 234, or amino acid residues selected from L298I, Y308L, L312M, R318I, A319M, A319T, D322A, V328I, L359V, I361L, M379T, W382M, L389A, C390I, K392R, Y394K, W398M, S404I, S404L, S404R, S405Y, D409G, D409R, D409S, D409Y, E410V, G411H, G411R, G411V, D412V, E414R, A415S, C417G, L419I, L419M, I420C, C457T, P458K, M466L, Q468H, Q468I, Q468M, M492C, M492G, M492H, M492R, M492S, N493R, H494Q, S495G, S495L, S495T, I513V, L517I, and F525I.

[0178] In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to carboxy terminal of SEQ ID NO: 256, or to a reference sequence corresponding to SEQ ID NO: 256, and one or more residue differences as compared to SEQ ID NO: 256 at amino acid positions 307, 319 / 410 / 495, 319 / 466 / 492 / 495, 319 / 492 / 495, 325 / 359 / 404 / 409, 359 / 404 / 405, 405, 405 / 409, 410, 412, 415, 419, 442 / 457, and 525. In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to carboxy terminal of SEQ ID NO: 256, or to a reference sequence corresponding to SEQ ID NO: 256, and one or more residue differences as compared to SEQ ID NO: 256, or amino acid residues selected from 307S, 319T / 410V / 495G, 319T / 466L / 492H / 495G, 319T / 492H / 495G, 325G / 359F / 404T / 409R, 359F / 404R / 405T, 405T, 405T / 409R, 410V, 412V, 415P, 419M, 442R / 457T, and 525T. In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to carboxy terminal of SEQ ID NO: 256, or to a reference sequence corresponding to SEQ ID NO: 256, and one or more residue differences as compared to SEQ ID NO: 256, or amino acid residues selected from T307S, A319T / E410V / S495G, A319T / M466L / M492H / S495G, A319T / M492H / S495G, W325G / L359F / S404T / D409R, L359F / S404R / S405T, S405T, S405T / D409R, E410V, D412V, A415P, L419M, G442R / C457T, and F525T.

[0179] In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to carboxy terminal of SEQ ID NO: 256, or to a reference sequence corresponding to SEQ IDDocket No. CX10-267WO3 NO: 256, and one or more residue differences as compared to SEQ ID NO: 256 at amino acid positions 323, 336, 339, 362, 363, 363 / 434, 368, 380, 383, 384, 402, 403, 426, 428, 430, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 459, 460, 497, 499, 501, 504, and 506. In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 660%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to carboxy terminal of SEQ ID NO: 256, or to a reference sequence corresponding to SEQ ID NO: 256, and one or more residue differences as compared to SEQ ID NO: 256, or amino acid residues selected from 323T, 336C, 336T, 339T, 362M, 363P, 363P / 434P, 368S, 380A, 380L, 380S, 380V, 383L, 384G, 384T, 402D, 402S, 402T, 403I, 426G, 428A, 430S, 432I, 432V, 433T, 434G, 434L, 434P, 435F, 435L, 435T, 436C, 436H, 436L, 437A, 437P, 438P, 439C, 439L, 439P, 440M, 441T, 442L, 443A, 443P, 443V, 459L, 460R, 460S, 460V, 497L, 499R, 501L, 504P, 506A, and 506L. In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to carboxy terminal of SEQ ID NO: 256, or to a reference sequence corresponding to SEQ ID NO: 256, and one or more residue differences as compared to SEQ ID NO: 256, or amino acid residues selected from A323T, D336C, D336T, A339T, S362M, S363P, S363P / N434P, K368S, R380A, R380L, R380S, R380V, K383L, K384G, K384T, G402D, G402S, G402T, L403I, H426G, V428A, K430S, K432I, K432V, H433T, N434G, N434L, N434P, M435F, M435L, M435T, P436C, P436H, P436L, T437A, T437P, G438P, S439C, S439L, S439P, N440M, M441T, G442L, E443A, E443P, E443V, R459L, G460R, G460S, G460V, Y497L, M499R, R501L, Q504P, P506A, and P506L.

[0180] In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to carboxy terminal of SEQ ID NO: 256, or to a reference sequence corresponding to SEQ ID NO: 256, and one or more residue differences as compared to SEQ ID NO: 256 at amino acid positions 329, 336, 339, 362, 363, 363 / 434, 368, 380, 381, 383, 384, 385, 397, 402, 425, 427, 428, 430, 432, 433, 435, 436, 437, 438, 439, 443, 459, 460, 497, 499, 500, 501, 506, 508, and 518. In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to carboxy terminal of SEQ ID NO: 256, or to a reference sequence corresponding to SEQ ID NO: 256, and one or more residue differences as compared to SEQ ID NO: 256, or amino acid residues selected from 329H, 336C, 339I, 339T, 362A, 363P / 434P, 363T, 368L, 368S, 380A, 380L, 380V, 381S, 383Q, 384T, 385V, 397T, 402A, 402D, 402M, 402P, 402S, 402T, 425R, 427A, 428T, 430W, 432I, 433V, 435T, 436C, 436G, 437A, 438P, 439P, 443V, 459L, 460L, 460S, 460V, 497L, 499L, 500A, 501L, 506A, 506L, 508L, and 518S. In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12Docket No. CX10-267WO3 to carboxy terminal of SEQ ID NO: 256, or to a reference sequence corresponding to SEQ ID NO: 256, and one or more residue differences as compared to SEQ ID NO: 256, or amino acid residues selected from Q329H, D336C, A339I, A339T, S362A, S363P / N434P, S363T, K368L, K368S, R380A, R380L, R380V, Y381S, K383Q, K384T, Q385V, K397T, G402A, G402D, G402M, G402P, G402S, G402T, D425R, L427A, V428T, K430W, K432I, H433V, M435T, P436C, P436G, T437A, G438P, S439P, E443V, R459L, G460L, G460S, G460V, Y497L, M499L, T500A, R501L, P506A, P506L, K508L, and G518S.

[0181] In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to carboxy terminal of SEQ ID NO: 478, or to a reference sequence corresponding to SEQ ID NO: 478, and one or more residue differences as compared to SEQ ID NO: 478 at amino acid positions 299 / 302 / 363 / 402 / 405 / 416, 299 / 363 / 402, 299 / 363 / 405 / 416, 299 / 363 / 459, 299 / 402 / 416 / 501, 299 / 459, 302 / 363 / 402 / 405, 302 / 402 / 459, 402 / 416 / 459, and 459. In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to carboxy terminal of SEQ ID NO: 478, or to a reference sequence corresponding to SEQ ID NO: 478, and one or more residue differences as compared to SEQ ID NO: 478, or amino acid residues selected from 299S / 302S / 363P / 402S / 405T / 416A, 299S / 363P / 402S, 299S / 363P / 405T / 416A, 299S / 363P / 459L, 299S / 402S / 416A / 501L, 299S / 459L, 302S / 363P / 402S / 405T, 302S / 402S / 459L, 402S / 416A / 459L, and 459L. In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to carboxy terminal of SEQ ID NO: 478, or to a reference sequence corresponding to SEQ ID NO: 478, and one or more residue differences as compared to SEQ ID NO: 478, or amino acid residues selected from T299S / A302S / S363P / G402S / S405T / S416A, T299S / S363P / G402S, T299S / S363P / S405T / S416A, T299S / S363P / R459L, T299S / G402S / S416A / R501L, T299S / R459L, A302S / S363P / G402S / S405T, A302S / G402S / R459L, G402S / S416A / R459L, and R459L.

[0182] In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to carboxy terminal of SEQ ID NO: 668, or to a reference sequence corresponding to SEQ ID NO: 668, and one or more residue differences as compared to SEQ ID NO: 668 at amino acid positions 302 / 416 / 497, 368, 368 / 416 / 435 / 436 / 501, and 416. In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to carboxy terminal of SEQ ID NO: 668, or to a reference sequence corresponding to SEQ ID NO: 668, and one or more residue differences as compared to SEQ ID NO: 668, or amino acid residues selected from 302S / 416A / 497L, 368S,Docket No. CX10-267WO3 368S / 416A / 435L / 436H / 501L, and 416A. In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to carboxy terminal of SEQ ID NO: 668, or to a reference sequence corresponding to SEQ ID NO: 668, and one or more residue differences as compared to SEQ ID NO: 668, or amino acid residues selected from A302S / S416A / Y497L, K368S, K368S / S416A / M435L / P436H / R501L, and S416A.

[0183] In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to carboxy terminal of SEQ ID NO: 668, or to a reference sequence corresponding to SEQ ID NO: 668, and one or more residue differences as compared to SEQ ID NO: 668 at amino acid positions 339 / 368 / 436 and 416. In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to carboxy terminal of SEQ ID NO: 668, or to a reference sequence corresponding to SEQ ID NO: 668, and one or more residue differences as compared to SEQ ID NO: 668, or amino acid residues selected from 339T / 368S / 436L, and 416A. In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to carboxy terminal of SEQ ID NO: 668, or to a reference sequence corresponding to SEQ ID NO: 668, and one or more residue differences as compared to SEQ ID NO: 668, or amino acid residues selected from A339T / K368S / P436L, and S416A.

[0184] In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to carboxy terminal of SEQ ID NO: 668, or to a reference sequence corresponding to SEQ ID NO: 668, and one or more residue differences as compared to SEQ ID NO: 668 at amino acid positions 184, 191, 222, 224, 232, 236, 246, 251, 288, 291, 319, 444, 485, and 490. In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to carboxy terminal of SEQ ID NO: 668, or to a reference sequence corresponding to SEQ ID NO: 668, and one or more residue differences as compared to SEQ ID NO: 668, or amino acid residues selected from 184R, 191F, 191W, 191Y, 222A, 224L, 232E, 236S, 246V, 251R, 288D, 291K, 319G, 319W, 444G, 485I, and 490I. In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12Docket No. CX10-267WO3 to carboxy terminal of SEQ ID NO: 668, or to a reference sequence corresponding to SEQ ID NO: 668, and one or more residue differences as compared to SEQ ID NO: 668, or amino acid residues selected from S184R, N191F, N191W, N191Y, K222A, T224L, D232E, G236S, E246V, Q251R, E288D, R291K, T319G, T319W, S444G, E485I, and M490I.

[0185] In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to carboxy terminal of SEQ ID NO: 682, or to a reference sequence corresponding to SEQ ID NO: 682, and one or more residue differences as compared to SEQ ID NO: 682 at amino acid positions 191, 368, 485, and 490. In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to carboxy terminal of SEQ ID NO: 682, or to a reference sequence corresponding to SEQ ID NO: 682, and one or more residue differences as compared to SEQ ID NO: 682, or amino acid residues selected from 191F, 368S, 485I, and 490I. In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to carboxy terminal of SEQ ID NO: 682, or to a reference sequence corresponding to SEQ ID NO: 682, and one or more residue differences as compared to SEQ ID NO: 682, or amino acid residues selected from N191F, K368S, E485I, and M490I.

[0186] In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to carboxy terminal of SEQ ID NO: 730, or to a reference sequence corresponding to SEQ ID NO: 730, and one or more residue differences as compared to SEQ ID NO: 730 at amino acid positions 191 / 222 / 236 / 246 / 291 / 368 / 444, 191 / 222 / 244 / 246 / 291 / 368 / 490, 191 / 222 / 244 / 246 / 368 / 490, 191 / 222 / 244 / 246 / 368 / 495, 191 / 222 / 244 / 291 / 368 / 444, 191 / 222 / 246 / 368 / 444, 191 / 222 / 291 / 368, 191 / 222 / 291 / 368 / 490, 191 / 222 / 368, 191 / 222 / 368 / 444 / 490, 191 / 222 / 368 / 490, 191 / 236 / 244 / 291 / 368 / 490, 191 / 236 / 291 / 368, 191 / 236 / 291 / 368 / 490, 191 / 246 / 291 / 368 / 490, 191 / 246 / 368 / 490, 191 / 291 / 368 / 444, 191 / 291 / 368 / 444 / 490, 191 / 368 / 444, 191 / 368 / 444 / 490, 191 / 368 / 490, 222 / 236 / 246 / 291 / 368 / 444 / 490, 222 / 244 / 291 / 368 / 444, 222 / 246 / 368 / 444, 222 / 246 / 490, 222 / 291 / 368, 222 / 291 / 368 / 444, 222 / 291 / 368 / 444 / 490, 222 / 368 / 444 / 490, 222 / 368 / 490, 236 / 246 / 291 / 368 / 444 / 490, 236 / 246 / 368, 244 / 246 / 291 / 368, 244 / 246 / 368 / 444, 244 / 246 / 368 / 490, 244 / 291 / 368 / 490, 246 / 291 / 296 / 368 / 444 / 490, 246 / 291 / 368, 246 / 291 / 368 / 490, 246 / 368 / 444, 368, 368 / 444, and 368 / 490. In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to carboxy terminal of SEQ ID NO: 730, or to a reference sequence corresponding to SEQ ID NO: 730, and one or more residue differences as compared to SEQ ID NO: 730, orDocket No. CX10-267WO3 amino acid residues selected from 191F / 222A / 236S / 246V / 291K / 368S / 444G, 191F / 222A / 244R / 246V / 291K / 368S / 490I, 191F / 222A / 244R / 246V / 368S / 490I, 191F / 222A / 244R / 246V / 368S / 495S, 191F / 222A / 244R / 291K / 368S / 444G, 191F / 222A / 246V / 368S / 444G, 191F / 222A / 291K / 368S, 191F / 222A / 291K / 368S / 490V, 191F / 222A / 368S, 191F / 222A / 368S / 444G / 490V, 191F / 222A / 368S / 490I, 191F / 236S / 244R / 291K / 368S / 490V, 191F / 236S / 291K / 368S, 191F / 236S / 291K / 368S / 490V, 191F / 246V / 291K / 368S / 490I, 191F / 246V / 368S / 490V, 191F / 291K / 368S / 444G, 191F / 291K / 368S / 444G / 490I, 191F / 368S / 444G, 191F / 368S / 444G / 490V, 191F / 368S / 490I, 191F / 368S / 490V, 222A / 236S / 246V / 291K / 368S / 444G / 490V, 222A / 244R / 291K / 368S / 444G, 222A / 246V / 368S / 444G, 222A / 246V / 490I, 222A / 291K / 368S, 222A / 291K / 368S / 444G, 222A / 291K / 368S / 444G / 490V, 222A / 368S / 444G / 490I, 222A / 368S / 444G / 490V, 222A / 368S / 490V, 236S / 246V / 291K / 368S / 444G / 490V, 236S / 246V / 368S, 244R / 246V / 291K / 368S, 244R / 246V / 368S / 444G, 244R / 246V / 368S / 490V, 244R / 291K / 368S / 490V, 246V / 291K / 296M / 368S / 444G / 490V, 246V / 291K / 368S, 246V / 291K / 368S / 490V, 246V / 368S / 444G, 368S, 368S / 444G, and 368S / 490V. In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to carboxy terminal of SEQ ID NO: 730, or to a reference sequence corresponding to SEQ ID NO: 730, and one or more residue differences as compared to SEQ ID NO: 730, or amino acid residues selected from N191F / K222A / G236S / E246V / R291K / K368S / S444G, N191F / K222A / V244R / E246V / R291K / K368S / M490I, N191F / K222A / V244R / E246V / K368S / M490I, N191F / K222A / V244R / E246V / K368S / G495S, N191F / K222A / V244R / R291K / K368S / S444G, N191F / K222A / E246V / K368S / S444G, N191F / K222A / R291K / K368S, N191F / K222A / R291K / K368S / M490V, N191F / K222A / K368S, N191F / K222A / K368S / S444G / M490V, N191F / K222A / K368S / M490I, N191F / G236S / V244R / R291K / K368S / M490V, N191F / G236S / R291K / K368S, N191F / G236S / R291K / K368S / M490V, N191F / E246V / R291K / K368S / M490I, N191F / E246V / K368S / M490V, N191F / R291K / K368S / S444G, N191F / R291K / K368S / S444G / M490I, N191F / K368S / S444G, N191F / K368S / S444G / M490V, N191F / K368S / M490I, N191F / K368S / M490V, K222A / G236S / E246V / R291K / K368S / S444G / M490V, K222A / V244R / R291K / K368S / S444G, K222A / E246V / K368S / S444G, K222A / E246V / M490I, K222A / R291K / K368S, K222A / R291K / K368S / S444G, K222A / R291K / K368S / S444G / M490V, K222A / K368S / S444G / M490I, K222A / K368S / S444G / M490V, K222A / K368S / M490V, G236S / E246V / R291K / K368S / S444G / M490V, G236S / E246V / K368S, V244R / E246V / R291K / K368S, V244R / E246V / K368S / S444G, V244R / E246V / K368S / M490V, V244R / R291K / K368S / M490V, E246V / R291K / V296M / K368S / S444G / M490V, E246V / R291K / K368S, E246V / R291K / K368S / M490V, E246V / K368S / S444G, K368S, K368S / S444G, and K368S / M490V.

[0187] In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to carboxy terminal of SEQ ID NO: 730, or to a reference sequence corresponding to SEQ ID NO: 730, and one or more residue differences as compared to SEQ ID NO: 730 at amino acid positions 161,Docket No. CX10-267WO3 173, 197, 201, 208, 262, 343, 379, 382, 416, 429, 494, 508, and 525. In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to carboxy terminal of SEQ ID NO: 730, or to a reference sequence corresponding to SEQ ID NO: 730, and one or more residue differences as compared to SEQ ID NO: 730, or amino acid residues selected from 161G, 161T, 173G, 197M, 201E, 208A, 262M, 343V, 379L, 382L, 416G, 429S, 494R, 508D, and 525W. In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to carboxy terminal of SEQ ID NO: 730, or to a reference sequence corresponding to SEQ ID NO: 730, and one or more residue differences as compared to SEQ ID NO: 730, or amino acid residues selected from K161G, K161T, T173G, N197M, A201E, E208A, F262M, L343V, M379L, W382L, A416G, R429S, H494R, K508D, and F525W.

[0188] In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to carboxy terminal of SEQ ID NO: 730, or to a reference sequence corresponding to SEQ ID NO: 730, and one or more residue differences as compared to SEQ ID NO: 730 at amino acid positions 161, 164, 201, 213, 223, 263, 333, 337, 411, 429, 463, 467, 479 / 485, 484 / 485, 502, 507, 522, 523, and 525. In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to carboxy terminal of SEQ ID NO: 730, or to a reference sequence corresponding to SEQ ID NO: 730, and one or more residue differences as compared to SEQ ID NO: 730, or amino acid residues selected from 161G, 161Q, 164Q, 201E, 213G, 223T, 263A, 333N, 333S, 337S, 411E, 429S, 463P, 467M, 479V / 485V, 484W / 485E, 502M, 507G, 522M, 522V, 523D, and 525R. In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to carboxy terminal of SEQ ID NO: 730, or to a reference sequence corresponding to SEQ ID NO: 730, and one or more residue differences as compared to SEQ ID NO: 730, or amino acid residues selected from K161G, K161Q, S164Q, A201E, S213G, D223T, H263A, A333N, A333S, P337S, G411E, R429S, A463P, L467M, L479V / I485V, R484W / I485E, G502M, A507G, L522M, L522V, E523D, and F525R.

[0189] In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to carboxy terminal of SEQ ID NO: 742, or to a reference sequence corresponding to SEQ ID NO: 742, and one or more residue differences as compared to SEQ ID NO: 742 at amino acid positions 161,Docket No. CX10-267WO3 161 / 197 / 208 / 262 / 416, 161 / 208, 161 / 208 / 262 / 411 / 416 / 429, 161 / 208 / 262 / 411 / 416 / 467, 161 / 208 / 467, 161 / 262, 161 / 262 / 411, 161 / 262 / 411 / 416, 161 / 262 / 411 / 416 / 467, 161 / 262 / 411 / 467, 161 / 262 / 416, 161 / 262 / 416 / 467, 161 / 262 / 429, 161 / 262 / 467, 161 / 416 / 442, 161 / 429, 197, 197 / 208, 197 / 208 / 262, 197 / 208 / 262 / 411 / 429 / 467, 197 / 208 / 411, 197 / 208 / 411 / 416 / 467, 197 / 208 / 416, 197 / 208 / 467, 197 / 262, 197 / 262 / 467, 208, 208 / 262, 208 / 262 / 411 / 416, 208 / 262 / 416 / 467, 208 / 262 / 429, 208 / 411 / 416, 208 / 411 / 467, 208 / 416, 208 / 416 / 429, 262, 262 / 411, 262 / 411 / 416, 262 / 411 / 416 / 429, 262 / 416, 262 / 416 / 467, 411 / 416, and 416. In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to carboxy terminal of SEQ ID NO: 742, or to a reference sequence corresponding to SEQ ID NO: 742, and one or more residue differences as compared to SEQ ID NO: 742, or amino acid residues selected from 161G, 161G / 197M / 208G / 262M / 416G, 161G / 208G, 161G / 208G / 262M / 411E / 416G / 429S, 161G / 208G / 262M / 411E / 416G / 467M, 161G / 208G / 467M, 161G / 262L / 429S, 161G / 262M, 161G / 262M / 411E, 161G / 262M / 411E / 416G, 161G / 262M / 411E / 416G / 467M, 161G / 262M / 411E / 467M, 161G / 262M / 416G, 161G / 262M / 416G / 467M, 161G / 262M / 467M, 161G / 416G / 442R, 161G / 429S, 197M, 197M / 208G, 197M / 208G / 262M, 197M / 208G / 262M / 411E / 429S / 467M, 197M / 208G / 411E, 197M / 208G / 411E / 416G / 467M, 197M / 208G / 416G, 197M / 208G / 467M, 197M / 262M, 197M / 262M / 467M, 208G, 208G / 262M, 208G / 262M / 411E / 416G, 208G / 262M / 416G / 467M, 208G / 262M / 429S, 208G / 411E / 416G, 208G / 411E / 467M, 208G / 416G, 208G / 416G / 429S, 262M, 262M / 411E, 262M / 411E / 416G, 262M / 411E / 416G / 429S, 262M / 416G, 262M / 416G / 467M, 411E / 416G, and 416G. In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to carboxy terminal of SEQ ID NO: 742, or to a reference sequence corresponding to SEQ ID NO: 742, and one or more residue differences as compared to SEQ ID NO: 742, or amino acid residues selected from K161G, K161G / N197M / E208G / F262M / A416G, K161G / E208G, K161G / E208G / F262M / G411E / A416G / R429S, K161G / E208G / F262M / G411E / A416G / L467M, K161G / E208G / L467M, K161G / F262L / R429S, K161G / F262M, K161G / F262M / G411E, K161G / F262M / G411E / A416G, K161G / F262M / G411E / A416G / L467M, K161G / F262M / G411E / L467M, K161G / F262M / A416G, K161G / F262M / A416G / L467M, K161G / F262M / L467M, K161G / A416G / G442R, K161G / R429S, N197M, N197M / E208G, N197M / E208G / F262M, N197M / E208G / F262M / G411E / R429S / L467M, N197M / E208G / G411E, N197M / E208G / G411E / A416G / L467M, N197M / E208G / A416G, N197M / E208G / L467M, N197M / F262M, N197M / F262M / L467M, E208G, E208G / F262M, E208G / F262M / G411E / A416G, E208G / F262M / A416G / L467M, E208G / F262M / R429S, E208G / G411E / A416G, E208G / G411E / L467M, E208G / A416G, E208G / A416G / R429S, F262M, F262M / G411E, F262M / G411E / A416G, F262M / G411E / A416G / R429S, F262M / A416G, F262M / A416G / L467M, G411E / A416G, and A416G.

[0190] In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,Docket No. CX10-267WO3 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to carboxy terminal of SEQ ID NO: 932, or to a reference sequence corresponding to SEQ ID NO: 932, and one or more residue differences as compared to SEQ ID NO: 932 at amino acid positions 262 / 317, 262 / 329, 262 / 339, 262 / 403, 262 / 462, 262 / 492, 262 / 526, and 387. In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to carboxy terminal of SEQ ID NO: 932, or to a reference sequence corresponding to SEQ ID NO: 932, and one or more residue differences as compared to SEQ ID NO: 932, or amino acid residues selected from 262M / 317R, 262M / 329L, 262M / 339V, 262M / 403E, 262M / 403F, 262M / 403P, 262M / 462L, 262M / 492A, 262M / 526E, and 387V. In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to carboxy terminal of SEQ ID NO: 932, or to a reference sequence corresponding to SEQ ID NO: 932, and one or more residue differences as compared to SEQ ID NO: 932, or amino acid residues selected from F262M / T317R, F262M / Q329L, F262M / A339V, F262M / L403E, F262M / L403F, F262M / L403P, F262M / M462L, F262M / H492A, F262M / Y526E, and L387V.

[0191] In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to carboxy terminal of SEQ ID NO: 734, or to a reference sequence corresponding to SEQ ID NO: 734, and one or more residue differences as compared to SEQ ID NO: 734 at amino acid positions 183, 186, 193, 264, 294, 297, 313, 314, 315, 347, 352, 353, 359, 389, 398, 405, 406, and 409. In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to carboxy terminal of SEQ ID NO: 734, or to a reference sequence corresponding to SEQ ID NO: 734, and one or more residue differences as compared to SEQ ID NO: 734, or amino acid residues selected from 183R, 183S, 186G, 186L, 186M, 186T, 186V, 193R, 264L, 294G, 297A, 297S, 313H, 313S, 314A, 314S, 315K, 315R, 347S, 352A, 352N, 353K, 359A, 359C, 389V, 398L, 405P, 406R, and 409P. In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to carboxy terminal of SEQ ID NO: 734, or to a reference sequence corresponding to SEQ ID NO: 734, and one or more residue differences as compared to SEQ ID NO: 734, or amino acid residues selected from D183R, D183S, E186G, E186L, E186M, E186T, E186V, S193R, A264L, K294G, K297A, K297S, A313H, A313S, R314A, R314S, G315K, G315R, Q347S, P352A, P352N, Q353K, L359A, L359C, L389V, W398L, S405P, G406R, and D409P.Docket No. CX10-267WO3

[0192] In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to carboxy terminal of SEQ ID NO: 932, or to a reference sequence corresponding to SEQ ID NO: 932, and one or more residue differences as compared to SEQ ID NO: 932 at amino acid positions 184 / 262, 262 / 340, 262 / 363, 262 / 371, 262 / 371 / 443, 262 / 377, and 262 / 387. In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to carboxy terminal of SEQ ID NO: 932, or to a reference sequence corresponding to SEQ ID NO: 932, and one or more residue differences as compared to SEQ ID NO: 932, or amino acid residues selected from 184K / 262M, 262M / 340V, 262M / 363G, 262M / 371G / 443D, 262M / 371R, 262M / 377R, and 262M / 387T. In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to carboxy terminal of SEQ ID NO: 932, or to a reference sequence corresponding to SEQ ID NO: 932, and one or more residue differences as compared to SEQ ID NO: 932, or amino acid residues selected from S184K / F262M, F262M / I340V, F262M / P363G, F262M / E371G / E443D, F262M / E371R, F262M / K377R, and F262M / L387T.

[0193] In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to carboxy terminal of SEQ ID NO: 992, or to a reference sequence corresponding to SEQ ID NO: 992, and one or more residue differences as compared to SEQ ID NO: 992 at amino acid positions 60 / 193 / 264 / 297, 60 / 263 / 264 / 297 / 300 / 525, 193 / 297, 208 / 273 / 294 / 407, 208 / 273 / 295, 273, and 297. In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to carboxy terminal of SEQ ID NO: 992, or to a reference sequence corresponding to SEQ ID NO: 992, and one or more residue differences as compared to SEQ ID NO: 992, or amino acid residues selected from 60H / 193T / 264L / 297S, 60H / 263A / 264L / 297S / 300S / 525W, 193T / 297A, 208E / 273L / 294T / 407G, 208E / 273M / 295N, 273L, and 297S. In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to carboxy terminal of SEQ ID NO: 992, or to a reference sequence corresponding to SEQ ID NO: 992, and one or more residue differences as compared to SEQ ID NO: 992, or amino acid residues selected from R60H / S193T / A264L / K297S, R60H / H263A / A264L / K297S / T300S / F525W, S193T / K297A, G208E / R273L / K294T / P407G, G208E / R273M / T295N, R273L, and K297S.Docket No. CX10-267WO3

[0194] In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to carboxy terminal of SEQ ID NO: 1144, or to a reference sequence corresponding to SEQ ID NO: 1144, and one or more residue differences as compared to SEQ ID NO: 1144 at amino acid positions 229, 230, 231, 233, 234, 237, 238, 241, 250, 251, 252, 260 / 264, and 264. In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to carboxy terminal of SEQ ID NO: 1144, or to a reference sequence corresponding to SEQ ID NO: 1144, and one or more residue differences as compared to SEQ ID NO: 1144, or amino acid residues selected from 229F, 230C, 230L, 231C, 231M, 231T, 233I, 234T, 237L, 238F, 238L, 238V, 241L, 250G, 251E, 251G, 251M, 251R, 251S, 251W, 252M, 252S, 260R / 264A, and 264A. In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to carboxy terminal of SEQ ID NO: 1144, or to a reference sequence corresponding to SEQ ID NO: 1144, and one or more residue differences as compared to SEQ ID NO: 1144, or amino acid residues selected from W229F, I230C, I230L, S231C, S231M, S231T, E233I, V234T, I237L, M238F, M238L, M238V, I241L, V250G, Q251E, Q251G, Q251M, Q251R, Q251S, Q251W, A252M, A252S, Q260R / L264A, and L264A.

[0195] In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to carboxy terminal of SEQ ID NO: 1144, or to a reference sequence corresponding to SEQ ID NO: 1144, and one or more residue differences as compared to SEQ ID NO: 1144 at amino acid positions 329, 329 / 339 / 340, 329 / 339 / 340 / 371 / 387, 329 / 339 / 340 / 403, 329 / 339 / 340 / 403 / 526, 329 / 339 / 371 / 387, 329 / 339 / 403 / 443, 329 / 526, 339, 339 / 340 / 387 / 526, 339 / 340 / 428, 339 / 403, 339 / 526, 371 / 403 / 526, and 443 / 526. In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to carboxy terminal of SEQ ID NO: 1144, or to a reference sequence corresponding to SEQ ID NO: 1144, and one or more residue differences as compared to SEQ ID NO: 1144, or amino acid residues selected from 329L, 329L / 339V / 340V, 329L / 339V / 340V / 371G / 387T, 329L / 339V / 340V / 403E, 329L / 339V / 340V / 403E / 526E, 329L / 339V / 371G / 387T, 329L / 339V / 403E / 443D, 329L / 526E, 339V, 339V / 340V / 387V / 526E, 339V / 340V / 428L, 339V / 403E, 339V / 526E, 371G / 403E / 526E, and 443D / 526E. In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to carboxy terminal of SEQ ID NO: 1144, or to a reference sequence corresponding to SEQ IDDocket No. CX10-267WO3 NO: 1144, and one or more residue differences as compared to SEQ ID NO: 1144, or amino acid residues selected from Q329L, Q329L / A339V / I340V, Q329L / A339V / I340V / E371G / L387T, Q329L / A339V / I340V / L403E, Q329L / A339V / I340V / L403E / Y526E, Q329L / A339V / E371G / L387T, Q329L / A339V / L403E / E443D, Q329L / Y526E, A339V, A339V / I340V / L387V / Y526E, A339V / I340V / V428L, A339V / L403E, A339V / Y526E, E371G / L403E / Y526E, and E443D / Y526E.

[0196] In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to carboxy terminal of SEQ ID NO: 1212, or to a reference sequence corresponding to SEQ ID NO: 1212, and one or more residue differences as compared to SEQ ID NO: 1212 at amino acid positions 184 / 329 / 403 / 404 / 492, 184 / 403 / 404 / 526, 329, 329 / 526, 340, 340 / 460, 376, and 492. In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to carboxy terminal of SEQ ID NO: 1212, or to a reference sequence corresponding to SEQ ID NO: 1212, and one or more residue differences as compared to SEQ ID NO: 1212, or amino acid residues selected from 184K / 329L / 403E / 404G / 492A, 184K / 403F / 404G / 526E, 329L, 329L / 526E, 340M, 340V / 460V, 376S, and 492A. In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to carboxy terminal of SEQ ID NO: 1212, or to a reference sequence corresponding to SEQ ID NO: 1212, and one or more residue differences as compared to SEQ ID NO: 1212, or amino acid residues selected from S184K / Q329L / L403E / S404G / H492A, S184K / L403F / S404G / Y526E, Q329L, Q329L / Y526E, I340M, I340V / G460V, Q376S, and H492A.

[0197] In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to carboxy terminal of SEQ ID NO: 1236, or to a reference sequence corresponding to SEQ ID NO: 1236, and one or more residue differences as compared to SEQ ID NO: 1236 at amino acid positions 174, 190, 198, 201, 202, 212, 263, 297, 318, 319, 322 / 329, 325 / 329, 329, 343, 379, 412, 415, 419, 448, 450, 456, 463, 477, 479, 480, 484, 523, and 525. In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to carboxy terminal of SEQ ID NO: 1236, or to a reference sequence corresponding to SEQ ID NO: 1236, and one or more residue differences as compared to SEQ ID NO: 1236, or amino acid residues selected from 174W, 190A, 190F, 190L, 190T, 190V, 198E, 201D, 202M, 202S, 212R, 263A, 263S, 263W, 297G, 297P, 318Q, 319Q, 322E / 329Q, 325G / 329Q, 325S / 329Q, 325Y / 329Q, 329Q, 343R, 343T, 343V, 379L, 412Q, 415G, 419A, 448R, 450V, 456M, 463S, 477T, 479V, 480M, 484A, 523D, 525I, 525R, and 525V. In some embodiments, the engineered TnT polypeptide comprisesDocket No. CX10-267WO3 an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to carboxy terminal of SEQ ID NO: 1236, or to a reference sequence corresponding to SEQ ID NO: 1236, and one or more residue differences as compared to SEQ ID NO: 1236, or amino acid residues selected from L174W, R190A, R190F, R190L, R190T, R190V, D198E, A201D, L202M, L202S, K212R, H263A, H263S, H263W, S297G, S297P, R318Q, T319Q, D322E / L329Q, W325G / L329Q, W325S / L329Q, W325Y / L329Q, L329Q, L343R, L343T, L343V, M379L, D412Q, A415G, L419A, K448R, I450V, R456M, A463S, Q477T, L479V, R480M, R484A, E523D, F525I, F525R, and F525V.

[0198] In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to carboxy terminal of SEQ ID NO: 1236, or to a reference sequence corresponding to SEQ ID NO: 1236, and one or more residue differences as compared to SEQ ID NO: 1236 at amino acid positions 175, 179, 183, 192, 194, 196, 199, 203, 208, 264, 280, 281, 295, 300, 313, 314, 352, 353, 354, 389, 404, 406, 407, 408, and 409. In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to carboxy terminal of SEQ ID NO: 1236, or to a reference sequence corresponding to SEQ ID NO: 1236, and one or more residue differences as compared to SEQ ID NO: 1236, or amino acid residues selected from 175V, 179L, 183V, 192A, 194L, 196H, 199F, 203M, 208Q, 264R, 280W, 281C, 281G, 281H, 281L, 281S, 295A, 300L, 300N, 313H, 313R, 314T, 352E, 352L, 353L, 353V, 354G, 389V, 404E, 404L, 404Q, 404V, 406N, 406T, 407L, 407V, 408L, and 409E. In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to carboxy terminal of SEQ ID NO: 1236, or to a reference sequence corresponding to SEQ ID NO: 1236, and one or more residue differences as compared to SEQ ID NO: 1236, or amino acid residues selected from N175V, Q179L, D183V, Y192A, F194L, G196H, D199F, E203M, G208Q, L264R, Y280W, Q281C, Q281G, Q281H, Q281L, Q281S, T295A, T300L, T300N, A313H, A313R, R314T, P352E, P352L, Q353L, Q353V, S354G, L389V, S404E, S404L, S404Q, S404V, G406N, G406T, P407L, P407V, A408L, and D409E.

[0199] In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to carboxy terminal of SEQ ID NO: 1264, or to a reference sequence corresponding to SEQ ID NO: 1264, and one or more residue differences as compared to SEQ ID NO: 1264 at amino acid positions 190 / 263 / 295 / 300 / 342 / 393, 190 / 300 / 525, 263 / 295 / 300 / 420, 263 / 295 / 420, 264 / 300 / 420, 264 / 379, 264 / 420, 264 / 420 / 525, 295 / 379 / 420, 295 / 420, 300 / 379 / 420, 300 / 420, 313, 313 / 379 / 525, 342, 342 / 420, 379, 379 / 420, 420, and 525. In some embodiments, the engineered TnT polypeptide comprises an amino acid sequenceDocket No. CX10-267WO3 having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to carboxy terminal of SEQ ID NO: 1264, or to a reference sequence corresponding to SEQ ID NO: 1264, and one or more residue differences as compared to SEQ ID NO: 1264, or amino acid residues selected from 190G / 263S / 295A / 300S / 342S / 393T, 190G / 300S / 525V, 263S / 295A / 300S / 420V, 263S / 295A / 420V, 264S / 300S / 420V, 264S / 379L, 264S / 420V, 264S / 420V / 525V, 295A / 379L / 420V, 295A / 420V, 300S / 379L / 420V, 300S / 420V, 313R, 313R / 379L / 525V, 342S, 342S / 420V, 379L, 379L / 420V, 420V, and 525V. In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to carboxy terminal of SEQ ID NO: 1264, or to a reference sequence corresponding to SEQ ID NO: 1264, and one or more residue differences as compared to SEQ ID NO: 1264, or amino acid residues selected from T190G / H263S / T295A / T300S / E342S / V393T, T190G / T300S / F525V, H263S / T295A / T300S / I420V, H263S / T295A / I420V, L264S / T300S / I420V, L264S / M379L, L264S / I420V, L264S / I420V / F525V, T295A / M379L / I420V, T295A / I420V, T300S / M379L / I420V, T300S / I420V, A313R, A313R / M379L / F525V, E342S, E342S / I420V, M379L, M379L / I420V, I420V, and F525V.

[0200] In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to carboxy terminal of SEQ ID NO: 1264, or to a reference sequence corresponding to SEQ ID NO: 1264, and one or more residue differences as compared to SEQ ID NO: 1264 at amino acid positions 174, 190, 190 / 193, 190 / 194, 260, 353, 392, 393, 395, 405, 407, 408, 411, and 418. In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to carboxy terminal of SEQ ID NO: 1264, or to a reference sequence corresponding to SEQ ID NO: 1264, and one or more residue differences as compared to SEQ ID NO: 1264, or amino acid residues selected from 174V, 190M, 190R / 193I, 190R / 194M, 260R, 353L, 353V, 392Q, 393T, 395E, 395M, 395T, 405Q, 407Q, 408N, 411K, and 418C. In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to carboxy terminal of SEQ ID NO: 1264, or to a reference sequence corresponding to SEQ ID NO: 1264, and one or more residue differences as compared to SEQ ID NO: 1264, or amino acid residues selected from L174V, T190M, T190R / T193I, T190R / F194M, Q260R, Q353L, Q353V, K392Q, V393T, W395E, W395M, W395T, S405Q, P407Q, A408N, G411K, and F418C.

[0201] In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to aminoDocket No. CX10-267WO3 acid residues 12 to carboxy terminal of SEQ ID NO: 1264, or to a reference sequence corresponding to SEQ ID NO: 1264, and one or more residue differences as compared to SEQ ID NO: 1264 at amino acid positions 190, 190 / 193, 190 / 194, 353, 405, 407, 408, and 409. In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to carboxy terminal of SEQ ID NO: 1264, or to a reference sequence corresponding to SEQ ID NO: 1264, and one or more residue differences as compared to SEQ ID NO: 1264, or amino acid residues selected from 190R, 190R / 193G, 190R / 193S, 190R / 193V, 190R / 194M, 353L, 405Q, 405V, 407G, 407Y, 408V, and 409V. In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to carboxy terminal of SEQ ID NO: 1264, or to a reference sequence corresponding to SEQ ID NO: 1264, and one or more residue differences as compared to SEQ ID NO: 1264, or amino acid residues selected from T190R, T190R / T193G, T190R / T193S, T190R / T193V, T190R / F194M, Q353L, S405Q, S405V, P407G, P407Y, A408V, and D409V.

[0202] In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to carboxy terminal of SEQ ID NO: 1414, or to a reference sequence corresponding to SEQ ID NO: 1414, and one or more residue differences as compared to SEQ ID NO: 1414 at amino acid positions 169 / 409, 194, 203 / 208 / 295 / 308 / 409, 203 / 208 / 295 / 389 / 409, 263 / 300 / 353, 295 / 308 / 409, 300, and 352 / 353. In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to carboxy terminal of SEQ ID NO: 1414, or to a reference sequence corresponding to SEQ ID NO: 1414, and one or more residue differences as compared to SEQ ID NO: 1414, or amino acid residues selected from 169L / 409E, 194L, 203M / 208Q / 295A / 308H / 409E, 203M / 208Q / 295D / 308H / 409E, 203M / 208Q / 295D / 389V / 409E, 263S / 300N / 353V, 295A / 308H / 409E, 300N, and 352A / 353V. In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to carboxy terminal of SEQ ID NO: 1414, or to a reference sequence corresponding to SEQ ID NO: 1414, and one or more residue differences as compared to SEQ ID NO: 1414, or amino acid residues selected from Q169L / D409E, F194L, E203M / G208Q / T295A / Y308H / D409E, E203M / G208Q / T295D / Y308H / D409E, E203M / G208Q / T295D / L389V / D409E, H263S / T300N / Q353V, T295A / Y308H / D409E, T300N, and P352A / Q353V.

[0203] In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to aminoDocket No. CX10-267WO3 acid residues 12 to carboxy terminal of SEQ ID NO: 1504, or to a reference sequence corresponding to SEQ ID NO: 1504, and one or more residue differences as compared to SEQ ID NO: 1504 at amino acid positions 174 / 260 / 263 / 353 / 405 / 408, 174 / 353, 174 / 405 / 407 / 420, 174 / 407 / 409 / 420, 260 / 353, 260 / 353 / 420, 263 / 352 / 353 / 405 / 408 / 420, 263 / 353 / 420, 263 / 420, 353 / 420, 405 / 408 / 409 / 420, 407 / 408 / 420, 407 / 409 / 420, and 420. In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 174V / 260R / 263S / 353L / 405Q / 408V, 174V / 353V, 174V / 405Q / 407Y / 420V, 174V / 407Y / 409D / 420V, 260R / 353V, 260R / 353V / 420V, 263S / 352A / 353V / 405Q / 408V / 420V, 263S / 353V / 420V, 263S / 420V, 353L / 420V, 353V / 420V, 405Q / 408V / 409D / 420V, 407S / 409D / 420V, 407Y / 408V / 420V, and 420V. In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to carboxy terminal of SEQ ID NO: 1504, or to a reference sequence corresponding to SEQ ID NO: 1504, and one or more residue differences as compared to SEQ ID NO: 1504, or amino acid residues selected from L174V / Q260R / H263S / Q353L / S405Q / A408V, L174V / Q353V, L174V / S405Q / P407Y / I420V, L174V / P407Y / E409D / I420V, Q260R / Q353V, Q260R / Q353V / I420V, H263S / P352A / Q353V / S405Q / A408V / I420V, H263S / Q353V / I420V, H263S / I420V, Q353L / I420V, Q353V / I420V, S405Q / A408V / E409D / I420V, P407S / E409D / I420V, P407Y / A408V / I420V, and I420V.

[0204] In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to carboxy terminal of SEQ ID NO: 1504, or to a reference sequence corresponding to SEQ ID NO: 1504, and one or more residue differences as compared to SEQ ID NO: 1504 at amino acid positions 133 / 197 / 203, 192, 197 / 203, 261, 263, 281, 281 / 440, 295 / 297, 295 / 301 / 308, 308, 308 / 314, 322, 328, 389, 390, 394, 404 / 409, 405 / 409, 461, and 484. In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to carboxy terminal of SEQ ID NO: 1504, or to a reference sequence corresponding to SEQ ID NO: 1504, and one or more residue differences as compared to SEQ ID NO: 1504, or amino acid residues selected from 133E / 197Q / 203E, 192H, 197R / 203E, 261G, 261R, 261V, 263K, 263R, 281G, 281S / 440D, 295T / 297G, 295T / 297P, 295T / 301V / 308Y, 308L, 308Y, 308Y / 314I, 322E, 328A, 389V, 390L, 390M, 394N, 394S, 404I / 409D, 404M / 409D, 405A / 409D, 405V / 409D, 461A, and 484W. In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to carboxy terminal of SEQ ID NO: 1504, or to a reference sequence corresponding to SEQ ID NO: 1504, and one or more residue differences as compared to SEQ ID NO: 1504, or amino acid residues selected from K133E / N197Q / M203E, Y192H, N197R / M203E, S261G, S261R, S261V, H263K, H263R, Q281G, Q281S / N440D, D295T / S297G, D295T / S297P, D295T / W301V / H308Y, H308L, H308Y,Docket No. CX10-267WO3 H308Y / R314I, D322E, V328A, L389V, C390L, C390M, Y394N, Y394S, S404I / E409D, S404M / E409D, S405A / E409D, S405V / E409D, S461A, and R484W.

[0205] In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to carboxy terminal of SEQ ID NO: 1522, or to a reference sequence corresponding to SEQ ID NO: 1522, and one or more residue differences as compared to SEQ ID NO: 1522 at amino acid positions 52, 175, 182, 193, 195, 196, 201, and 278. In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to carboxy terminal of SEQ ID NO: 1522, or to a reference sequence corresponding to SEQ ID NO: 1522, and one or more residue differences as compared to SEQ ID NO: 1522, or amino acid residues selected from 52G, 175E, 175S, 175T, 182V, 193A, 193G, 193V, 195R, 195T, 196V, 201S, and 278R. In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to carboxy terminal of SEQ ID NO: 1522, or to a reference sequence corresponding to SEQ ID NO: 1522, and one or more residue differences as compared to SEQ ID NO: 1522, or amino acid residues selected from E52G, N175E, N175S, N175T, T182V, T193A, T193G, T193V, K195R, K195T, G196V, A201S, and K278R.

[0206] In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to carboxy terminal of SEQ ID NO: 1522, or to a reference sequence corresponding to SEQ ID NO: 1522, and one or more residue differences as compared to SEQ ID NO: 1522 at amino acid positions 173, 193, 195, 196, 201, 272, 275, 278, 282, 297, 299, 301, 314, 412, 418, 461, 462, 479, and 494. In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to carboxy terminal of SEQ ID NO: 1522, or to a reference sequence corresponding to SEQ ID NO: 1522, and one or more residue differences as compared to SEQ ID NO: 1522, or amino acid residues selected from 173P, 193R, 195F, 195W, 196R, 201P, 201R, 272R, 275R, 275S, 278R, 282N, 297A, 297P, 297T, 297V, 297W, 299G, 299H, 299P, 301A, 314A, 412S, 412V, 418S, 418T, 461V, 462F, 479M, 479T, 494L, 494M, 494Q, and 494V. In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to carboxy terminal of SEQ ID NO: 1522, or to a reference sequence corresponding to SEQ ID NO: 1522, and one or more residue differences as compared to SEQ ID NO: 1522, or amino acid residues selected from T173P, T193R, K195F, K195W, G196R, A201P, A201R, G272R,Docket No. CX10-267WO3 T275R, T275S, K278R, M282N, S297A, S297P, S297T, S297V, S297W, S299G, S299H, S299P, W301A, R314A, D412S, D412V, F418S, F418T, S461V, L462F, L479M, L479T, H494L, H494M, H494Q, and H494V.

[0207] In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to carboxy terminal of SEQ ID NO: 1522, or to a reference sequence corresponding to SEQ ID NO: 1522, and one or more residue differences as compared to SEQ ID NO: 1522 at amino acid positions 174 / 194 / 195 / 197 / 319 / 361 / 420, 174 / 194 / 197 / 260 / 263 / 319 / 353 / 409 / 420, 174 / 194 / 197 / 260 / 263 / 353 / 409 / 420, 194 / 195 / 197 / 319, 194 / 195 / 319 / 420, 194 / 195 / 361, 194 / 197, 194 / 197 / 319 / 361 / 420, 194 / 319, 194 / 319 / 361 / 420, 194 / 319 / 420, 195 / 319 / 361 / 420, 195 / 319 / 420, 197 / 420, 300 / 325 / 406, 300 / 353 / 420 / 525, 300 / 406 / 409, 319 / 420, 325 / 406, and 361 / 420. In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to carboxy terminal of SEQ ID NO: 1522, or to a reference sequence corresponding to SEQ ID NO: 1522, and one or more residue differences as compared to SEQ ID NO: 1522, or amino acid residues selected from 174L / 194L / 195Q / 197M / 319V / 361V / 420V, 174L / 194L / 197M / 260Q / 263H / 319V / 353V / 409D / 420V, 174L / 194L / 197M / 260Q / 263H / 353V / 409D / 420V, 194L / 195Q / 197M / 319V, 194L / 195Q / 319V / 420V, 194L / 195Q / 361V, 194L / 197M, 194L / 197M / 319V / 361V / 420V, 194L / 319V, 194L / 319V / 361V / 420V, 194L / 319V / 420V, 195Q / 319V / 361V / 420V, 195Q / 319V / 420V, 197M / 420V, 300N / 325V / 406D, 300N / 353V / 420V / 525T, 300N / 406D / 409D, 319V / 420V, 325V / 406D, and 361V / 420V. In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to carboxy terminal of SEQ ID NO: 1522, or to a reference sequence corresponding to SEQ ID NO: 1522, and one or more residue differences as compared to SEQ ID NO: 1522, or amino acid residues selected from V174L / F194L / K195Q / N197M / T319V / I361V / I420V, V174L / F194L / N197M / R260Q / S263H / T319V / L353V / E409D / I420V, V174L / F194L / N197M / R260Q / S263H / L353V / E409D / I420V, F194L / K195Q / N197M / T319V, F194L / K195Q / T319V / I420V, F194L / K195Q / I361V, F194L / N197M, F194L / N197M / T319V / I361V / I420V, F194L / T319V, F194L / T319V / I361V / I420V, F194L / T319V / I420V, K195Q / T319V / I361V / I420V, K195Q / T319V / I420V, N197M / I420V, T300N / W325V / G406D, T300N / L353V / I420V / V525T, T300N / G406D / E409D, T319V / I420V, W325V / G406D, and I361V / I420V.

[0208] In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to carboxy terminal of SEQ ID NO: 1702, or to a reference sequence corresponding to SEQ ID NO: 1702, and one or more residue differences as compared to SEQ ID NO: 1702 at amino acid positionsDocket No. CX10-267WO3 261 / 409, 297, 300, and 409. In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to carboxy terminal of SEQ ID NO: 1702, or to a reference sequence corresponding to SEQ ID NO: 1702, and one or more residue differences as compared to SEQ ID NO: 1702, or amino acid residues selected from 261V / 409D, 297P, 300N, and 409D. In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to carboxy terminal of SEQ ID NO: 1702, or to a reference sequence corresponding to SEQ ID NO: 1702, and one or more residue differences as compared to SEQ ID NO: 1702, or amino acid residues selected from S261V / E409D, S297P, T300N, and E409D.

[0209] In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to carboxy terminal of SEQ ID NO: 1742, or to a reference sequence corresponding to SEQ ID NO: 1742, and one or more residue differences as compared to SEQ ID NO: 1742 at amino acid positions 52 / 201 / 390, 175 / 193 / 297 / 300, 175 / 297 / 300, 175 / 297 / 300 / 462, 175 / 300, 175 / 300 / 314, 175 / 300 / 442, 193 / 195 / 297 / 300 / 314, 193 / 297 / 300 / 314 / 442, 195 / 297 / 300 / 462, 221 / 297 / 300, and 297 / 300. In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to carboxy terminal of SEQ ID NO: 1742, or to a reference sequence corresponding to SEQ ID NO: 1742, and one or more residue differences as compared to SEQ ID NO: 1742, or amino acid residues selected from 52G / 201S / 390M, 175S / 193G / 297V / 300N, 175S / 297V / 300N, 175S / 297V / 300N / 462F, 175S / 300N, 175S / 300N / 314A, 175S / 300N / 442R, 193G / 195T / 297V / 300N / 314A, 193G / 297V / 300N / 314A / 442R, 195T / 297A / 300N / 462F, 195T / 297V / 300N / 462F, 221Q / 297A / 300N, 297A / 300N, and 297V / 300N. In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to carboxy terminal of SEQ ID NO: 1742, or to a reference sequence corresponding to SEQ ID NO: 1742, and one or more residue differences as compared to SEQ ID NO: 1742, or amino acid residues selected from E52G / A201S / C390M, N175S / T193G / S297V / T300N, N175S / S297V / T300N, N175S / S297V / T300N / L462F, N175S / T300N, N175S / T300N / R314A, N175S / T300N / G442R, T193G / K195T / S297V / T300N / R314A, T193G / S297V / T300N / R314A / G442R, K195T / S297A / T300N / L462F, K195T / S297V / T300N / L462F, L221Q / S297A / T300N, S297A / T300N, and S297V / T300N.

[0210] In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to aminoDocket No. CX10-267WO3 acid residues 12 to carboxy terminal of SEQ ID NO: 1742, or to a reference sequence corresponding to SEQ ID NO: 1742, and one or more residue differences as compared to SEQ ID NO: 1742 at amino acid positions 152 / 281, 182, 196, 197, 261, 281, 297, 303, 307, 319, 397, 404 / 409, 405 / 409, 406 / 409, 409, 463, 474, and 477. In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to carboxy terminal of SEQ ID NO: 1742, or to a reference sequence corresponding to SEQ ID NO: 1742, and one or more residue differences as compared to SEQ ID NO: 1742, or amino acid residues selected from 152H / 281S, 182V, 196D, 196L, 196S, 197I, 261T, 281S, 297T, 303R, 307N, 319P, 397S, 404E / 409E, 404G / 409E, 405S / 409E, 406A / 409E, 406P / 409E, 409E, 463P, 474L, and 477S. In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to carboxy terminal of SEQ ID NO: 1742, or to a reference sequence corresponding to SEQ ID NO: 1742, and one or more residue differences as compared to SEQ ID NO: 1742, or amino acid residues selected from R152H / Q281S, T182V, G196D, G196L, G196S, M197I, V261T, Q281S, S297T, K303R, T307N, V319P, K397S, S404E / D409E, S404G / D409E, Q405S / D409E, G406A / D409E, G406P / D409E, D409E, A463P, M474L, and Q477S.

[0211] In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to carboxy terminal of SEQ ID NO: 1742, or to a reference sequence corresponding to SEQ ID NO: 1742, and one or more residue differences as compared to SEQ ID NO: 1742 at amino acid positions 152 / 281, 190, 197, 208, 274, 281, 297, 299, 303, 307, 308, 313, 319, 321, 360, 404 / 409, 406 / 409, 417, 460, 461, 477, 479, 492, and 495. In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to carboxy terminal of SEQ ID NO: 1742, or to a reference sequence corresponding to SEQ ID NO: 1742, and one or more residue differences as compared to SEQ ID NO: 1742, or amino acid residues selected from 152H / 281S, 190R, 197I, 208E, 208T, 274S, 281A, 281G, 281H, 281S, 297T, 299N, 303R, 303T, 307D, 307E, 307M, 308Y, 313A, 319G, 319L, 319M, 319P, 321T, 360V, 404E / 409E, 404M / 409E, 404V / 409E, 406A / 409E, 406M / 409E, 406P / 409E, 417G, 460D, 461A, 477E, 479M, 479T, 492G, and 495A. In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to carboxy terminal of SEQ ID NO: 1742, or to a reference sequence corresponding to SEQ ID NO: 1742, and one or more residue differences as compared to SEQ ID NO: 1742, or amino acid residues selected from R152H / Q281S, T190R, M197I, Q208E, Q208T, P274S, Q281A, Q281G, Q281H, Q281S, S297T, S299N, K303R, K303T, T307D, T307E, T307M, H308Y, R313A, V319G, V319L,Docket No. CX10-267WO3 V319M, V319P, A321T, L360V, S404E / D409E, S404M / D409E, S404V / D409E, G406A / D409E, G406M / D409E, G406P / D409E, C417G, G460D, S461A, Q477E, L479M, L479T, H492G, and G495A.

[0212] In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to carboxy terminal of SEQ ID NO: 1928, or to a reference sequence corresponding to SEQ ID NO: 1928, and one or more residue differences as compared to SEQ ID NO: 1928 at amino acid positions 161 / 251 / 408, 183 / 522, 196 / 231 / 522, 231 / 416 / 474, 273, 273 / 274 / 325 / 416 / 522, 416, or 416 / 474. In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to carboxy terminal of SEQ ID NO: 1928, or to a reference sequence corresponding to SEQ ID NO: 1928, and one or more residue differences as compared to SEQ ID NO: 1928, or amino acid residues selected from 161G / 251M / 408A, 183T / 522V, 196Q / 231M / 522V, 231M / 416S / 474L, 273M, 273M / 274R / 325V / 416G / 522V, 416S, or 416S / 474L. In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to carboxy terminal of SEQ ID NO: 1928, or to a reference sequence corresponding to SEQ ID NO: 1928, and one or more residue differences as compared to SEQ ID NO: 1928, or amino acid residues selected from K161G / Q251M / V408A, D183T / L522V, G196Q / S231M / L522V, S231M / A416S / M474L, R273M, R273M / P274R / W325V / A416G / L522V, A416S, or A416S / M474L.

[0213] In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to carboxy terminal of SEQ ID NO: 1944, or to a reference sequence corresponding to SEQ ID NO: 1944, and one or more residue differences as compared to SEQ ID NO: 1944 at amino acid positions 231 / 408 and 406 / 463. In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to carboxy terminal of SEQ ID NO: 1944, or to a reference sequence corresponding to SEQ ID NO: 1944, and one or more residue differences as compared to SEQ ID NO: 1944, or amino acid residues selected from 231M / 408A and 406P / 463P. In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to carboxy terminal of SEQ ID NO: 1944, or to a reference sequence corresponding to SEQ ID NO: 1944, and one or more residue differences as compared to SEQ ID NO: 1944, or amino acid residues selected from S231M / V408A and G406P / A463P.Docket No. CX10-267WO3

[0214] In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to carboxy terminal of SEQ ID NO: 1944, or to a reference sequence corresponding to SEQ ID NO: 1944, and one or more residue differences as compared to SEQ ID NO: 1944 at amino acid positions 231, 273, 367, 407, 424, 427, 429, 430, or 499. In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to carboxy terminal of SEQ ID NO: 1944, or to a reference sequence corresponding to SEQ ID NO: 1944, and one or more residue differences as compared to SEQ ID NO: 1944, or amino acid residues selected from 231N, 273I, 273V, 367N, 367Q, 407D, 424P, 427M, 429S, 430G, 499N, and 499R. In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to carboxy terminal of SEQ ID NO: 1944, or to a reference sequence corresponding to SEQ ID NO: 1944, and one or more residue differences as compared to SEQ ID NO: 1944, or amino acid residues selected from S231N, M273I, M273V, E367N, E367Q, P407D, Y424P, L427M, R429S, K430G, M499N, and M499R.

[0215] In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to carboxy terminal of SEQ ID NO: 1946, or to a reference sequence corresponding to SEQ ID NO: 1946, and one or more residue differences as compared to SEQ ID NO: 1946 at amino acid positions 196, 196 / 273, 196 / 273 / 325, 196 / 273 / 325 / 335, 196 / 273 / 325 / 335 / 367, 196 / 273 / 430, 196 / 335, 196 / 335 / 367, 196 / 335 / 367 / 430, 196 / 367 / 479, 196 / 430, 273, 273 / 274, 273 / 274 / 367 / 430, 273 / 325 / 335 / 367, 273 / 325 / 430, 273 / 335 / 430, 273 / 367, 273 / 430, 273 / 430 / 479, 274, 274 / 325 / 335 / 479, 274 / 430, 325 / 335, 325 / 335 / 417, 325 / 335 / 417 / 430 / 479, 325 / 367, 335, 335 / 367, 335 / 367 / 479, 335 / 430, 335 / 479, 367, 367 / 430, 367 / 479, 417, 417 / 430 / 479, 430, and 479. In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to carboxy terminal of SEQ ID NO: 1946, or to a reference sequence corresponding to SEQ ID NO: 1946, and one or more residue differences as compared to SEQ ID NO: 1946, or amino acid residues selected from 196Q, 196Q / 273I / 325V, 196Q / 273I / 325V / 335V, 196Q / 273I / 430G, 196Q / 335V, 196Q / 335V / 367R / 430G, 196Q / 367R / 479M, 196Q / 430G, 196S / 273I, 196S / 273I / 325V / 335V / 367Q, 196S / 335V / 367R, 196S / 367R / 479M, 273I, 273I / 274R, 273I / 274R / 367R / 430G, 273I / 325V / 335V / 367Q, 273I / 325V / 430G, 273I / 335V / 430G, 273I / 367R, 273I / 430G, 273I / 430G / 479M, 274R, 274R / 325V / 335V / 479M, 274R / 430G, 325V / 335V, 325V / 335V / 417S, 325V / 335V / 417S / 430G / 479M, 325V / 367Q, 325V / 367R, 335V, 335V / 367Q, 335V / 367R / 479M, 335V / 430G, 335V / 479M, 367Q, 367Q / 479M, 367R, 367R / 430G, 417S, 417S / 430G / 479M, 430G, and 479M. In some embodiments, theDocket No. CX10-267WO3 engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to carboxy terminal of SEQ ID NO: 1946, or to a reference sequence corresponding to SEQ ID NO: 1946, and one or more residue differences as compared to SEQ ID NO: 1946, or amino acid residues selected from G196Q, G196Q / M273I / W325V, G196Q / M273I / W325V / F335V, G196Q / M273I / K430G, G196Q / F335V, G196Q / F335V / E367R / K430G, G196Q / E367R / L479M, G196Q / K430G, G196S / M273I, G196S / M273I / W325V / F335V / E367Q, G196S / F335V / E367R, G196S / E367R / L479M, M273I, M273I / P274R, M273I / P274R / E367R / K430G, M273I / W325V / F335V / E367Q, M273I / W325V / K430G, M273I / F335V / K430G, M273I / E367R, M273I / K430G, M273I / K430G / L479M, P274R, P274R / W325V / F335V / L479M, P274R / K430G, W325V / F335V, W325V / F335V / C417S, W325V / F335V / C417S / K430G / L479M, W325V / E367Q, W325V / E367R, F335V, F335V / E367Q, F335V / E367R / L479M, F335V / K430G, F335V / L479M, E367Q, E367Q / L479M, E367R, E367R / K430G, C417S, C417S / K430G / L479M, K430G, and L479M.

[0216] In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to carboxy terminal of SEQ ID NO: 1984, or to a reference sequence corresponding to SEQ ID NO: 1984, and one or more residue differences as compared to SEQ ID NO: 1984at amino acid positions 231, 231 / 273, 231 / 273 / 380 / 417 / 500, 231 / 273 / 506, 231 / 380 / 417 / 501, 231 / 500 / 501 / 506, or 231 / 501. In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to carboxy terminal of SEQ ID NO: 1984, or to a reference sequence corresponding to SEQ ID NO: 1984, and one or more residue differences as compared to SEQ ID NO: 1984, or amino acid residues selected from 231N, 231N / 273K, 231N / 273K / 380K / 417S / 500N, 231N / 273K / 506K, 231N / 273M, 231N / 380K / 417S / 501K, 231N / 500N / 501K / 506K, and 231N / 501K. In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to carboxy terminal of SEQ ID NO: 1984, or to a reference sequence corresponding to SEQ ID NO: 1984, and one or more residue differences as compared to SEQ ID NO: 1984, or amino acid residues selected from M231N, M231N / I273K, M231N / I273K / R380K / C417S / T500N, M231N / I273K / P506K, M231N / I273M, M231N / R380K / C417S / R501K, M231N / T500N / R501K / P506K, and M231N / R501K.

[0217] In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to carboxy terminal of SEQ ID NO: 1984, or to a reference sequence corresponding to SEQ ID NO: 1984, and one or more residue differences as compared to SEQ ID NO: 1984at amino acid positionsDocket No. CX10-267WO3 187, 190, 259, 260, 263, 273, 296, 300, 314, 343, 361, 367, 376, 384, 389, 390, 393, 395, 416, 417, 428, or 446. In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to carboxy terminal of SEQ ID NO: 1984, or to a reference sequence corresponding to SEQ ID NO: 1984, and one or more residue differences as compared to SEQ ID NO: 1984, or amino acid residues selected from 187L, 187T, 187V, 187Y, 190Q, 190V, 259F, 260K, 260S, 260T, 263R, 273M, 296L, 300A, 300K, 314E, 343M, 343V, 361I, 367E, 376E, 384S, 389V, 390S, 390T, 393T, 395F, 395S, 416G, 417A, 428L, 446D, 446G, 446H, 446S, and 446T. In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to carboxy terminal of SEQ ID NO: 1984, or to a reference sequence corresponding to SEQ ID NO: 1984, and one or more residue differences as compared to SEQ ID NO: 1984, or amino acid residues selected from I187L, I187T, I187V, I187Y, T190Q, T190V, Y259F, R260K, R260S, R260T, S263R, I273M, V296L, N300A, N300K, A314E, L343M, L343V, V361I, Q367E, Q376E, K384S, L389V, M390S, M390T, V393T, W395F, W395S, A416G, C417A, V428L, P446D, P446G, P446H, P446S, and P446T.

[0218] In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to carboxy terminal of SEQ ID NO: 2064, or to a reference sequence corresponding to SEQ ID NO: 2064, and one or more residue differences as compared to SEQ ID NO: 2064 at amino acid positions 161, 162, 174, 201, 219, 223, 244, 246, 252, 254, 318, 433, 437, 503, or 525. In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to carboxy terminal of SEQ ID NO: 2064, or to a reference sequence corresponding to SEQ ID NO: 2064, and one or more residue differences as compared to SEQ ID NO: 2064, or amino acid residues selected from 161L, 162P, 174W, 201Q, 219D, 223H, 244R, 246L, 252L, 254N, 254Q, 254R, 318K, 433A, 437H, 503V, and 525T. In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to carboxy terminal of SEQ ID NO: 2064, or to a reference sequence corresponding to SEQ ID NO: 2064, and one or more residue differences as compared to SEQ ID NO: 2064, or amino acid residues selected from K161L, T162P, V174W, A201Q, R219D, D223H, V244R, E246L, A252L, L254N, L254Q, L254R, R318K, H433A, T437H, I503V, and V525T.

[0219] In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to aminoDocket No. CX10-267WO3 acid residues 12 to carboxy terminal of SEQ ID NO: 2104, or to a reference sequence corresponding to SEQ ID NO: 2104, and one or more residue differences as compared to SEQ ID NO: 2104 at amino acid positions 174 / 199 / 389 / 390, 174 / 231, 174 / 231 / 260 / 273 / 389 / 390, 174 / 231 / 263 / 273 / 343, 174 / 231 / 263 / 389, 174 / 231 / 389 / 390 / 446, 174 / 231 / 390, 174 / 259 / 260 / 263 / 273 / 361 / 389 / 390 / 446, 174 / 259 / 273 / 343 / 361 / 389, 174 / 263 / 389 / 390, 174 / 273 / 343 / 389 / 390 / 446, 187 / 190 / 231 / 252 / 273 / 296 / 300 / 353 / 417, 187 / 190 / 252 / 273 / 300 / 417, 187 / 190 / 273 / 353, 187 / 231 / 252 / 273 / 296 / 300 / 353, 187 / 231 / 273 / 296 / 300 / 353, 187 / 231 / 296 / 300 / 353, 187 / 231 / 300 / 353, 187 / 273 / 353, 190 / 231 / 273, 196 / 199 / 231 / 263 / 273 / 389, 199 / 231 / 260 / 263 / 361 / 389 / 446, 231, 231 / 259 / 263 / 273, 231 / 273, 259 / 263 / 273 / 389 / 390, 260 / 273 / 446, 263 / 273 / 343 / 446, 273, 273 / 353, 273 / 389 / 446, or 273 / 390. In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to carboxy terminal of SEQ ID NO: 2104, or to a reference sequence corresponding to SEQ ID NO: 2104, and one or more residue differences as compared to SEQ ID NO: 2104, or amino acid residues selected from 174L / 199V / 389V / 390T, 174L / 231N, 174L / 231N / 260K / 273K / 389V / 390T, 174L / 231N / 263R / 389V, 174L / 231N / 263T / 273K / 343V, 174L / 231N / 263T / 389V, 174L / 231N / 389V / 390T / 446S, 174L / 231N / 390T, 174L / 259F / 260K / 263R / 273K / 361I / 389V / 390T / 446S, 174L / 259F / 273K / 343V / 361I / 389V, 174L / 263R / 389V / 390T, 174L / 273K / 343V / 389V / 390T / 446S, 187V / 190Q / 252M / 273K / 300K / 417S, 187V / 190Q / 273K / 353V, 187V / 231N / 252E / 273K / 296L / 300K / 353R, 187V / 231N / 300K / 353V, 187V / 273K / 353V, 187Y / 190Q / 231N / 252M / 273K / 296L / 300K / 353V / 417S, 187Y / 231N / 273K / 296L / 300K / 353R, 187Y / 231N / 296L / 300K / 353R, 190Q / 231N / 273K, 196Q / 199V / 231N / 263T / 273K / 389V, 199V / 231N / 260K / 263T / 361I / 389V / 446S, 231N, 231N / 259F / 263T / 273K, 231N / 273K, 259F / 263R / 273K / 389V / 390T, 260K / 273K / 446S, 263T / 273K / 343V / 446S, 273K, 273K / 353V, 273K / 389V / 446S, and 273K / 390T. In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to carboxy terminal of SEQ ID NO: 2104, or to a reference sequence corresponding to SEQ ID NO: 2104, and one or more residue differences as compared to SEQ ID NO: 2104, or amino acid residues selected from V174L / D199V / L389V / S390T, V174L / M231N, V174L / M231N / R260K / I273K / L389V / S390T, V174L / M231N / S263R / L389V, V174L / M231N / S263T / I273K / L343V, V174L / M231N / S263T / L389V, V174L / M231N / L389V / S390T / P446S, V174L / M231N / S390T, V174L / Y259F / R260K / S263R / I273K / V361I / L389V / S390T / P446S, V174L / Y259F / I273K / L343V / V361I / L389V, V174L / S263R / L389V / S390T, V174L / I273K / L343V / L389V / S390T / P446S, I187V / T190Q / A252M / I273K / N300K / C417S, I187V / T190Q / I273K / L353V, I187V / M231N / A252E / I273K / V296L / N300K / L353R, I187V / M231N / N300K / L353V, I187V / I273K / L353V, I187Y / T190Q / M231N / A252M / I273K / V296L / N300K / L353V / C417S, I187Y / M231N / I273K / V296L / N300K / L353R, I187Y / M231N / V296L / N300K / L353R, T190Q / M231N / I273K,Docket No. CX10-267WO3 G196Q / D199V / M231N / S263T / I273K / L389V, D199V / M231N / R260K / S263T / V361I / L389V / P446S, M231N, M231N / Y259F / S263T / I273K, M231N / I273K, Y259F / S263R / I273K / L389V / S390T, R260K / I273K / P446S, S263T / I273K / L343V / P446S, I273K, I273K / L353V, I273K / L389V / P446S, and I273K / S390T.

[0220] In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to carboxy terminal of SEQ ID NO: 2210, or to a reference sequence corresponding to SEQ ID NO: 2210, and one or more residue differences as compared to SEQ ID NO: 2210 at amino acid positions 161 / 162 / 254 / 417, 161 / 162 / 318 / 446, 174 / 244 / 273 / 390 / 437 / 503, 174 / 244 / 437 / 525, 174 / 273 / 503, 201 / 376, 433, or 525. In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to carboxy terminal of SEQ ID NO: 2210, or to a reference sequence corresponding to SEQ ID NO: 2210, and one or more residue differences as compared to SEQ ID NO: 2210, or amino acid residues selected from 161L / 162P / 254R / 417A, 161L / 162P / 318K / 446S, 174W / 244G / 437H / 525T, 174W / 244R / 273K / 390T / 437H / 503L, 174W / 273K / 503L, 201Q / 376E, 433P, and 525T. In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to carboxy terminal of SEQ ID NO: 2210, or to a reference sequence corresponding to SEQ ID NO: 2210, and one or more residue differences as compared to SEQ ID NO: 2210, or amino acid residues selected from K161L / T162P / L254R / C417A, K161L / T162P / R318K / P446S, V174W / V244G / T437H / V525T, V174W / V244R / I273K / S390T / T437H / I503L, V174W / I273K / I503L, A201Q / Q376E, H433P, and V525T.

[0221] In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to carboxy terminal of SEQ ID NO: 2210, or to a reference sequence corresponding to SEQ ID NO: 2210, and one or more residue differences as compared to SEQ ID NO: 2210 at amino acid positions 303, 397, or 466. In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to carboxy terminal of SEQ ID NO: 2210, or to a reference sequence corresponding to SEQ ID NO: 2210, and one or more residue differences as compared to SEQ ID NO: 2210, or amino acid residues selected from 303E, 397S, and 466I. In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to carboxy terminal of SEQ ID NO: 2210, or to a reference sequence corresponding to SEQ ID NO: 2210, and one or more residueDocket No. CX10-267WO3 differences as compared to SEQ ID NO: 2210, or amino acid residues selected from K303E, K397S, and M466I.

[0222] In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to carboxy terminal of SEQ ID NO: 2210, or to a reference sequence corresponding to SEQ ID NO: 2210, and one or more residue differences as compared to SEQ ID NO: 2210 at amino acid positions 278, 290, 297, 303, 397, or 525. In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to carboxy terminal of SEQ ID NO: 2210, or to a reference sequence corresponding to SEQ ID NO: 2210, and one or more residue differences as compared to SEQ ID NO: 2210, or amino acid residues selected from 278S, 290L, 297L, 297Y, 303E, 397G, 397S, and 525G. In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to carboxy terminal of SEQ ID NO: 2210, or to a reference sequence corresponding to SEQ ID NO: 2210, and one or more residue differences as compared to SEQ ID NO: 2210, or amino acid residues selected from K278S, V290L, S297L, S297Y, K303E, K397G, K397S, and V525G.

[0223] In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to carboxy terminal of SEQ ID NO: 2256, or to a reference sequence corresponding to SEQ ID NO: 2256, and one or more residue differences as compared to SEQ ID NO: 2256 at amino acid positions 22, 22 / 162 / 201 / 244 / 254 / 318 / 376 / 417 / 433 / 437 / 525, 22 / 201 / 376, 22 / 244, 22 / 244 / 254, 22 / 244 / 433 / 525, 22 / 318 / 376 / 433 / 525, 22 / 318 / 376 / 446, 22 / 376 / 417 / 437 / 446 / 525, 161 / 162 / 201 / 244 / 318 / 376 / 417 / 437, 161 / 162 / 201 / 244 / 376, 162, 162 / 201 / 318 / 376 / 417 / 433 / 525, 162 / 201 / 318 / 376 / 433 / 437 / 446, 162 / 244 / 254 / 433, 162 / 244 / 318 / 376 / 417 / 433 / 437, 162 / 254, 162 / 254 / 318 / 376 / 417 / 433, 162 / 254 / 376 / 417 / 433, 162 / 376 / 525, 162 / 433, 201 / 244 / 318 / 376, 201 / 244 / 376 / 433 / 525, 201 / 318 / 376 / 446, 244 / 318 / 376 / 417, 244 / 318 / 376 / 417 / 433 / 437 / 525, 318 / 376, 318 / 376 / 433, 433, or 437. In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to carboxy terminal of SEQ ID NO: 2256, or to a reference sequence corresponding to SEQ ID NO: 2256, and one or more residue differences as compared to SEQ ID NO: 2256, or amino acid residues selected from 22P, 22P / 162P / 201Q / 244G / 254R / 318K / 376E / 417A / 433P / 437H / 525T, 22P / 201Q / 376E, 22P / 244G, 22P / 244G / 254R, 22P / 244G / 433P / 525T, 22P / 318K / 376E / 433P / 525T, 22P / 318K / 376E / 446S, 22P / 376E / 417A / 437H / 446S / 525T, 161L / 162P / 201Q / 244G / 318K / 376E / 417A / 437H, 161L / 162P / 201Q / 244G / 376E, 162P, 162P / 201Q / 318K / 376E / 417A / 433P / 525T,Docket No. CX10-267WO3 162P / 201Q / 318K / 376E / 433P / 437H / 446S, 162P / 244G / 254R / 433P, 162P / 244G / 318K / 376E / 417A / 433P / 437H, 162P / 254R, 162P / 254R / 318K / 376E / 417A / 433P, 162P / 254R / 376E / 417A / 433P, 162P / 376E / 525T, 162P / 433P, 201Q / 244G / 318K / 376E, 201Q / 244G / 376E / 433P / 525T, 201Q / 318K / 376E / 446S, 244G / 318K / 376E / 417A, 244G / 318K / 376E / 417A / 433P / 437H / 525T, 318K / 376E, 318K / 376E / 433P, 433P, and 437H. In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to carboxy terminal of SEQ ID NO: 2256, or to a reference sequence corresponding to SEQ ID NO: 2256, and one or more residue differences as compared to SEQ ID NO: 2256, or amino acid residues selected from F22P, F22P / T162P / A201Q / V244G / L254R / R318K / Q376E / C417A / H433P / T437H / V525T, F22P / A201Q / Q376E, F22P / V244G, F22P / V244G / L254R, F22P / V244G / H433P / V525T,at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to carboxy terminal of SEQ ID NO: 2280, or to a reference sequence corresponding to SEQ ID NO: 2280, and one or more residue differences as compared to SEQ ID NO: 2280 at amino acid positions 22 / 27 / 437 / 466, 22 / 34 / 278 / 318 / 437 / 466, 22 / 466, 41 / 42 / 43 / 397, 43 / 244 / 397, 45 / 397, or 397. In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to carboxy terminal of SEQ ID NO: 2280, or to a reference sequence corresponding to SEQ ID NO: 2280, and one or more residue differences as compared to SEQ ID NO: 2280, or amino acid residues selected from 22P / 27P / 437H / 466I, 22P / 34Y / 278S / 318K / 437H / 466I, 22P / 466I, 41T / 42T / 43- / 397S, 43T / 244G / 397S, 45T / 397S, and 397S. In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to carboxy terminal of SEQ ID NO: 2280, or to a reference sequence corresponding to SEQ ID NO: 2280, and one or more residue differences as compared to SEQ ID NO: 2280,Docket No. CX10-267WO3 or amino acid residues selected from F22P / Q27P / T437H / M466I, F22P / D34Y / K278S / R318K / T437H / M466I, F22P / M466I, I41T / K42T / F43- / K397S, F43T / V244G / K397S, E45T / K397S, and K397S.

[0225] In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to carboxy terminal of SEQ ID NO: 2280, or to a reference sequence corresponding to SEQ ID NO: 2280, and one or more residue differences as compared to SEQ ID NO: 2280 at amino acid positions 173, 175, 176, 179, 182, 196, 197, 208, 263, 273, 274, 277, 308, 310, 319, 328, 341, 376, 388, 405, 408, 411, 462, 477, or 480. In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to carboxy terminal of SEQ ID NO: 2280, or to a reference sequence corresponding to SEQ ID NO: 2280, and one or more residue differences as compared to SEQ ID NO: 2280, or amino acid residues selected from 173P, 175L, 175R, 176H, 179C, 179K, 179R, 182I, 196D, 197L, 208T, 263R, 263T, 273M, 274L, 277D, 308Y, 310S, 319A, 328A, 341T, 376T, 388V, 405L, 408G, 411T, 462Y, 477L, 477W, and 480K. In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to carboxy terminal of SEQ ID NO: 2280, or to a reference sequence corresponding to SEQ ID NO: 2280, and one or more residue differences as compared to SEQ ID NO: 2280, or amino acid residues selected from T173P, S175L, S175R, N176H, Q179C, Q179K, Q179R, T182I, G196D, M197L, Q208T, S263R, S263T, K273M, P274L, S277D, H308Y, E310S, V319A, V328A, V341T, E376T, L388V, Q405L, A408G, G411T, L462Y, Q477L, Q477W, and R480K.

[0226] In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to carboxy terminal of SEQ ID NO: 2280, or to a reference sequence corresponding to SEQ ID NO: 2280, and one or more residue differences as compared to SEQ ID NO: 2280 at amino acid positions 195, 203, 273, 274, 277, 278, 291, 299, 300, 324, or 477. In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to carboxy terminal of SEQ ID NO: 2280, or to a reference sequence corresponding to SEQ ID NO: 2280, and one or more residue differences as compared to SEQ ID NO: 2280, or amino acid residues selected from 195T, 203Q, 273E, 273G, 273M, 273N, 273Q, 274L, 277D, 278D, 291L, 299G, 299H, 300S, 324T, and 477W. In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to carboxy terminal of SEQ ID NO: 2280, or to a reference sequence corresponding to SEQ ID NO: 2280, and one orDocket No. CX10-267WO3 more residue differences as compared to SEQ ID NO: 2280, or amino acid residues selected from K195T, M203Q, K273E, K273G, K273M, K273N, K273Q, P274L, S277D, K278D, R291L, S299G, S299H, K300S, I324T, and Q477W.

[0227] In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to carboxy terminal of SEQ ID NO: 2342, or to a reference sequence corresponding to SEQ ID NO: 2342, and one or more residue differences as compared to SEQ ID NO: 2342 at amino acid positions 34 / 273 / 300 / 477 / 480, 34 / 396, or 300 / 396 / 477 / 480. In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to carboxy terminal of SEQ ID NO: 2342, or to a reference sequence corresponding to SEQ ID NO: 2342, and one or more residue differences as compared to SEQ ID NO: 2342, or amino acid residues selected from 34D / 273M / 300S / 477L / 480K, 34D / 396T, and 300S / 396T / 477L / 480K. In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to carboxy terminal of SEQ ID NO: 2342, or to a reference sequence corresponding to SEQ ID NO: 2342, and one or more residue differences as compared to SEQ ID NO: 2342, or amino acid residues selected from Y34D / K273M / K300S / Q477L / R480K, Y34D / S396T, and K300S / S396T / Q477L / R480K.

[0228] In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to carboxy terminal of SEQ ID NO: 2438, or to a reference sequence corresponding to SEQ ID NO: 2438, and one or more residue differences as compared to SEQ ID NO: 2438 at amino acid positions 27 / 34 / 173 / 176 / 179 / 195 / 277, 27 / 173, 27 / 173 / 176, 27 / 173 / 176 / 179 / 273 / 277, 27 / 173 / 176 / 179 / 277, 27 / 173 / 179 / 273, 27 / 173 / 179 / 273 / 277, 27 / 173 / 179 / 277, 27 / 176 / 179 / 273, 27 / 179, 27 / 179 / 273 / 277, 27 / 179 / 277, 34, 34 / 173 / 176 / 197 / 277, 105 / 173 / 176 / 197 / 277, 173 / 176 / 179, 173 / 176 / 179 / 197, 173 / 176 / 179 / 277, 173 / 179, 173 / 179 / 273, 173 / 179 / 273 / 277, 173 / 277, 176 / 179, 176 / 179 / 197, 176 / 179 / 273, 176 / 179 / 273 / 277, 179, 179 / 197, 179 / 273, or 179 / 273 / 277. In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to carboxy terminal of SEQ ID NO: 2438, or to a reference sequence corresponding to SEQ ID NO: 2438, and one or more residue differences as compared to SEQ ID NO: 2438, or amino acid residues selected from 27P / 34D / 173P / 176H / 179K / 195T / 277D, 27P / 173P, 27P / 173P / 176H, 27P / 173P / 176H / 179K / 273M / 277D, 27P / 173P / 176H / 179R / 277D, 27P / 173P / 179K / 273M, 27P / 173P / 179K / 273M / 277D, 27P / 173P / 179K / 277D, 27P / 176H / 179R / 273M, 27P / 179K, 27P / 179K / 273M / 277D, 27P / 179R / 277D, 34D, 34D / 173P / 176H / 197L / 277D, 105G / 173P / 176H / 197L / 277D,Docket No. CX10-267WO3 173P / 176H / 179K / 277D, 173P / 176H / 179R, 173P / 176H / 179R / 197L, 173P / 179K / 273M / 277D, 173P / 179R, 173P / 179R / 273M, 173P / 179R / 273M / 277D, 173P / 277D, 176H / 179K / 273M, 176H / 179K / 273M / 277D, 176H / 179R, 176H / 179R / 197L, 176H / 179R / 273M / 277D, 179K, 179K / 197L, 179K / 273M, 179K / 273M / 277D, 179R, and 179R / 197L. In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to carboxy terminal of SEQ ID NO: 2438, or to a reference sequence corresponding to SEQ ID NO: 2438, and one or more residue differences as compared to SEQ ID NO: 2438, or amino acid residues selected from Q27P / Y34D / T173P / N176H / Q179K / K195T / S277D, Q27P / T173P, Q27P / T173P / N176H, Q27P / T173P / N176H / Q179K / K273M / S277D, Q27P / T173P / N176H / Q179R / S277D, Q27P / T173P / Q179K / K273M, Q27P / T173P / Q179K / K273M / S277D, Q27P / T173P / Q179K / S277D, Q27P / N176H / Q179R / K273M, Q27P / Q179K, Q27P / Q179K / K273M / S277D, Q27P / Q179R / S277D, Y34D, Y34D / T173P / N176H / M197L / S277D, E105G / T173P / N176H / M197L / S277D, T173P / N176H / Q179K / S277D, T173P / N176H / Q179R, T173P / N176H / Q179R / M197L, T173P / Q179K / K273M / S277D, T173P / Q179R, T173P / Q179R / K273M, T173P / Q179R / K273M / S277D, T173P / S277D, N176H / Q179K / K273M, N176H / Q179K / K273M / S277D, N176H / Q179R, N176H / Q179R / M197L, N176H / Q179R / K273M / S277D, Q179K, Q179K / M197L, Q179K / K273M, Q179K / K273M / S277D, Q179R, and Q179R / M197L.

[0229] In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to carboxy terminal of SEQ ID NO: 2438, or to a reference sequence corresponding to SEQ ID NO: 2438, and one or more residue differences as compared to SEQ ID NO: 2438 at amino acid positions 177, 199, 202, 229, 292, 339, 342, 352, 363, 376, 406, 407, 430, 487, 508, or 525. In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to carboxy terminal of SEQ ID NO: 2438, or to a reference sequence corresponding to SEQ ID NO: 2438, and one or more residue differences as compared to SEQ ID NO: 2438, or amino acid residues selected from 177C, 199E, 202V, 229L, 292A, 339T, 342T, 352A, 352T, 363G, 376H, 406D, 406P, 407N, 430G, 430S, 487R, 508C, and 525T. In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to carboxy terminal of SEQ ID NO: 2438, or to a reference sequence corresponding to SEQ ID NO: 2438, and one or more residue differences as compared to SEQ ID NO: 2438, or amino acid residues selected from R177C, D199E, L202V, W229L, S292A, V339T, E342T, P352A, P352T, P363G, E376H, G406D, G406P, P407N, K430G, K430S, K487R, K508C, and V525T.

[0230] In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to aminoDocket No. CX10-267WO3 acid residues 12 to carboxy terminal of SEQ ID NO: 2452, or to a reference sequence corresponding to SEQ ID NO: 2452, and one or more residue differences as compared to SEQ ID NO: 2452 at amino acid positions 176 / 183, 176 / 183 / 205, 183, 183 / 205, 183 / 205 / 408, or 205. In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to carboxy terminal of SEQ ID NO: 2452, or to a reference sequence corresponding to SEQ ID NO: 2452, and one or more residue differences as compared to SEQ ID NO: 2452, or amino acid residues selected from 176N / 183R, 176N / 183R / 205L, 183G, 183G / 205L, 183R, 183R / 205L, 183R / 205L / 408V, and 205L. In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to carboxy terminal of SEQ ID NO: 2452, or to a reference sequence corresponding to SEQ ID NO: 2452, and one or more residue differences as compared to SEQ ID NO: 2452, or amino acid residues selected from H176N / D183R, H176N / D183R / R205L, D183G, D183G / R205L, D183R, D183R / R205L, D183R / R205L / A408V, and R205L.

[0231] In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to carboxy terminal of SEQ ID NO: 2562, or to a reference sequence corresponding to SEQ ID NO: 2562, and one or more residue differences as compared to SEQ ID NO: 2562 at amino acid positions 177, 177 / 292 / 339 / 342 / 406, 177 / 292 / 339 / 406, 177 / 292 / 342 / 363 / 376 / 406, 177 / 292 / 342 / 406, 177 / 339 / 342, 177 / 339 / 342 / 376, 177 / 339 / 342 / 406, 177 / 339 / 342 / 406 / 487, 177 / 342 / 376 / 407, 177 / 342 / 406 / 430, 177 / 407, 177 / 487, 292 / 339 / 342 / 376 / 406 / 487, 292 / 342, 292 / 342 / 406, 292 / 406, 339 / 342, 339 / 342 / 407, 339 / 342 / 407 / 487, or 406. In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to carboxy terminal of SEQ ID NO: 2562, or to a reference sequence corresponding to SEQ ID NO: 2562, and one or more residue differences as compared to SEQ ID NO: 2562, or amino acid residues selected from 177K, 177K / 292A / 339T / 342T / 406D, 177K / 292A / 339T / 406D, 177K / 292A / 339T / 406P, 177K / 292A / 342T / 363G / 376H / 406D, 177K / 292A / 342T / 406P, 177K / 339T / 342T, 177K / 339T / 342T / 376H, 177K / 339T / 342T / 406D, 177K / 339T / 342T / 406P / 487R, 177K / 342T / 376H / 407N, 177K / 342T / 406P / 430G, 177K / 407N, 177K / 487R, 292A / 339T / 342T / 376H / 406P / 487R, 292A / 342T, 292A / 342T / 406D, 292A / 406P, 339T / 342T, 339T / 342T / 407N, 339T / 342T / 407N / 487R, and 406P. In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to carboxy terminal of SEQ ID NO: 2562, or to a reference sequence corresponding to SEQ ID NO: 2562, and one or more residue differences as compared to SEQ ID NO: 2562, or amino acid residues selected from R177K,Docket No. CX10-267WO3 R177K / S292A / V339T / E342T / G406D, R177K / S292A / V339T / G406D, R177K / S292A / V339T / G406P, R177K / S292A / E342T / P363G / E376H / G406D, R177K / S292A / E342T / G406P, R177K / V339T / E342T, R177K / V339T / E342T / E376H, R177K / V339T / E342T / G406D, R177K / V339T / E342T / G406P / K487R, R177K / E342T / E376H / P407N, R177K / E342T / G406P / K430G, R177K / P407N, R177K / K487R, S292A / V339T / E342T / E376H / G406P / K487R, S292A / E342T, S292A / E342T / G406D, S292A / G406P, V339T / E342T, V339T / E342T / P407N, V339T / E342T / P407N / K487R, and G406P.

[0232] In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to carboxy terminal of SEQ ID NO: 2562, or to a reference sequence corresponding to SEQ ID NO: 2562, and one or more residue differences as compared to SEQ ID NO: 2562 at amino acid positions 174, 187, 190, 198, 267, 274, 277, 307, 316, 343, 359, 379, 402 / 407, 407, 409, 412, 414, 481, 494, or 523. In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to carboxy terminal of SEQ ID NO: 2562, or to a reference sequence corresponding to SEQ ID NO: 2562, and one or more residue differences as compared to SEQ ID NO: 2562, or amino acid residues selected from 174I, 187T, 190G, 198P, 198S, 198T, 267G, 274V, 277S, 307K, 316L, 343R, 359F, 379M, 402P / 407A, 407S, 409S, 412P, 414Q, 481H, 494T, 494Y, 523A, and 523H. In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to carboxy terminal of SEQ ID NO: 2562, or to a reference sequence corresponding to SEQ ID NO: 2562, and one or more residue differences as compared to SEQ ID NO: 2562, or amino acid residues selected from W174I, Y187T, T190G, D198P, D198S, D198T, S267G, P274V, D277S, T307K, V316L, L343R, L359F, L379M, S402P / P407A, P407S, D409S, D412P, E414Q, D481H, H494T, H494Y, E523A, and E523H.

[0233] In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to carboxy terminal of SEQ ID NO: 2576, or to a reference sequence corresponding to SEQ ID NO: 2576, and one or more residue differences as compared to SEQ ID NO: 2576 at amino acid positions 202, 208, 221, 224, 235, 236, 238, 239, 242, 249, 251, 252, 259, 263, 405 / 406, 406, 477, 484 / 487, or 487. In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to carboxy terminal of SEQ ID NO: 2576, or to a reference sequence corresponding to SEQ ID NO: 2576, and one or more residue differences as compared to SEQ ID NO: 2576, or amino acid residues selected from 202M, 208M, 221I, 224V, 235R, 236R, 238I, 238L, 239G, 239L, 239R, 239S, 242L, 249L, 249Q, 251A, 252R, 259F, 263R, 405G / 406G, 405V / 406G, 406E, 406M, 406T, 477I, 484L / 487K, 484Y / 487K,Docket No. CX10-267WO3 and 487K. In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to carboxy terminal of SEQ ID NO: 2576, or to a reference sequence corresponding to SEQ ID NO: 2576, and one or more residue differences as compared to SEQ ID NO: 2576, or amino acid residues selected from L202M, Q208M, L221I, T224V, K235R, G236R, M238I, M238L, E239G, E239L, E239R, E239S, I242L, E249L, E249Q, Q251A, A252R, Y259F, S263R, Q405G / P406G, Q405V / P406G, P406E, P406M, P406T, L477I, R484L / R487K, R484Y / R487K, and R487K.

[0234] In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to carboxy terminal of SEQ ID NO: 2576, or to a reference sequence corresponding to SEQ ID NO: 2576, and one or more residue differences as compared to SEQ ID NO: 2576 at amino acid positions 174 / 190, 174 / 190 / 202 / 204 / 254, 174 / 190 / 202 / 254, 174 / 190 / 204, 174 / 190 / 236, 174 / 202 / 204 / 254, 174 / 202 / 204 / 254 / 353, 174 / 204 / 236 / 254 / 353, 187, 187 / 224, 187 / 224 / 321 / 436, 187 / 224 / 379 / 436, 187 / 249, 187 / 249 / 321 / 379 / 436, 204 / 254 / 353, 249 / 379, 359 / 407, 359 / 407 / 412, 379, or 436. In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to carboxy terminal of SEQ ID NO: 2576, or to a reference sequence corresponding to SEQ ID NO: 2576, and one or more residue differences as compared to SEQ ID NO: 2576, or amino acid residues selected from 174I / 190G, 174I / 190G / 202H / 204M / 254I, 174I / 190G / 202H / 254I, 174I / 190G / 204M, 174I / 190G / 236R, 174I / 202H / 204M / 254I, 174I / 202H / 204M / 254I / 353V, 174I / 204M / 236R / 254I / 353V, 187T, 187T / 224V, 187T / 224V / 321R / 436S, 187T / 224V / 379M / 436S, 187T / 249M, 187T / 249M / 321R / 379M / 436S, 204V / 254I / 353V, 249M / 379M, 359F / 407A, 359F / 407S / 412P, 379M, and 436S. In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to carboxy terminal of SEQ ID NO: 2576, or to a reference sequence corresponding to SEQ ID NO: 2576, and one or more residue differences as compared to SEQ ID NO: 2576, or amino acid residues selected from W174I / T190G, W174I / T190G / L202H / F204M / R254I, W174I / T190G / L202H / R254I, W174I / T190G / F204M, W174I / T190G / G236R, W174I / L202H / F204M / R254I, W174I / L202H / F204M / R254I / R353V, W174I / F204M / G236R / R254I / R353V, Y187T, Y187T / T224V, Y187T / T224V / A321R / P436S, Y187T / T224V / L379M / P436S, Y187T / E249M, Y187T / E249M / A321R / L379M / P436S, F204V / R254I / R353V, E249M / L379M, L359F / P407A, L359F / P407S / D412P, L379M, and P436S.

[0235] In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to carboxy terminal of SEQ ID NO: 2576, or to a reference sequence corresponding to SEQDocket No. CX10-267WO3 ID NO: 2576, and one or more residue differences as compared to SEQ ID NO: 2576 at amino acid positions 219, 293, 300, 310, 313, 318, 340, 364, or 392. In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to carboxy terminal of SEQ ID NO: 2576, or to a reference sequence corresponding to SEQ ID NO: 2576, and one or more residue differences as compared to SEQ ID NO: 2576, or amino acid residues selected from 219Q, 293R, 300R, 310D, 313Q, 318P, 340L, 340T, 364S, 392Q, and 392S. In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to carboxy terminal of SEQ ID NO: 2576, or to a reference sequence corresponding to SEQ ID NO: 2576, and one or more residue differences as compared to SEQ ID NO: 2576, or amino acid residues selected from R219Q, S293R, S300R, E310D, R313Q, K318P, I340L, I340T, P364S, K392Q, and K392S.

[0236] In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to carboxy terminal of SEQ ID NO: 2576, or to a reference sequence corresponding to SEQ ID NO: 2576, and one or more residue differences as compared to SEQ ID NO: 2576 at amino acid positions 160, 161, 173, 175, 222, 244, 278, 293, 300, 301, 310, 318, 319, 324, 334, 339, 341, 342, 366, 367, 368, 370, 376, or 378. In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to carboxy terminal of SEQ ID NO: 2576, or to a reference sequence corresponding to SEQ ID NO: 2576, and one or more residue differences as compared to SEQ ID NO: 2576, or amino acid residues selected from 160A, 160D, 160T, 160W, 161A, 161L, 173H, 175H, 175P, 222N, 244I, 244S, 278A, 278L, 293R, 300R, 301F, 310D, 318P, 318R, 318S, 319P, 324A, 334S, 339A, 339S, 341L, 342S, 366F, 366H, 366P, 367V, 368R, 370L, 370R, 376A, 376G, 376R, 376S, 376V, and 378M. In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to carboxy terminal of SEQ ID NO: 2576, or to a reference sequence corresponding to SEQ ID NO: 2576, and one or more residue differences as compared to SEQ ID NO: 2576, or amino acid residues selected from M160A, M160D, M160T, M160W, K161A, K161L, P173H, S175H, S175P, A222N, V244I, V244S, S278A, S278L, S293R, S300R, W301F, E310D, K318P, K318R, K318S, V319P, I324A, T334S, T339A, T339S, V341L, T342S, A366F, A366H, A366P, Q367V, S368R, Q370L, Q370R, E376A, E376G, E376R, E376S, E376V, and V378M.

[0237] In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,Docket No. CX10-267WO3 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to carboxy terminal of SEQ ID NO: 2714, or to a reference sequence corresponding to SEQ ID NO: 2714, and one or more residue differences as compared to SEQ ID NO: 2714 at amino acid positions 187, 187 / 224, 187 / 224 / 263, 187 / 224 / 407, 187 / 224 / 407 / 412, 187 / 224 / 412, 187 / 254 / 263 / 359 / 379 / 407, 187 / 263, 187 / 359, 187 / 379, 224 / 359 / 407, 249 / 263, 249 / 263 / 407, 263, or 263 / 407 / 412. In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to carboxy terminal of SEQ ID NO: 2714, or to a reference sequence corresponding to SEQ ID NO: 2714, and one or more residue differences as compared to SEQ ID NO: 2714, or amino acid residues selected from 187T, 187T / 224V, 187T / 224V / 263R, 187T / 224V / 407A, 187T / 224V / 407A / 412P, 187T / 224V / 412P, 187T / 254R / 263R / 359F / 379M / 407S, 187T / 263R, 187T / 359F, 187T / 379M, 224V / 359F / 407A, 249M / 263R, 249M / 263R / 407S, 263R, and 263R / 407S / 412P. In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to carboxy terminal of SEQ ID NO: 2714, or to a reference sequence corresponding to SEQ ID NO: 2714, and one or more residue differences as compared to SEQ ID NO: 2714, or amino acid residues selected from Y187T, Y187T / T224V, Y187T / T224V / S263R, Y187T / T224V / P407A, Y187T / T224V / P407A / D412P, Y187T / T224V / D412P, Y187T / I254R / S263R / L359F / L379M / P407S, Y187T / S263R, Y187T / L359F, Y187T / L379M, T224V / L359F / P407A, E249M / S263R, E249M / S263R / P407S, S263R, and S263R / P407S / D412P.

[0238] In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to carboxy terminal of SEQ ID NO: 2876, or to a reference sequence corresponding to SEQ ID NO: 2876, and one or more residue differences as compared to SEQ ID NO: 2876 at amino acid positions 160 / 263 / 318 / 339, 175 / 244 / 293 / 364 / 407, 219 / 310, 219 / 310 / 340, 244 / 293 / 313, 244 / 293 / 334, 244 / 313, 244 / 313 / 334 / 364 / 407, 263 / 310 / 318 / 366, 263 / 318, 293 / 313 / 334 / 364, 293 / 334 / 364 / 407, 310 / 318 / 340, 318, or 334 / 364. In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to carboxy terminal of SEQ ID NO: 2876, or to a reference sequence corresponding to SEQ ID NO: 2876, and one or more residue differences as compared to SEQ ID NO: 2876, or amino acid residues selected from 60T / 263R / 318P / 339S, 175H / 244I / 293R / 364S / 407P, 219S / 310D, 219S / 310D / 340T, 244I / 293R / 313Q, 244I / 313Q, 244I / 313Q / 334S / 364S / 407S, 244S / 293R / 334S, 263R / 310D / 318S / 366H, 263R / 318P, 293R / 313Q / 334S / 364S, 293R / 334S / 364S / 407P, 310D / 318P / 340T, 318P, and 334S / 364S. In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12Docket No. CX10-267WO3 to carboxy terminal of SEQ ID NO: 2876, or to a reference sequence corresponding to SEQ ID NO: 2876, and one or more residue differences as compared to SEQ ID NO: 2876, or amino acid residues selected from M160T / S263R / K318P / T339S, S175H / V244I / S293R / P364S / A407P, R219S / E310D, R219S / E310D / I340T, V244I / S293R / R313Q, V244I / R313Q, V244I / R313Q / T334S / P364S / A407S, V244S / S293R / T334S, S263R / E310D / K318S / A366H, S263R / K318P, S293R / R313Q / T334S / P364S, S293R / T334S / P364S / A407P, E310D / K318P / I340T, K318P, or T334S / P364S.

[0239] In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to carboxy terminal of SEQ ID NO: 2884, or to a reference sequence corresponding to SEQ ID NO: 2884, and one or more residue differences as compared to SEQ ID NO: 2884 at amino acid positions 254 / 293 / 310, 254 / 293 / 310 / 334 / 340 / 407, 310 / 334, 334, 334 / 340, 334 / 407, 340, or 407. In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to carboxy terminal of SEQ ID NO: 2884, or to a reference sequence corresponding to SEQ ID NO: 2884, and one or more residue differences as compared to SEQ ID NO: 2884, or amino acid residues selected from 254R / 293R / 310D, 254R / 293R / 310D / 334S / 340T / 407P, 310D / 334S, 334S, 334S / 340T, 334S / 407P, 340T, and 407P. In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to carboxy terminal of SEQ ID NO: 2884, or to a reference sequence corresponding to SEQ ID NO: 2884, and one or more residue differences as compared to SEQ ID NO: 2884, or amino acid residues selected from I254R / S293R / E310D, I254R / S293R / E310D / T334S / I340T / A407P, E310D / T334S, T334S, T334S / I340T, T334S / A407P, I340T, and A407P.

[0240] In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to carboxy terminal of SEQ ID NO: 2884, or to a reference sequence corresponding to SEQ ID NO: 2884, and one or more residue differences as compared to SEQ ID NO: 2884 at amino acid positions 183, 190, 203, 232, 252, 259 / 263, 261 / 263, 397, 408, or 413. In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to carboxy terminal of SEQ ID NO: 2884, or to a reference sequence corresponding to SEQ ID NO: 2884, and one or more residue differences as compared to SEQ ID NO: 2884, or amino acid residues selected from 183V, 190N, 203L, 232G, 252H, 259F / 263S, 261S / 263S, 397Q, 408D, and 413T. In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or moreDocket No. CX10-267WO3 sequence identity to a reference sequence corresponding to amino acid residues 12 to carboxy terminal of SEQ ID NO: 2884, or to a reference sequence corresponding to SEQ ID NO: 2884, and one or more residue differences as compared to SEQ ID NO: 2884, or amino acid residues selected from D183V, T190N, M203L, D232G, A252H, Y259F / R263S, V261S / R263S, K397Q, A408D, and G413T.

[0241] In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to carboxy terminal of SEQ ID NO: 2884, or to a reference sequence corresponding to SEQ ID NO: 2884, and one or more residue differences as compared to SEQ ID NO: 2884 at amino acid positions 179, 200, 201, 208, 218, 223, 227, 229, 230, 235, 297, or 408. In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to carboxy terminal of SEQ ID NO: 2884, or to a reference sequence corresponding to SEQ ID NO: 2884, and one or more residue differences as compared to SEQ ID NO: 2884, or amino acid residues selected from 179V, 200Q, 201R, 208G, 218I, 223L, 227G, 227S, 229G, 229S, 230V, 235N, 297Q, and 408D. In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to carboxy terminal of SEQ ID NO: 2884, or to a reference sequence corresponding to SEQ ID NO: 2884, and one or more residue differences as compared to SEQ ID NO: 2952, or amino acid residues selected from K179V, K200Q, A201R, Q208G, V218I, D223L, L227G, L227S, W229G, W229S, I230V, K235N, S297Q, and A408D.

[0242] In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to carboxy terminal of SEQ ID NO: 2910, or to a reference sequence corresponding to SEQ ID NO: 2910, and one or more residue differences as compared to SEQ ID NO: 2910 at amino acid positions 307 / 429 / 474, or 508. In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to carboxy terminal of SEQ ID NO: 2910, or to a reference sequence corresponding to SEQ ID NO: 2910, and one or more residue differences as compared to SEQ ID NO: 2910, or amino acid residues selected from 307K / 429A / 474L and 508Q. In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to carboxy terminal of SEQ ID NO: 2910, or to a reference sequence corresponding to SEQ ID NO: 2910, and one or more residueDocket No. CX10-267WO3 differences as compared to SEQ ID NO: 2910, or amino acid residues selected from T307K / R429A / M474L and K508Q.

[0243] In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to carboxy terminal of SEQ ID NO: 2974, or to a reference sequence corresponding to SEQ ID NO: 2974, and one or more residue differences as compared to SEQ ID NO: 2974 at amino acid positions 190, 190 / 203 / 205 / 508, 205 / 208, 205 / 223, 205 / 230, 205 / 239 / 403 / 508, 205 / 297 / 508, 205 / 370 / 403, 205 / 403, 205 / 413, 205 / 508, or 218 / 319 / 508. In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to carboxy terminal of SEQ ID NO: 2974, or to a reference sequence corresponding to SEQ ID NO: 2974, and one or more residue differences as compared to SEQ ID NO: 2974, or amino acid residues selected from 190N, 190N / 203F / 205R / 508K, 205R / 208G, 205R / 223L, 205R / 230V, 205R / 239S / 403F / 508K, 205R / 297Q / 508K, 205R / 370R / 403F, 205R / 403F, 205R / 413T, 205R / 508K, and 218I / 319M / 508K. In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to carboxy terminal of SEQ ID NO: 2974, or to a reference sequence corresponding to SEQ ID NO: 2974, and one or more residue differences as compared to SEQ ID NO: 2974, or amino acid residues selected from T190N, T190N / M203F / L205R / Q508K, L205R / Q208G, L205R / D223L, L205R / I230V, L205R / E239S / L403F / Q508K, L205R / S297Q / Q508K, L205R / Q370R / L403F, L205R / L403F, L205R / G413T, L205R / Q508K, and V218I / V319M / Q508K.

[0244] In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to carboxy terminal of SEQ ID NO: 2998, or to a reference sequence corresponding to SEQ ID NO: 2998, and one or more residue differences as compared to SEQ ID NO: 2998 at amino acid positions 230 / 251 / 352 / 357, 238 / 297 / 403, 238 / 297 / 403 / 466, 297, and 403 / 466. In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to carboxy terminal of SEQ ID NO: 2998, or to a reference sequence corresponding to SEQ ID NO: 2998, and one or more residue differences as compared to SEQ ID NO: 2998, or amino acid residues selected from 230I / 251A / 352T / 357I, 238L / 297V / 403F, 238L / 297V / 403F / 466L, 297V, and 403F / 466L. In some embodiments, the engineered TnT polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to carboxy terminal of SEQ ID NO: 2998, or to a reference sequence corresponding to SEQ ID NO: 2998, and one or more residueDocket No. CX10-267WO3 differences as compared to SEQ ID NO: 2998, or amino acid residues selected from V230I / Q251A / P352T / V357I, M238L / S297V / L403F, M238L / S297V / L403F / I466L, S297V, and L403F / I466L.

[0245] As will be appreciated by the skilled artisan, in some embodiments, one or a combination of residue differences above that is selected can be kept constant (i.e., maintained) in the engineered TnT as a core feature, and additional residue differences at other residue positions incorporated into the sequence to generate additional engineered TnT polypeptides with improved properties. Accordingly, it is to be understood for any engineered TnT containing one or a subset of the residue differences above, the present invention contemplates other engineered TnTs that comprise the one or subset of the residue differences, and additionally one or more residue differences at the other residue positions disclosed herein.

[0246] As noted above, the engineered TnT polypeptides are also capable of converting substrates (e.g., NTP-3’-O-RBG or a natural or modified NTP and an oligo acceptor substrate) to products (e.g., an oligo acceptor substrate with an added nucleotide-3’-O-RBG). In some embodiments, the engineered TnT polypeptide is capable of converting the substrate compounds to the product compound with at least 1.1, 1.2 fold, 1.5 fold, 2 fold, 3 fold, 4 fold, 5 fold, 10 fold, 20 fold, 30 fold, 40 fold, 50 fold, 60 fold, 70 fold, 80 fold, 90 fold, 100 fold, or more activity relative to the activity of the reference polypeptide of SEQ ID NOs: 2, 8, 70, 148, 184, 234, 256, 478, 668, 682, 730, 734, 742, 932, 992, 1144, 1212, 1236, 1264, 1414, 1504, 1522, 1702, 1742, 1928, 1944, 1946, 1984, 2064, 2104, 2210, 2256, 2280, 2342, 2438, 2452, 2562, 2576, 2714, 2876, 2884, 2910, 2974, 2998, or 3002.

[0247] In some embodiments, the engineered TnT capable of converting the substrate compounds to the product compounds with at least 1.2 fold or at least 2 fold the activity relative to SEQ ID NOs: 2, 8, 70, 148, 184, 234, 256, 478, 668, 682, 730, 734, 742, 932, 992, 1144, 1212, 1236, 1264, 1414, 1504, 1522, 1702, 1742, 1928, 1944, 1946, 1984, 2064, 2104, 2210, 2256, 2280, 2342, 2438, 2452, 2562, 2576, 2714, 2876, 2884, 2910, 2974, 2998, or 3002, comprises an amino acid sequence selected from the even-numbered sequences in SEQ ID NO: 2-1056, 1130-1880, and 1928-3008.

[0248] In some embodiments, the engineered TnT capable of converting an oligo acceptor substrate of 4 to 7 basepairs to the oligo acceptor substrate extended by one nucleotide with at least 1.3 fold, 1.4 fold, or 1.5 fold the activity relative to SEQ ID NO: 148 or 256, comprises an amino acid sequence with at least 80% sequence identity to SEQ ID NO: 682.

[0249] In some embodiments, the engineered TnT capable of converting an oligo acceptor substrate of 4 to 7 basepairs to the oligo acceptor substrate extended by one nucleotide, has activity in the conversion of a variety of oligo acceptor substrates and comprises an amino acid sequence with at least 80% sequence identity to SEQ ID NO: 148, 256, or 682.

[0250] In some embodiments, the engineered TnT capable of converting an oligo acceptor substrate of 3 to 4 basepairs to the oligo acceptor substrate extended by one nucleotide with at least 31% or 91% conversion comprises an amino acid sequence with at least 80% sequence identity to SEQ ID NO: 932.Docket No. CX10-267WO3

[0251] In some embodiments, the engineered TnT capable of converting an oligo acceptor substrate to the oligo acceptor substrate extended by a glycol nucleic acid nucleotide ((S)-GNA) or a locked nucleic acid nucleotide (LNA) with at least 75% conversion comprises an amino acid sequence with at least 80% sequence identity to SEQ ID NO: 992.

[0252] In some embodiments, the engineered TnT with an amino acid sequence comprising one or more residue differences as compared to SEQ ID NO: 2, that increases substrate promiscuity for NTP-3’-O-RBG substrates of 2'-methoxyadenosine, 2'-methoxycytidine, 2'-methoxyguanosine, 2'-methoxyuridine, 2'- fluoroadenosine, 2'-fluorocytidine, 2'-fluoroguanosine, 2'-fluorouridine, adenosine ribonucleotide, cytidine ribonucleotide, guanosine ribonucleotide, uridine ribonucleotide, phosphorothioate linkage, 5'- phosphorylation modification, adenosine (S)-glycol nucleic acid, cytidine (S)-glycol nucleic acid, guanosine (S)-glycol nucleic acid, thymidine (S)-glycol nucleic acid, uridine (S)-glycol nucleic acid, C2'-endo locked adenosine ribonucleotide, C2'-endo locked cytidine ribonucleotide, C2'-endo locked guanosine ribonucleotide, C2'-endo locked thymidine ribonucleotide, C2'-endo locked uridine ribonucleotide, 2'-O-(2- methoxyethyl)adenosine, 2'-O-(2-methoxyethyl)cytidine, 2'-O-(2-methoxyethyl)guanosine, 2'-O-(2- methoxyethyl)uridine, 2',3'-dideoxyadenosine, 2',3'-dideoxycytidine, 2',3'-dideoxyguanosine, 2',3'- dideoxythymidine, 2',3'-dideoxyuridine and vinylphosphonate 2'O-methoxy uridine, 1-((2S))-1-(2,3- dihydroxypropyl))thymine, 2'O-hexadecyl cytidine, as compared to a wild-type or engineered reference TnT, comprises an amino acid sequence selected from the even-numbered sequences in SEQ ID NO: 2-1056, 1130- 1880, and 1928-3008.

[0253] In some embodiments, the engineered TnT with an amino acid sequence comprising one or more residue differences as compared to SEQ ID NO: 2, that increases substrate promiscuity for NTP-3’-O-RBG substrates of 2'-methoxyadenosine, 2'-methoxycytidine, 2'-methoxyguanosine, 2'-methoxyuridine, 2'- fluoroadenosine, 2'-fluorocytidine, 2'-fluoroguanosine, 2'-fluorouridine, adenosine ribonucleotide, cytidine ribonucleotide, guanosine ribonucleotide, uridine ribonucleotide, phosphorothioate linkage, 5'- phosphorylation modification, adenosine (S)-glycol nucleic acid, cytidine (S)-glycol nucleic acid, guanosine (S)-glycol nucleic acid, thymidine (S)-glycol nucleic acid, uridine (S)-glycol nucleic acid, C2'-endo locked adenosine ribonucleotide, C2'-endo locked cytidine ribonucleotide, C2'-endo locked guanosine ribonucleotide, C2'-endo locked thymidine ribonucleotide, C2'-endo locked uridine ribonucleotide, 2'-O-(2- methoxyethyl)adenosine, 2'-O-(2-methoxyethyl)cytidine, 2'-O-(2-methoxyethyl)guanosine, 2'-O-(2- methoxyethyl)uridine, 2',3'-dideoxyadenosine, 2',3'-dideoxycytidine, 2',3'-dideoxyguanosine, 2',3'- dideoxythymidine, 2',3'-dideoxyuridine and vinylphosphonate 2'O-methoxy uridine, 1-((2S))-1-(2,3- dihydroxypropyl))thymine, 2'O-hexadecyl cytidine, as compared to a wild-type or engineered reference TnT, comprises an amino acid sequence with at least 80% sequence identity to SEQ ID NO: 992.

[0254] In some embodiments, the engineered TnT with an amino acid sequence comprising one ...

Claims

Docket No. CX10-267WO3 CLAIMS What is claimed is:

1. An engineered terminal nucleotidyl transferase, or a functional fragment thereof, comprising an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to the carboxy terminus of an even-numbered SEQ ID NO. of SEQ ID NOs: 2, 4-1056, 1130-1880, and 1928-3008, or to a reference sequence corresponding to an even- numbered SEQ ID NO. of SEQ ID NOs: 2, 4-1056, 1130-1880, and 1928-3008, wherein the amino acid sequence comprises one or more amino differences relative to the reference sequence corresponding to amino acid residues 12 to the carboxy terminus of SEQ ID NO: 2, 8, 70, 148, 184, 234, 256, 478, 668, 682, 730, 734, 742, 932, 992, 1144, 1212, 1236, 1264, 1414, 1504, 1522, 1702, 1742, 1928, 1944, 1946, 1984, 2064, 2104, 2210, 2256, 2280, 2342, 2438, 2452, 2562, 2576, 2562, 2714, 2876, 2884, 2910, 2974, 2998, or 3002, or to the reference sequence corresponding to SEQ ID NO: 2, 8, 70, 148, 184, 234, 256, 478, 668, 682, 730, 734, 742, 932, 992, 1144, 1212, 1236, 1264, 1414, 1504, 1522, 1702, 1742, 1928, 1944, 1946, 1984, 2064, 2104, 2210, 2256, 2280, 2342, 2438, 2452, 2562, 2576, 2562, 2714, 2876, 2884, 2910, 2974, 2998, or 3002.

2. The engineered terminal nucleotidyl transferase of Claim 1, comprising an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to the carboxy terminus of SEQ ID NO: 2, 8, 70, 148, 184, 234, 256, 478, 668, 682, 730, 734, 742, 932, 992, 1144, 1212, 1236, 1264, 1414, 1504, 1522, 1702, 1742, 1928, 1944, 1946, 1984, 2064, 2104, 2210, 2256, 2280, 2342, 2438, 2452, 2562, 2576, 2562, 2714, 2876, 2884, 2910, 2974, 2998, or 3002, or to a reference sequence corresponding to SEQ ID NO: 2, 8, 70, 148, 184, 234, 256, 478, 668, 682, 730, 734, 742, 932, 992, 1144, 1212, 1236, 1264, 1414, 1504, 1522, 1702, 1742, 1928, 1944, 1946, 1984, 2064, 2104, 2210, 2256, 2280, 2342, 2438, 2452, 2562, 2576, 2562, 2714, 2876, 2884, 2910, 2974, 2998, or 3002, wherein the amino acid sequence comprises one or more amino differences relative to the reference sequence corresponding to amino acid residues 12 to the carboxy terminus of SEQ ID NO: 2, 8, 70, 148, 184, 234, 256, 478, 668, 682, 730, 734, 742, 932, 992, 1144, 1212, 1236, 1264, 1414, 1504, 1522, 1702, 1742, 1928, 1944, 1946, 1984, 2064, 2104, 2210, 2256, 2280, 2342, 2438, 2452, 2562, 2576, 2562, 2714, 2876, 2884, 2910, 2974, 2998, or 3002, or to the reference sequence corresponding to SEQ ID NO: 2, 8, 70, 148, 184, 234, 256, 478, 668, 682, 730, 734, 742, 932, 992, 1144, 1212, 1236, 1264, 1414, 1504, 1522, 1702, 1742, 1928, 1944, 1946, 1984, 2064, 2104, 2210, 2256, 2280, 2342, 2438, 2452, 2562, 2576, 2562, 2714, 2876, 2884, 2910, 2974, 2998, or 3002.

3. The engineered terminal nucleotidyl transferase of Claim 1 or 2, wherein the amino acid sequence of the engineered terminal nucleotidyl transferase comprises at least an amino acid residue difference at amino acid position 22, 27, 34, 41, 42, 43, 45, 52, 60, 92, 105, 133, 152, 160, 161, 162, 164, 169, 173, 174, 175, 176, 177, 179, 182, 183, 184, 186, 187, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 208, 212, 213, 218, 219, 221, 222, 223, 224, 227, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 241, 242, 244, 246, 249, 250, 251, 252, 254, 259, 260, 261, 262, 263, 264, 267, 268, 272,Docket No. CX10-267WO3 273, 274, 275, 277, 278, 280, 281, 282, 288, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 307, 308, 309, 310, 312, 313, 314, 315, 316, 317, 318, 319, 321, 322, 323, 324, 325, 328, 329, 333, 334, 335, 336, 337, 339, 340, 341, 342, 343, 347, 352, 353, 354, 357, 359, 360, 361, 362, 363, 364, 366, 367, 368, 370, 371, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 387, 388, 389, 390, 392, 393, 394, 395, 396, 397, 398, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 446, 448, 450, 451, 456, 457, 458, 459, 460, 461, 462, 463, 465, 466, 467, 468, 474, 477, 479, 480, 481, 484, 485, 487, 490, 492, 493, 494, 495, 497, 499, 500, 501, 502, 503, 504, 506, 507, 508, 513, 515, 517, 518, 522, 523, 525, or 526, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12 to the carboxy terminus of SEQ ID NO: 2, 8, 70, 148, 184, 234, 256, 478, 668, 682, 730, 734, 742, 932, 992, 1144, 1212, 1236, 1264, 1414, 1504, 1522, 1702, 1742, 1928, 1944, 1946, 1984, 2064, 2104, 2210, 2256, 2280, 2342, 2438, 2452, 2562, 2576, 2562, 2714, 2876, 2884, 2910, 2974, 2998, or 3002, or to a reference sequence corresponding to SEQ ID NO: 2, 8, 70, 148, 184, 234, 256, 478, 668, 682, 730, 734, 742, 932, 992, 1144, 1212, 1236, 1264, 1414, 1504, 1522, 1702, 1742, 1928, 1944, 1946, 1984, 2064, 2104, 2210, 2256, 2280, 2342, 2438, 2452, 2562, 2576, 2562, 2714, 2876, 2884, 2910, 2974, 2998, or 3002.

4. The engineered terminal nucleotidyl transferase of Claim 1, wherein the amino acid sequence of the engineered terminal nucleotidyl transferase comprises at least an amino acid residue difference or amino acid residue 22P, 27P, 34Y, 34D, 41T, 42T, 43- / T, 45T, 52G, 60H, 92C, 105G, 133E, 152H, 160A / D / T / W, 161A / G / L / Q / T, 162P, 164Q, 169L, 173H, 173G / P, 174V / W, 174L / W, 174I, 175E / S / T / V, 175H / L / P / R, 176N, 176H, 177C / K, 179V, 179C / K / L / R, 182I / V, 183G / R / S / T / V, 184K / R, 186G / L / M / R / T / V, 187L / T / V / Y, 187T, 190A / R, 190A / F / H / L / T / V, 190G / M / N / Q / R / V, 191F / W / Y, 192A / H, 193R / T, 193A / G / I / R / S / V, 193Q / S / T / W, 194L / M, 195F / Q / R / T / W, 196D / H / K / L / Q / R / S / V, 197I / L, 197M / Q / R, 198E / H / P / Q / R / S / T, 199E / F / V, 200Q, 201D / E / P / Q / R / S, 202H / M / S / V, 203M, 203E / F / L / Q, 204M / V, 205R, 205L, 208A / G, 208E / Q, 208D / E / G, 208E / G / M / T, 212R, 213G, 218I, 219D / Q / S, 221I / Q, 222N, 222A, 223H / L / T, 224L / V, 227G / S, 229F / G / L / S, 230C / L / V, 230I, 231N, 231C / M / N / T, 232E / G, 233I / P, 234T, 235N / R, 236R / S, 237L, 238F / I / L / V, 239G / L / R / S, 241L, 242L, 244G / I / R / S, 246L / V, 249L / M / Q, 250G, 251A / E / G / M / R / S / W, 252E / H / L / M / R / S, 254R, 254N / Q / R, 254I, 259F, 260R, 260K / Q / S / T, 261G / R / V, 261S / T, 262L / M, 263A / K / R / S / W, 263S, 263H / R / T, 264L, 264A / R / S, 267S, 267G, 268L, 272R, 273K / M, 273E / G / M / N / Q, 273I / V, 273L / M / V, 273R, 274P / R, 274L / N / R / S / V, 275I / L / R / S, 277S / T, 277D, 278D / R / S, 278A / L, 280W, 281A / C / G / H / L / S, 282N, 288D, 290L, 291K / L, 292A, 293R, 294G / T, 295T, 295A / D / N, 296V, 296L / M, 297A / S, 297A / G / L / P / Q / T / V / W / Y, 298I, 299G / H / N / P, 299A / G / N / P / R / S, 300S, 300A / K, 300R, 300A / E / F / G / I / K / L / M / N / Q / R / S / V / Y, 301A / F / V, 302S, 303E / R / T, 304L, 307D / E / K / M / N / S, 308L / Y, 308H / L, 309H, 310D / S, 312M, 313H / R / S / T, 313A / Q, 314E, 314A / I / S / T, 315K / R, 316L, 317R, 318P / R / S, 318I / K / L / Q, 319M / T, 319G / Q / V / W, 319A / G / L / M / P, 321R / T, 322A / E, 323T, 324A / T, 325G / S / V / Y, 328A / I, 329Q, 329H / L, 333N / S, 334S, 335V, 336C / T, 337S, 339I / T / V, 339A / S, 339T, 340L / M / T / V, 341L / T, 342S / T, 342S, 343L, 343I / M / R / T / V, 347A / S, 352A / E / L / M / N / T, 353R / V, 353K / L / T / V, 353V, 354G, 357I, 359A / C / F / V, 360V, 361L / V, 361I, 362A / M, 363G, 363P / T, 364S, 366F / H / P, 367N / Q / R / S, 367E / V, 368L / S, 368R, 370L / R, 371G / R, 376A / G / H / R / S / T / V, 376E / S, 377R, 378M, 379M, 379L / T, 380A / K / L / S / V, 381Y, 381S, 382L / M,Docket No. CX10-267WO3 383L / Q, 384A / G / S / T, 385V, 387T / V, 388V, 389A / V, 390I / L / M, 390S / T, 390T, 392E / H / N / Q / R / S, 393A / G / I / L / M / S / T / Y, 394K / N / S, 395E / F / I / L / M / Q / S / T, 396T, 397G / Q / S / T, 397E / F / H / I / K / L / M / R / V / W / Y, 398L / M, 402A / D / K / M / P / S / T, 402P, 403E / F / I / P, 404A / S, 404E / G / I / L / M / Q / R / T / V, 405G / L / S / V, 405A / P / Q / T / V / Y, 406A / D / H / M / N / P / R / T, 406E / G / M / T, 407P / S, 407P, 407A / D / G / L / N / Q / S / V / Y, 408D / G / L / N / V, 408A, 409E / G / P / R / S / V / Y, 409D, 410V, 411E / H / K / R / T / V, 412M / N / P / Q / S / V, 413T, 414Q / R, 415G / P / S, 416G / S, 416A / H, 417A / G / S, 418C / S / T, 419A / I / M, 420C / L / V, 424P, 425R, 426G, 427A / M, 428A / L / T, 429A / S, 430G / Q / S / W, 431G, 432I / V, 433A / P / T / V, 434G / L / P, 435F / L / T, 436C / G / H / L / S, 437A / H / P, 438P, 439C / L / P, 440D / M, 441E / T, 442L / R, 443A / D / P / V, 444G, 446D / G / H / S / T, 448R, 450V, 451R, 456M, 457T, 458K, 459L, 460A / D / L / R / S / V, 461A / V, 462M, 462F / Y, 462H / L, 463P / S, 465G, 466L, 466I / L, 467M, 468H / I / M, 474L, 477I, 477E / L / R / S / T / W, 477Q / S, 479M / T / V, 480K / M / W, 481H, 484A / L / W / Y, 485I / L, 485E / V, 487R, 487K, 490I / V, 492M / S / Y, 492A / G, 492C / G / H / R / S, 493R, 494L / M / Q / R / T / V / Y, 495A / S, 495G / L / T, 497L, 499L / N / R, 500A / N, 501K / L, 502M, 503L / R / V, 504P, 506A / K / L, 507G, 508C / D / L / Q, 508K, 513V, 515G, 517I, 518S, 522M / V, 523A / D / H, 525H / I / R / T / V / W, 525G / T, or 526E, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12 to the carboxy terminus of SEQ ID NO: 2, or to a reference sequence corresponding to SEQ ID NO:

2.

5. The engineered terminal nucleotidyl transferase of Claim 1 or 3, wherein the amino acid sequence of the engineered terminal nucleotidyl transferase comprises at least an amino acid residue difference at amino acid position(s) 233 / 402, 296 / 384 / 412 / 415 / 451 / 477 / 480, 296 / 415 / 451 / 477 / 480, 296 / 451 / 477, 296 / 451 / 480, 384 / 412 / 451 / 480, 384 / 451 / 477, 402 / 404 / 460, 404, 404 / 431, 415 / 451 / 477 / 480 / 503, 451, 451 / 477, or 477, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12 to the carboxy terminus of SEQ ID NO: 2, or to a reference sequence corresponding to SEQ ID NO:

2.

6. The engineered terminal nucleotidyl transferase of Claim 1 or 3, wherein the amino acid sequence of the engineered terminal nucleotidyl transferase comprises at least an amino acid residue difference(s) 233P / 402K, 296V / 384S / 412M / 415P / 451R / 477Q / 480W, 296V / 415P / 451R / 477Q / 480W, 296V / 451R / 477Q, 296V / 451R / 480W, 384A / 412M / 451R / 480W, 384S / 451R / 477S, 402K / 404A / 460A, 402K / 404A / 460S, 404A, 404A / 431G, 415P / 451R / 477Q / 480W / 503R, 451R, 451R / 477Q, or 477Q, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12 to the carboxy terminus of SEQ ID NO: 2, or to a reference sequence corresponding to SEQ ID NO:

2.

7. The engineered terminal nucleotidyl transferase of Claim 1 or 3, wherein the amino acid sequence of the engineered terminal nucleotidyl transferase comprises at least an amino acid residue difference at amino acid position 22, 27, 34, 41, 42, 43, 45, 52, 60, 92, 105, 133, 152, 160, 161, 162, 164, 169, 173, 174, 175, 176, 177, 179, 182, 183, 184, 186, 187, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 208, 212, 213, 218, 219, 221, 222, 223, 224, 227, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 241, 242, 244, 246, 249, 250, 251, 252, 254, 259, 260, 261, 262, 263, 264, 267, 268, 272, 273, 274, 275, 277, 278, 280, 281, 282, 288, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302,Docket No. CX10-267WO3 303, 304, 307, 308, 309, 310, 312, 313, 314, 315, 316, 317, 318, 319, 321, 322, 323, 324, 325, 328, 329, 333, 334, 335, 336, 337, 339, 340, 341, 342, 343, 347, 352, 353, 354, 357, 359, 360, 361, 362, 363, 364, 366, 367, 368, 370, 371, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 387, 388, 389, 390, 392, 393, 394, 395, 396, 397, 398, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 446, 448, 450, 451, 456, 457, 458, 459, 460, 461, 462, 463, 465, 466, 467, 468, 474, 477, 479, 480, 481, 484, 485, 487, 490, 492, 493, 494, 495, 497, 499, 500, 501, 502, 503, 504, 506, 507, 508, 513, 515, 517, 518, 522, 523, 525, or 526, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12 to the carboxy terminus of SEQ ID NO: 8, 70, 148, 184, 234, 256, 478, 668, 682, 730, 734, 742, 932, 992, 1144, 1212, 1236, 1264, 1414, 1504, 1522, 1702, 1742, 1928, 1944, 1946, 1984, 2064, 2104, 2210, 2256, 2280, 2342, 2438, 2452, 2562, 2576, 2562, 2714, 2876, 2884, 2910, 2974, 2998, or 3002, or to the reference sequence corresponding to SEQ ID NO: 8, 70, 148, 184, 234, 256, 478, 668, 682, 730, 734, 742, 932, 992, 1144, 1212, 1236, 1264, 1414, 1504, 1522, 1702, 1742, 1928, 1944, 1946, 1984, 2064, 2104, 2210, 2256, 2280, 2342, 2438, 2452, 2562, 2576, 2562, 2714, 2876, 2884, 2910, 2974, 2998, or 3002.

8. The engineered terminal nucleotidyl transferase of any one of Claims 1, 3, and 7, wherein the amino acid sequence of the engineered terminal nucleotidyl transferase comprises at least an amino acid residue difference or amino acid residue 22P, 27P, 34Y, 34D, 41T, 42T, 43- / T, 45T, 52G, 60H, 92C, 105G, 133E, 152H, 160A / D / T / W, 161A / G / L / Q / T, 162P, 164Q, 169L, 173H, 173G / P, 174V / W, 174L / W, 174I, 175E / S / T / V, 175H / L / P / R, 176N, 176H, 177C / K, 179V, 179C / K / L / R, 182I / V, 183G / R / S / T / V, 184K / R, 186G / L / M / R / T / V, 187L / T / V / Y, 187T, 190A / R, 190A / F / H / L / T / V, 190G / M / N / Q / R / V, 191F / W / Y, 192A / H, 193R / T, 193A / G / I / R / S / V, 193Q / S / T / W, 194L / M, 195F / Q / R / T / W, 196D / H / K / L / Q / R / S / V, 197I / L, 197M / Q / R, 198E / H / P / Q / R / S / T, 199E / F / V, 200Q, 201D / E / P / Q / R / S, 202H / M / S / V, 203M, 203E / F / L / Q, 204M / V, 205R, 205L, 208A / G, 208E / Q, 208D / E / G, 208E / G / M / T, 212R, 213G, 218I, 219D / Q / S, 221I / Q, 222N, 222A, 223H / L / T, 224L / V, 227G / S, 229F / G / L / S, 230C / L / V, 230I, 231N, 231C / M / N / T, 232E / G, 233I / P, 234T, 235N / R, 236R / S, 237L, 238F / I / L / V, 239G / L / R / S, 241L, 242L, 244G / I / R / S, 246L / V, 249L / M / Q, 250G, 251A / E / G / M / R / S / W, 252E / H / L / M / R / S, 254R, 254N / Q / R, 254I, 259F, 260R, 260K / Q / S / T, 261G / R / V, 261S / T, 262L / M, 263A / K / R / S / W, 263S, 263H / R / T, 264L, 264A / R / S, 267S, 267G, 268L, 272R, 273K / M, 273E / G / M / N / Q, 273I / V, 273L / M / V, 273R, 274P / R, 274L / N / R / S / V, 275I / L / R / S, 277S / T, 277D, 278D / R / S, 278A / L, 280W, 281A / C / G / H / L / S, 282N, 288D, 290L, 291K / L, 292A, 293R, 294G / T, 295T, 295A / D / N, 296V, 296L / M, 297A / S, 297A / G / L / P / Q / T / V / W / Y, 298I, 299G / H / N / P, 299A / G / N / P / R / S, 300S, 300A / K, 300R, 300A / E / F / G / I / K / L / M / N / Q / R / S / V / Y, 301A / F / V, 302S, 303E / R / T, 304L, 307D / E / K / M / N / S, 308L / Y, 308H / L, 309H, 310D / S, 312M, 313H / R / S / T, 313A / Q, 314E, 314A / I / S / T, 315K / R, 316L, 317R, 318P / R / S, 318I / K / L / Q, 319M / T, 319G / Q / V / W, 319A / G / L / M / P, 321R / T, 322A / E, 323T, 324A / T, 325G / S / V / Y, 328A / I, 329Q, 329H / L, 333N / S, 334S, 335V, 336C / T, 337S, 339I / T / V, 339A / S, 339T, 340L / M / T / V, 341L / T, 342S / T, 342S, 343L, 343I / M / R / T / V, 347A / S, 352A / E / L / M / N / T, 353R / V, 353K / L / T / V, 353V, 354G, 357I, 359A / C / F / V, 360V, 361L / V, 361I, 362A / M, 363G, 363P / T, 364S, 366F / H / P, 367N / Q / R / S, 367E / V, 368L / S, 368R, 370L / R, 371G / R, 376A / G / H / R / S / T / V, 376E / S, 377R, 378M, 379M, 379L / T, 380A / K / L / S / V, 381Y, 381S, 382L / M, 383L / Q, 384A / G / S / T, 385V, 387T / V, 388V, 389A / V, 390I / L / M, 390S / T, 390T,Docket No. CX10-267WO3 392E / H / N / Q / R / S, 393A / G / I / L / M / S / T / Y, 394K / N / S, 395E / F / I / L / M / Q / S / T, 396T, 397G / Q / S / T, 397E / F / H / I / K / L / M / R / V / W / Y, 398L / M, 402A / D / K / M / P / S / T, 402P, 403E / F / I / P, 404A / S, 404E / G / I / L / M / Q / R / T / V, 405G / L / S / V, 405A / P / Q / T / V / Y, 406A / D / H / M / N / P / R / T, 406E / G / M / T, 407P / S, 407P, 407A / D / G / L / N / Q / S / V / Y, 408D / G / L / N / V, 408A, 409E / G / P / R / S / V / Y, 409D, 410V, 411E / H / K / R / T / V, 412M / N / P / Q / S / V, 413T, 414Q / R, 415G / P / S, 416G / S, 416A / H, 417A / G / S, 418C / S / T, 419A / I / M, 420C / L / V, 424P, 425R, 426G, 427A / M, 428A / L / T, 429A / S, 430G / Q / S / W, 431G, 432I / V, 433A / P / T / V, 434G / L / P, 435F / L / T, 436C / G / H / L / S, 437A / H / P, 438P, 439C / L / P, 440D / M, 441E / T, 442L / R, 443A / D / P / V, 444G, 446D / G / H / S / T, 448R, 450V, 451R, 456M, 457T, 458K, 459L, 460A / D / L / R / S / V, 461A / V, 462M, 462F / Y, 462H / L, 463P / S, 465G, 466L, 466I / L, 467M, 468H / I / M, 474L, 477I, 477E / L / R / S / T / W, 477Q / S, 479M / T / V, 480K / M / W, 481H, 484A / L / W / Y, 485I / L, 485E / V, 487R, 487K, 490I / V, 492M / S / Y, 492A / G, 492C / G / H / R / S, 493R, 494L / M / Q / R / T / V / Y, 495A / S, 495G / L / T, 497L, 499L / N / R, 500A / N, 501K / L, 502M, 503L / R / V, 504P, 506A / K / L, 507G, 508C / D / L / Q, 508K, 513V, 515G, 517I, 518S, 522M / V, 523A / D / H, 525H / I / R / T / V / W, 525G / T, or 526E, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12 to the carboxy terminus of SEQ ID NO: 8, 70, 148, 184, 234, 256, 478, 668, 682, 730, 734, 742, 932, 992, 1144, 1212, 1236, 1264, 1414, 1504, 1522, 1702, 1742, 1928, 1944, 1946, 1984, 2064, 2104, 2210, 2256, 2280, 2342, 2438, 2452, 2562, 2576, 2562, 2714, 2876, 2884, 2910, 2974, 2998, or 3002, or to the reference sequence corresponding to SEQ ID NO: 8, 70, 148, 184, 234, 256, 478, 668, 682, 730, 734, 742, 932, 992, 1144, 1212, 1236, 1264, 1414, 1504, 1522, 1702, 1742, 1928, 1944, 1946, 1984, 2064, 2104, 2210, 2256, 2280, 2342, 2438, 2452, 2562, 2576, 2562, 2714, 2876, 2884, 2910, 2974, 2998, or 3002.

9. The engineered terminal nucleotidyl transferase of any one of Claims 1, 3, and 7, wherein the amino acid sequence of the engineered terminal nucleotidyl transferase comprises (a) at least an amino acid residue difference at amino acid position(s) 92 / 273 / 277 / 462, 267 / 313 / 314, 267 / 313 / 314 / 408 / 485, 267 / 313 / 367 / 485, 267 / 313 / 406, 267 / 314 / 408 / 441, 267 / 367 / 485, 267 / 406 / 408 / 485, 273 / 277, 273 / 277 / 381, 273 / 277 / 381 / 404 / 462 / 492, 273 / 277 / 381 / 429, 273 / 277 / 381 / 462, 273 / 277 / 381 / 462 / 492, 273 / 277 / 381 / 492, 273 / 277 / 429, 273 / 277 / 429 / 492, 273 / 277 / 462, 273 / 381, 273 / 381 / 492, 273 / 462, 273 / 462 / 492, 277 / 381, 277 / 381 / 429 / 462 / 492, 277 / 381 / 429 / 492, 277 / 381 / 462 / 492, 277 / 381 / 492, 277 / 429 / 462 / 492, 277 / 429 / 492, 277 / 492, 309 / 313 / 314 / 412 / 485, 381 / 462 / 492, or 381 / 492, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12 to the carboxy terminus of SEQ ID NO: 8, or to the reference sequence corresponding to SEQ ID NO: 8; or (b) at least an amino acid residue difference(s) 92C / 273R / 277T / 462M, 267S / 313S / 314A / 408V / 485L, 267S / 313S / 367S / 485L, 267S / 313S / 406H, 267S / 313T / 314A, 267S / 314A / 408V / 441E, 267S / 367S / 485L, 267S / 406H / 408V / 485L, 273R / 277S, 273R / 277S / 381Y, 273R / 277S / 381Y / 404S / 462M / 492M, 273R / 277S / 381Y / 429A, 273R / 277S / 381Y / 462M, 273R / 277S / 381Y / 462M / 492M, 273R / 277S / 381Y / 462M / 492Y, 273R / 277S / 429A, 273R / 277S / 429A / 492S, 273R / 277S / 462M, 273R / 277T, 273R / 277T / 381Y, 273R / 277T / 381Y / 429A, 273R / 277T / 381Y / 492M, 273R / 277T / 429A, 273R / 277T / 462M, 273R / 381Y, 273R / 381Y / 492M, 273R / 462M, 273R / 462M / 492S, 273R / 462M / 492Y, 277S / 381Y, 277S / 381Y / 429A / 462M / 492M, 277S / 381Y / 429A / 492M, 277S / 381Y / 462M / 492M, 277S / 381Y / 462M / 492S, 277S / 429A / 462M / 492S, 277S / 429A / 492Y, 277S / 492S, 277T / 381Y, 277T / 381Y / 429A / 462M / 492M,Docket No. CX10-267WO3 277T / 381Y / 492M, 277T / 381Y / 492S, 309H / 313S / 314A / 412N / 485L, 381Y / 462M / 492Y, or 381Y / 492S, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12 to the carboxy terminus of SEQ ID NO: 8, or to the reference sequence corresponding to SEQ ID NO: 8; or (c) at least an amino acid residue difference at an amino acid positions(s) 186, 193, 196, 198, 198 / 515, 274, 394, or 409, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12 to the carboxy terminus of SEQ ID NO: 8, or to the reference sequence corresponding to SEQ ID NO: 8; or (d) at least an amino acid residue difference 186R, 193Q, 193T, 193W, 196K, 198H, 198Q, 198R, 198R / 515G, 198S, 274R, 394S, or 409R, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12 to the carboxy terminus of SEQ ID NO: 8, or to the reference sequence corresponding to SEQ ID NO:

8.

10. The engineered terminal nucleotidyl transferase of any one of Claims 1, 3, and 7, wherein the amino acid sequence of the engineered terminal nucleotidyl transferase comprises (a) at least an amino acid residue difference at an amino acid positions(s) 190, 190 / 208 / 267 / 274, 190 / 208 / 267 / 274 / 309 / 417, 190 / 208 / 267 / 274 / 417 / 462, 190 / 208 / 274 / 309 / 313 / 417 / 462, 190 / 208 / 309 / 462, 190 / 267 / 274, 190 / 274 / 417, 190 / 274 / 417 / 477, 208 / 274 / 309 / 313 / 430, 267 / 274 / 477, 273, 274, 274 / 309, 274 / 309 / 313 / 417 / 462, 274 / 309 / 417 / 477, 274 / 313, or 274 / 462, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12 to the carboxy terminus of SEQ ID NO: 70, or to the reference sequence corresponding to SEQ ID NO: 70; or (b) at least an amino acid residue difference(s) 190A, 190A / 208E / 267S / 274P, 190A / 208E / 309H / 462H, 190A / 208G / 267S / 274P / 417A / 462H, 190A / 274P / 417A, 190R / 208G / 267S / 274P / 309H / 417A, 190R / 208G / 274P / 309H / 313S / 417A / 462H, 190R / 267S / 274P, 190R / 274P / 417A / 477R, 208E / 274P / 309H / 313S / 430Q, 267S / 274P / 477R, 273V, 274P, 274P / 309H, 274P / 309H / 313S / 417A / 462H, 274P / 309H / 417A / 477R, 274P / 313S, or 274P / 462H, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12 to the carboxy terminus of SEQ ID NO: 70, or to the reference sequence corresponding to SEQ ID NO:

70.

11. The engineered terminal nucleotidyl transferase of any one of Claims 1, 3, and 7, wherein the amino acid sequence of the engineered terminal nucleotidyl transferase comprises (a) at least an amino acid residue difference at an amino acid positions(s) 183 / 208 / 309 / 313 / 407 / 462, 208 / 343 / 407, 208 / 407, or 309, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12 to the carboxy terminus of SEQ ID NO: 148, or to the reference sequence corresponding to SEQ ID NO: 148; or (b) at least an amino acid residue difference(s) 183R / 208E / 309H / 313S / 407P / 462H, 208D / 343L / 407P, 208E / 343L / 407P, 208E / 407P, or 309H, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12 to the carboxy terminus of SEQ ID NO: 148, or to the reference sequence corresponding to SEQ ID NO: 148; or (c) at least an amino acid residue difference at an amino acid positions(s) 190, 268, 275, 303, 397, 409, 415, 417, and 477, or combinations thereof, wherein the amino acid positions are relative to the referenceDocket No. CX10-267WO3 sequence corresponding to amino acid residues 12 to the carboxy terminus of SEQ ID NO: 148, or to the reference sequence corresponding to SEQ ID NO: 148; or (d) at least an amino acid residue difference(s) 190H, 268L, 275L, 275S, 303T, 397V, 409P, 415G, 415P, 417S, and 477R, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12 to the carboxy terminus of SEQ ID NO: 148, or to the reference sequence corresponding to SEQ ID NO: 148; or (e) at least an amino acid residue difference at an amino acid positions(s) 186, 304, 318, 347, 353, 409, 416, 417, and 420, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12 to the carboxy terminus of SEQ ID NO: 148, or to the reference sequence corresponding to SEQ ID NO: 148; or (f) at least an amino acid residue difference(s) 186L, 186T, 186V, 304L, 318L, 347A, 353L, 409P, 416H, 417S, and 420L, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12 to the carboxy terminus of SEQ ID NO: 148, or to the reference sequence corresponding to SEQ ID NO:

148.

12. The engineered terminal nucleotidyl transferase of any one of Claims 1, 3, and 7, wherein the amino acid sequence of the engineered terminal nucleotidyl transferase comprises (a) at least an amino acid residue difference at an amino acid positions(s) 193 / 274 / 411 / 465 / 525, 193 / 408 / 465, 193 / 465, 274 / 352 / 353 / 465, 281 / 411 / 465, 343 / 408 / 465, 343 / 411, 411, 411 / 525, and 525, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12 to the carboxy terminus of SEQ ID NO: 184, or to the reference sequence corresponding to SEQ ID NO: 184; or (b) at least an amino acid residue difference(s) 193S / 274N / 411T / 465G / 525H, 193S / 408L / 465G, 193S / 465G, 274N / 352M / 353T / 465G, 281S / 411T / 465G, 343I / 408L / 465G, 343I / 411T, 411T, 411T / 525H, and 525H, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12 to the carboxy terminus of SEQ ID NO: 184, or to the reference sequence corresponding to SEQ ID NO:

184.

13. The engineered terminal nucleotidyl transferase of any one of Claims 1, 3, and 7, wherein the amino acid sequence of the engineered terminal nucleotidyl transferase comprises (a) at least an amino acid residue difference at an amino acid positions(s) 275, 299, 300, 392, 393, or 397, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12 to the carboxy terminus of SEQ ID NO: 234, or to the reference sequence corresponding to SEQ ID NO: 234; or (b) at least an amino acid residue difference(s) 275I, 299G, 299P, 299R, 299S, 300F, 300G, 300I, 300K, 300L, 300M, 300N, 300Q, 300R, 300Y, 392H, 392N, 393G, 393Y, 397F, 397H, 397I, 397K, 397M, 397R, 397V, 397W, or 397Y, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12 to the carboxy terminus of SEQ ID NO: 234, or to the reference sequence corresponding to SEQ ID NO: 234; or (c) at least an amino acid residue difference at an amino acid positions(s) 299, 300, 392, 393, 395, or 397, or combinations thereof, wherein the amino acid positions are relative to the reference sequenceDocket No. CX10-267WO3 corresponding to amino acid residues 12 to the carboxy terminus of SEQ ID NO: 234, or to the reference sequence corresponding to SEQ ID NO: 234; or (d) at least an amino acid residue difference(s) 299A, 299G, 299N, 299R, 299S, 300A, 300E, 300F, 300L, 300M, 300N, 300S, 300V, 300Y, 392E, 392N, 392Q, 392S, 393A, 393I, 393L, 393M, 393S, 393T, 395F, 395I, 395L, 395M, 395Q, 395T, 397E, 397L, or 397M, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12 to the carboxy terminus of SEQ ID NO: 234, or to the reference sequence corresponding to SEQ ID NO: 234; or (e) at least an amino acid residue difference at an amino acid positions(s) 307, 359, 405, 420, 492, or 525, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12 to the carboxy terminus of SEQ ID NO: 234, or to the reference sequence corresponding to SEQ ID NO: 234 or (f) at least an amino acid residue difference(s) 307S, 359F, 405T, 420V, 492H, or 525T, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12 to the carboxy terminus of SEQ ID NO: 234, or to the reference sequence corresponding to SEQ ID NO: 234; or (g) at least an amino acid residue difference at an amino acid positions(s) 298, 308, 312, 318, 319, 322, 328, 359, 361, 379, 382, 389, 390, 392, 394, 398, 404, 405, 409, 410, 411, 412, 414, 415, 417, 419, 420, 457, 458, 466, 468, 492, 493, 494, 495, 513, 517, or 525, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12 to the carboxy terminus of SEQ ID NO: 234, or to the reference sequence corresponding to SEQ ID NO: 234; or (h) at least an amino acid residue difference(s) 298I, 308L, 312M, 318I, 319M, 319T, 322A, 328I, 359V, 361L, 379T, 382M, 389A, 390I, 392R, 394K, 398M, 404I, 404L, 404R, 405Y, 409G, 409R, 409S, 409Y, 410V, 411H, 411R, 411V, 412V, 414R, 415S, 417G, 419I, 419M, 420C, 457T, 458K, 466L, 468H, 468I, 468M, 492C, 492G, 492H, 492R, 492S, 493R, 494Q, 495G, 495L, 495T, 513V, 517I, or 525I, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12 to the carboxy terminus of SEQ ID NO: 234, or to the reference sequence corresponding to SEQ ID NO:

234.

14. The engineered terminal nucleotidyl transferase of any one of Claims 1, 3, and 7, wherein the amino acid sequence of the engineered terminal nucleotidyl transferase comprises (a) at least an amino acid residue difference at an amino acid positions(s) 307, 319 / 410 / 495, 319 / 466 / 492 / 495, 319 / 492 / 495, 325 / 359 / 404 / 409, 359 / 404 / 405, 405, 405 / 409, 410, 412, 415, 419, 442 / 457, or 525, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12 to the carboxy terminus of SEQ ID NO: 256, or to the reference sequence corresponding to SEQ ID NO: 256; or (b) at least an amino acid residue difference(s) 307S, 319T / 410V / 495G, 319T / 466L / 492H / 495G, 319T / 492H / 495G, 325G / 359F / 404T / 409R, 359F / 404R / 405T, 405T, 405T / 409R, 410V, 412V, 415P, 419M, 442R / 457T, or 525T, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12 to the carboxy terminus of SEQ ID NO: 256, or to the reference sequence corresponding to SEQ ID NO: 256;Docket No. CX10-267WO3 (c) at least an amino acid residue difference at an amino acid positions(s) 323, 336, 339, 362, 363, 363 / 434, 368, 380, 383, 384, 402, 403, 426, 428, 430, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 459, 460, 497, 499, 501, 504, or 506, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12 to the carboxy terminus of SEQ ID NO: 256, or to the reference sequence corresponding to SEQ ID NO: 256; or (d) at least an amino acid residue difference(s) 323T, 336C, 336T, 339T, 362M, 363P, 363P / 434P, 368S, 380A, 380L, 380S, 380V, 383L, 384G, 384T, 402D, 402S, 402T, 403I, 426G, 428A, 430S, 432I, 432V, 433T, 434G, 434L, 434P, 435F, 435L, 435T, 436C, 436H, 436L, 437A, 437P, 438P, 439C, 439L, 439P, 440M, 441T, 442L, 443A, 443P, 443V, 459L, 460R, 460S, 460V, 497L, 499R, 501L, 504P, 506A, or 506L, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12 to the carboxy terminus of SEQ ID NO: 256, or to the reference sequence corresponding to SEQ ID NO: 256; or (e) at least an amino acid residue difference at an amino acid positions(s) 329, 336, 339, 362, 363, 363 / 434, 368, 380, 381, 383, 384, 385, 397, 402, 425, 427, 428, 430, 432, 433, 435, 436, 437, 438, 439, 443, 459, 460, 497, 499, 500, 501, 506, 508, or 518, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12 to the carboxy terminus of SEQ ID NO: 256, or to the reference sequence corresponding to SEQ ID NO: 256; or (f) at least an amino acid residue difference(s) 329H, 336C, 339I, 339T, 362A, 363P / 434P, 363T, 368L, 368S, 380A, 380L, 380V, 381S, 383Q, 384T, 385V, 397T, 402A, 402D, 402M, 402P, 402S, 402T, 425R, 427A, 428T, 430W, 432I, 433V, 435T, 436C, 436G, 437A, 438P, 439P, 443V, 459L, 460L, 460S, 460V, 497L, 499L, 500A, 501L, 506A, 506L, 508L, or 518S, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12 to the carboxy terminus of SEQ ID NO: 256, or to the reference sequence corresponding to SEQ ID NO:

256.

15. The engineered terminal nucleotidyl transferase of any one of Claims 1, 3, and 7, wherein the amino acid sequence of the engineered terminal nucleotidyl transferase comprises (a) at least an amino acid residue difference at an amino acid positions(s) 299 / 302 / 363 / 402 / 405 / 416, 299 / 363 / 402, 299 / 363 / 405 / 416, 299 / 363 / 459, 299 / 402 / 416 / 501, 299 / 459, 302 / 363 / 402 / 405, 302 / 402 / 459, 402 / 416 / 459, or 459, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12 to the carboxy terminus of SEQ ID NO: 478, or to the reference sequence corresponding to SEQ ID NO: 478; or (b) at least an amino acid residue difference(s) 299S / 302S / 363P / 402S / 405T / 416A, 299S / 363P / 402S, 299S / 363P / 405T / 416A, 299S / 363P / 459L, 299S / 402S / 416A / 501L, 299S / 459L, 302S / 363P / 402S / 405T, 302S / 402S / 459L, 402S / 416A / 459L, or 459L, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12 to the carboxy terminus of SEQ ID NO: 478, or to the reference sequence corresponding to SEQ ID NO:

478.

16. The engineered terminal nucleotidyl transferase of any one of Claims 1, 3, and 7, wherein the amino acid sequence of the engineered terminal nucleotidyl transferase comprises (a) at least an amino acid residue difference at an amino acid positions(s) 302 / 416 / 497, 368, 368 / 416 / 435 / 436 / 501, or 416, or combinations thereof, wherein the amino acid positions are relative to theDocket No. CX10-267WO3 reference sequence corresponding to amino acid residues 12 to the carboxy terminus of SEQ ID NO: 668, or to the reference sequence corresponding to SEQ ID NO: 668; or (b) at least an amino acid residue difference(s) 302S / 416A / 497L, 368S, 368S / 416A / 435L / 436H / 501L, or 416A, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12 to the carboxy terminus of SEQ ID NO: 668, or to the reference sequence corresponding to SEQ ID NO: 668; or (c) at least an amino acid residue difference at an amino acid positions(s) 339 / 368 / 436, and 416, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12 to the carboxy terminus of SEQ ID NO: 668, or to the reference sequence corresponding to SEQ ID NO: 668; or (d) at least an amino acid residue difference(s) 339T / 368S / 436L, or 416A, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12 to the carboxy terminus of SEQ ID NO: 668, or to the reference sequence corresponding to SEQ ID NO: 668; or (e) at least an amino acid residue difference at an amino acid positions(s) 184, 191, 222, 224, 232, 236, 246, 251, 288, 291, 319, 444, 485, or 490, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12 to the carboxy terminus of SEQ ID NO: 668, or to the reference sequence corresponding to SEQ ID NO: 668; or (f) at least an amino acid residue difference(s) 184R, 191F, 191W, 191Y, 222A, 224L, 232E, 236S, 246V, 251R, 288D, 291K, 319G, 319W, 444G, 485I, or 490I, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12 to the carboxy terminus of SEQ ID NO: 668, or to the reference sequence corresponding to SEQ ID NO:

668.

17. The engineered terminal nucleotidyl transferase of any one of Claims 1, 3, and 7, wherein the amino acid sequence of the engineered terminal nucleotidyl transferase comprises (a) at least an amino acid residue difference at an amino acid positions 191, 368, 485, or 490, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12 to the carboxy terminus of SEQ ID NO: 682, or to the reference sequence corresponding to SEQ ID NO: 682; or (b) at least an amino acid residue difference 191F, 368S, 485I, or 490I, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12 to the carboxy terminus of SEQ ID NO: 682, or to the reference sequence corresponding to SEQ ID NO:

682.

18. The engineered terminal nucleotidyl transferase of any one of Claims 1, 3, and 7, wherein the amino acid sequence of the engineered terminal nucleotidyl transferase comprises (a) at least an amino acid residue difference at an amino acid positions(s) 191 / 222 / 236 / 246 / 291 / 368 / 444, 191 / 222 / 244 / 246 / 291 / 368 / 490, 191 / 222 / 244 / 246 / 368 / 490, 191 / 222 / 244 / 246 / 368 / 495, 191 / 222 / 244 / 291 / 368 / 444, 191 / 222 / 246 / 368 / 444, 191 / 222 / 291 / 368, 191 / 222 / 291 / 368 / 490, 191 / 222 / 368, 191 / 222 / 368 / 444 / 490, 191 / 222 / 368 / 490, 191 / 236 / 244 / 291 / 368 / 490, 191 / 236 / 291 / 368, 191 / 236 / 291 / 368 / 490, 191 / 246 / 291 / 368 / 490, 191 / 246 / 368 / 490, 191 / 291 / 368 / 444,Docket No. CX10-267WO3 191 / 291 / 368 / 444 / 490, 191 / 368 / 444, 191 / 368 / 444 / 490, 191 / 368 / 490, 222 / 236 / 246 / 291 / 368 / 444 / 490, 222 / 244 / 291 / 368 / 444, 222 / 246 / 368 / 444, 222 / 246 / 490, 222 / 291 / 368, 222 / 291 / 368 / 444, 222 / 291 / 368 / 444 / 490, 222 / 368 / 444 / 490, 222 / 368 / 490, 236 / 246 / 291 / 368 / 444 / 490, 236 / 246 / 368, 244 / 246 / 291 / 368, 244 / 246 / 368 / 444, 244 / 246 / 368 / 490, 244 / 291 / 368 / 490, 246 / 291 / 296 / 368 / 444 / 490, 246 / 291 / 368, 246 / 291 / 368 / 490, 246 / 368 / 444, 368, 368 / 444, or 368 / 490, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12 to the carboxy terminus of SEQ ID NO: 730, or to the reference sequence corresponding to SEQ ID NO: 730; or (b) at least an amino acid residue difference(s) 191F / 222A / 236S / 246V / 291K / 368S / 444G, 191F / 222A / 244R / 246V / 291K / 368S / 490I, 191F / 222A / 244R / 246V / 368S / 490I, 191F / 222A / 244R / 246V / 368S / 495S, 191F / 222A / 244R / 291K / 368S / 444G, 191F / 222A / 246V / 368S / 444G, 191F / 222A / 291K / 368S, 191F / 222A / 291K / 368S / 490V, 191F / 222A / 368S, 191F / 222A / 368S / 444G / 490V, 191F / 222A / 368S / 490I, 191F / 236S / 244R / 291K / 368S / 490V, 191F / 236S / 291K / 368S, 191F / 236S / 291K / 368S / 490V, 191F / 246V / 291K / 368S / 490I, 191F / 246V / 368S / 490V, 191F / 291K / 368S / 444G, 191F / 291K / 368S / 444G / 490I, 191F / 368S / 444G, 191F / 368S / 444G / 490V, 191F / 368S / 490I, 191F / 368S / 490V, 222A / 236S / 246V / 291K / 368S / 444G / 490V, 222A / 244R / 291K / 368S / 444G, 222A / 246V / 368S / 444G, 222A / 246V / 490I, 222A / 291K / 368S, 222A / 291K / 368S / 444G, 222A / 291K / 368S / 444G / 490V, 222A / 368S / 444G / 490I, 222A / 368S / 444G / 490V, 222A / 368S / 490V, 236S / 246V / 291K / 368S / 444G / 490V, 236S / 246V / 368S, 244R / 246V / 291K / 368S, 244R / 246V / 368S / 444G, 244R / 246V / 368S / 490V, 244R / 291K / 368S / 490V, 246V / 291K / 296M / 368S / 444G / 490V, 246V / 291K / 368S, 246V / 291K / 368S / 490V, 246V / 368S / 444G, 368S, 368S / 444G, or 368S / 490V, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12 to the carboxy terminus of SEQ ID NO: 730, or to the reference sequence corresponding to SEQ ID NO: 730; or (c) at least an amino acid residue difference at an amino acid positions(s) 161, 173, 197, 201, 208, 262, 343, 379, 382, 416, 429, 494, 508, or 525, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12 to the carboxy terminus of SEQ ID NO: 730, or to the reference sequence corresponding to SEQ ID NO: 730; or (d) at least an amino acid residue difference(s) 161G, 161T, 173G, 197M, 201E, 208A, 262M, 343V, 379L, 382L, 416G, 429S, 494R, 508D, or 525W, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12 to the carboxy terminus of SEQ ID NO: 730, or to the reference sequence corresponding to SEQ ID NO: 730; or (e) at least an amino acid residue difference at an amino acid positions(s) 161, 164, 201, 213, 223, 263, 333, 337, 411, 429, 463, 467, 479 / 485, 484 / 485, 502, 507, 522, 523, or 525, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12 to the carboxy terminus of SEQ ID NO: 730, or to the reference sequence corresponding to SEQ ID NO: 730; or (f) at least an amino acid residue difference(s) 161G, 161Q, 164Q, 201E, 213G, 223T, 263A, 333N, 333S, 337S, 411E, 429S, 463P, 467M, 479V / 485V, 484W / 485E, 502M, 507G, 522M, 522V, 523D, or 525R, or combinations thereof, wherein the amino acid positions are relative to the reference sequence correspondingDocket No. CX10-267WO3 to amino acid residues 12 to the carboxy terminus of SEQ ID NO: 730, or to the reference sequence corresponding to SEQ ID NO:

730.

19. The engineered terminal nucleotidyl transferase of any one of Claims 1, 3, and 7, wherein the amino acid sequence of the engineered terminal nucleotidyl transferase comprises (a) at least an amino acid residue difference at an amino acid positions(s) 161, 161 / 197 / 208 / 262 / 416, 161 / 208, 161 / 208 / 262 / 411 / 416 / 429, 161 / 208 / 262 / 411 / 416 / 467, 161 / 208 / 467, 161 / 262, 161 / 262 / 411, 161 / 262 / 411 / 416, 161 / 262 / 411 / 416 / 467, 161 / 262 / 411 / 467, 161 / 262 / 416, 161 / 262 / 416 / 467, 161 / 262 / 429, 161 / 262 / 467, 161 / 416 / 442, 161 / 429, 197, 197 / 208, 197 / 208 / 262, 197 / 208 / 262 / 411 / 429 / 467, 197 / 208 / 411, 197 / 208 / 411 / 416 / 467, 197 / 208 / 416, 197 / 208 / 467, 197 / 262, 197 / 262 / 467, 208, 208 / 262, 208 / 262 / 411 / 416, 208 / 262 / 416 / 467, 208 / 262 / 429, 208 / 411 / 416, 208 / 411 / 467, 208 / 416, 208 / 416 / 429, 262, 262 / 411, 262 / 411 / 416, 262 / 411 / 416 / 429, 262 / 416, 262 / 416 / 467, 411 / 416, or 416, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12 to the carboxy terminus of SEQ ID NO: 742, or to the reference sequence corresponding to SEQ ID NO: 742; or (b) at least an amino acid residue difference(s) 161G, 161G / 197M / 208G / 262M / 416G, 161G / 208G, 161G / 208G / 262M / 411E / 416G / 429S, 161G / 208G / 262M / 411E / 416G / 467M, 161G / 208G / 467M, 161G / 262L / 429S, 161G / 262M, 161G / 262M / 411E, 161G / 262M / 411E / 416G, 161G / 262M / 411E / 416G / 467M, 161G / 262M / 411E / 467M, 161G / 262M / 416G, 161G / 262M / 416G / 467M, 161G / 262M / 467M, 161G / 416G / 442R, 161G / 429S, 197M, 197M / 208G, 197M / 208G / 262M, 197M / 208G / 262M / 411E / 429S / 467M, 197M / 208G / 411E, 197M / 208G / 411E / 416G / 467M, 197M / 208G / 416G, 197M / 208G / 467M, 197M / 262M, 197M / 262M / 467M, 208G, 208G / 262M, 208G / 262M / 411E / 416G, 208G / 262M / 416G / 467M, 208G / 262M / 429S, 208G / 411E / 416G, 208G / 411E / 467M, 208G / 416G, 208G / 416G / 429S, 262M, 262M / 411E, 262M / 411E / 416G, 262M / 411E / 416G / 429S, 262M / 416G, 262M / 416G / 467M, 411E / 416G, or 416G, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12 to the carboxy terminus of SEQ ID NO: 742, or to the reference sequence corresponding to SEQ ID NO:

742.

20. The engineered terminal nucleotidyl transferase of any one of Claims 1, 3, and 7, wherein the amino acid sequence of the engineered terminal nucleotidyl transferase comprises (a) at least an amino acid residue difference at an amino acid positions(s) 262 / 317, 262 / 329, 262 / 339, 262 / 403, 262 / 462, 262 / 492, 262 / 526, or 387, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12 to the carboxy terminus of SEQ ID NO: 932, or to the reference sequence corresponding to SEQ ID NO: 932; or (b) at least an amino acid residue difference(s) 262M / 317R, 262M / 329L, 262M / 339V, 262M / 403E, 262M / 403F, 262M / 403P, 262M / 462L, 262M / 492A, 262M / 526E, or 387V, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12 to the carboxy terminus of SEQ ID NO: 932, or to the reference sequence corresponding to SEQ ID NO: 932; or (c) at least an amino acid residue difference at an amino acid positions(s) 184 / 262, 262 / 340, 262 / 363, 262 / 371, 262 / 371 / 443, 262 / 377, or 262 / 387, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12 to the carboxy terminus of SEQ ID NO: 932, or to the reference sequence corresponding to SEQ ID NO: 932; orDocket No. CX10-267WO3 (d) at least an amino acid residue difference(s) 184K / 262M, 262M / 340V, 262M / 363G, 262M / 371G / 443D, 262M / 371R, 262M / 377R, or 262M / 387T, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12 to the carboxy terminus of SEQ ID NO: 932, or to the reference sequence corresponding to SEQ ID NO:

932.

21. The engineered terminal nucleotidyl transferase of any one of Claims 1, 3, and 7, wherein the amino acid sequence of the engineered terminal nucleotidyl transferase comprises (a) at least an amino acid residue difference at an amino acid positions(s) 183, 186, 193, 264, 294, 297, 313, 314, 315, 347, 352, 353, 359, 389, 398, 405, 406, and 409, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12 to the carboxy terminus of SEQ ID NO: 734, or to the reference sequence corresponding to SEQ ID NO: 734; or (b) at least an amino acid residue difference(s) 183R, 183S, 186G, 186L, 186M, 186T, 186V, 193R, 264L, 294G, 297A, 297S, 313H, 313S, 314A, 314S, 315K, 315R, 347S, 352A, 352N, 353K, 359A, 359C, 389V, 398L, 405P, 406R, and 409P, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12 to the carboxy terminus of SEQ ID NO: 734, or to the reference sequence corresponding to SEQ ID NO:

734.

22. The engineered terminal nucleotidyl transferase of any one of Claims 1, 3, and 7, wherein the amino acid sequence of the engineered terminal nucleotidyl transferase comprises (a) at least an amino acid residue difference at an amino acid positions(s) 60 / 193 / 264 / 297, 60 / 263 / 264 / 297 / 300 / 525, 193 / 297, 208 / 273 / 294 / 407, 208 / 273 / 295, 273, and 297, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12 to the carboxy terminus of SEQ ID NO: 992, or to the reference sequence corresponding to SEQ ID NO: 992; or (b) at least an amino acid residue difference(s) 60H / 193T / 264L / 297S, 60H / 263A / 264L / 297S / 300S / 525W, 193T / 297A, 208E / 273L / 294T / 407G, 208E / 273M / 295N, 273L, or 297S, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12 to the carboxy terminus of SEQ ID NO: 992, or to the reference sequence corresponding to SEQ ID NO:

992.

23. The engineered terminal nucleotidyl transferase of any one of Claims 1, 3, and 7, wherein the amino acid sequence of the engineered terminal nucleotidyl transferase comprises at least an amino acid residue difference at an amino acid positions(s) 229, 230, 231, 233, 234, 237, 238, 241, 250, 251, 252, 260 / 264, or 264, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12 to the carboxy terminus of SEQ ID NO: 1144, or to the reference sequence corresponding to SEQ ID NO: 1144; or (b) at least an amino acid residue difference(s) 229F, 230C, 230L, 231C, 231M, 231T, 233I, 234T, 237L, 238F, 238L, 238V, 241L, 250G, 251E, 251G, 251M, 251R, 251S, 251W, 252M, 252S, 260R / 264A, and 264A, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12 to the carboxy terminus of SEQ ID NO: 1144, or to the reference sequence corresponding to SEQ ID NO: 1144; orDocket No. CX10-267WO3 (c) at least an amino acid residue difference at an amino acid positions(s) 329, 329 / 339 / 340, 329 / 339 / 340 / 371 / 387, 329 / 339 / 340 / 403, 329 / 339 / 340 / 403 / 526, 329 / 339 / 371 / 387, 329 / 339 / 403 / 443, 329 / 526, 339, 339 / 340 / 387 / 526, 339 / 340 / 428, 339 / 403, 339 / 526, 371 / 403 / 526, and 443 / 526, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12 to the carboxy terminus of SEQ ID NO: 1144, or to the reference sequence corresponding to SEQ ID NO: 1144; or (d) least an amino acid residue difference(s) 329L, 329L / 339V / 340V, 329L / 339V / 340V / 371G / 387T, 329L / 339V / 340V / 403E, 329L / 339V / 340V / 403E / 526E, 329L / 339V / 371G / 387T, 329L / 339V / 403E / 443D, 329L / 526E, 339V, 339V / 340V / 387V / 526E, 339V / 340V / 428L, 339V / 403E, 339V / 526E, 371G / 403E / 526E, and 443D / 526E, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12 to the carboxy terminus of SEQ ID NO: 1144, or to the reference sequence corresponding to SEQ ID NO: 1144.

24. The engineered terminal nucleotidyl transferase of any one of Claims 1, 3, and 7, wherein the amino acid sequence of the engineered terminal nucleotidyl transferase comprises (a) at least an amino acid residue difference at an amino acid positions(s) 184 / 329 / 403 / 404 / 492, 184 / 403 / 404 / 526, 329, 329 / 526, 340, 340 / 460, 376, and 492, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12 to the carboxy terminus of SEQ ID NO: 1212, or to the reference sequence corresponding to SEQ ID NO: 1212; or (b) at least an amino acid residue difference(s) 184K / 329L / 403E / 404G / 492A, 184K / 403F / 404G / 526E, 329L, 329L / 526E, 340M, 340V / 460V, 376S, and 492A, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12 to the carboxy terminus of SEQ ID NO: 1212, or to the reference sequence corresponding to SEQ ID NO: 1212.

25. The engineered terminal nucleotidyl transferase of any one of Claims 1, 3, and 7, wherein the amino acid sequence of the engineered terminal nucleotidyl transferase comprises (a) at least an amino acid residue difference at an amino acid positions(s) 174, 190, 198, 201, 202, 212, 263, 297, 318, 319, 322 / 329, 325 / 329, 329, 343, 379, 412, 415, 419, 448, 450, 456, 463, 477, 479, 480, 484, 523, and 525, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12 to the carboxy terminus of SEQ ID NO: 1236, or to the reference sequence corresponding to SEQ ID NO: 1236; or (b) at least an amino acid residue difference(s) 174W, 190A, 190F, 190L, 190T, 190V, 198E, 201D, 202M, 202S, 212R, 263A, 263S, 263W, 297G, 297P, 318Q, 319Q, 322E / 329Q, 325G / 329Q, 325S / 329Q, 325Y / 329Q, 329Q, 343R, 343T, 343V, 379L, 412Q, 415G, 419A, 448R, 450V, 456M, 463S, 477T, 479V, 480M, 484A, 523D, 525I, 525R, and 525V, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12 to the carboxy terminus of SEQ ID NO: 1236, or to the reference sequence corresponding to SEQ ID NO: 1236; or (c) at least an amino acid residue difference at an amino acid positions(s) 175, 179, 183, 192, 194, 196, 199, 203, 208, 264, 280, 281, 295, 300, 313, 314, 352, 353, 354, 389, 404, 406, 407, 408, and 409, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding toDocket No. CX10-267WO3 amino acid residues 12 to the carboxy terminus of SEQ ID NO: 1236, or to the reference sequence corresponding to SEQ ID NO: 1236; or (d) at least an amino acid residue difference(s) 175V, 179L, 183V, 192A, 194L, 196H, 199F, 203M, 208Q, 264R, 280W, 281C, 281G, 281H, 281L, 281S, 295A, 300L, 300N, 313H, 313R, 314T, 352E, 352L, 353L, 353V, 354G, 389V, 404E, 404L, 404Q, 404V, 406N, 406T, 407L, 407V, 408L, and 409E, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12 to the carboxy terminus of SEQ ID NO: 1236, or to the reference sequence corresponding to SEQ ID NO: 1236.

26. The engineered terminal nucleotidyl transferase of any one of Claims 1, 3, and 7, wherein the amino acid sequence of the engineered terminal nucleotidyl transferase comprises (a) at least an amino acid residue difference at an amino acid positions(s) 190 / 263 / 295 / 300 / 342 / 393, 190 / 300 / 525, 263 / 295 / 300 / 420, 263 / 295 / 420, 264 / 300 / 420, 264 / 379, 264 / 420, 264 / 420 / 525, 295 / 379 / 420, 295 / 420, 300 / 379 / 420, 300 / 420, 313, 313 / 379 / 525, 342, 342 / 420, 379, 379 / 420, 420, and 525, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12 to the carboxy terminus of SEQ ID NO: 1264, or to the reference sequence corresponding to SEQ ID NO: 1264; or (b) at least an amino acid residue difference(s) 190G / 263S / 295A / 300S / 342S / 393T, 190G / 300S / 525V, 263S / 295A / 300S / 420V, 263S / 295A / 420V, 264S / 300S / 420V, 264S / 379L, 264S / 420V, 264S / 420V / 525V, 295A / 379L / 420V, 295A / 420V, 300S / 379L / 420V, 300S / 420V, 313R, 313R / 379L / 525V, 342S, 342S / 420V, 379L, 379L / 420V, 420V, and 525V, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12 to the carboxy terminus of SEQ ID NO: 1264, or to the reference sequence corresponding to SEQ ID NO: 1264; (c) at least an amino acid residue difference at an amino acid positions(s) 174, 190, 190 / 193, 190 / 194, 260, 353, 392, 393, 395, 405, 407, 408, 411, and 418, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12 to the carboxy terminus of SEQ ID NO: 1264, or to the reference sequence corresponding to SEQ ID NO: 1264; or (d) at least an amino acid residue difference(s) 174V, 190M, 190R / 193I, 190R / 194M, 260R, 353L, 353V, 392Q, 393T, 395E, 395M, 395T, 405Q, 407Q, 408N, 411K, and 418C, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12 to the carboxy terminus of SEQ ID NO: 1264, or to the reference sequence corresponding to SEQ ID NO: 1264; or (e) at least an amino acid residue difference at an amino acid positions(s) 190, 190 / 193, 190 / 194, 353, 405, 407, 408, and 409, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12 to the carboxy terminus of SEQ ID NO: 1264, or to the reference sequence corresponding to SEQ ID NO: 1264; or (f) at least an amino acid residue difference(s) 190R, 190R / 193G, 190R / 193S, 190R / 193V, 190R / 194M, 353L, 405Q, 405V, 407G, 407Y, 408V, and 409V, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12 to the carboxy terminus of SEQ ID NO: 1264, or to the reference sequence corresponding to SEQ ID NO: 1264.Docket No. CX10-267WO3 27. The engineered terminal nucleotidyl transferase of any one of Claims 1, 3, and 7, wherein the amino acid sequence of the engineered terminal nucleotidyl transferase comprises (a) at least an amino acid residue difference at an amino acid positions(s) 169 / 409, 194, 203 / 208 / 295 / 308 / 409, 203 / 208 / 295 / 389 / 409, 263 / 300 / 353, 295 / 308 / 409, 300, and 352 / 353, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12 to the carboxy terminus of SEQ ID NO: 1414, or to the reference sequence corresponding to SEQ ID NO: 1414; or (b) at least an amino acid residue difference(s) 169L / 409E, 194L, 203M / 208Q / 295A / 308H / 409E, 203M / 208Q / 295D / 308H / 409E, 203M / 208Q / 295D / 389V / 409E, 263S / 300N / 353V, 295A / 308H / 409E, 300N, and 352A / 353V, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12 to the carboxy terminus of SEQ ID NO: 1414, or to the reference sequence corresponding to SEQ ID NO: 1414.

28. The engineered terminal nucleotidyl transferase of any one of Claims 1, 3, and 7, wherein the amino acid sequence of the engineered terminal nucleotidyl transferase comprises (a) at least an amino acid residue difference at an amino acid positions(s) 174 / 260 / 263 / 353 / 405 / 408, 174 / 353, 174 / 405 / 407 / 420, 174 / 407 / 409 / 420, 260 / 353, 260 / 353 / 420, 263 / 352 / 353 / 405 / 408 / 420, 263 / 353 / 420, 263 / 420, 353 / 420, 405 / 408 / 409 / 420, 407 / 408 / 420, 407 / 409 / 420, and 420, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12 to the carboxy terminus of SEQ ID NO: 1504, or to the reference sequence corresponding to SEQ ID NO: 1504; or (b) at least an amino acid residue difference(s) 174V / 260R / 263S / 353L / 405Q / 408V, 174V / 353V, 174V / 405Q / 407Y / 420V, 174V / 407Y / 409D / 420V, 260R / 353V, 260R / 353V / 420V, 263S / 352A / 353V / 405Q / 408V / 420V, 263S / 353V / 420V, 263S / 420V, 353L / 420V, 353V / 420V, 405Q / 408V / 409D / 420V, 407S / 409D / 420V, 407Y / 408V / 420V, and 420V, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12 to the carboxy terminus of SEQ ID NO: 1504, or to the reference sequence corresponding to SEQ ID NO: 1504; or (c) at least an amino acid residue difference at an amino acid positions(s) 133 / 197 / 203, 192, 197 / 203, 261, 263, 281, 281 / 440, 295 / 297, 295 / 301 / 308, 308, 308 / 314, 322, 328, 389, 390, 394, 404 / 409, 405 / 409, 461, and 484, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12 to the carboxy terminus of SEQ ID NO: 1504, or to the reference sequence corresponding to SEQ ID NO: 1504; or (d) at least an amino acid residue difference(s) 133E / 197Q / 203E, 192H, 197R / 203E, 261G, 261R, 261V, 263K, 263R, 281G, 281S / 440D, 295T / 297G, 295T / 297P, 295T / 301V / 308Y, 308L, 308Y, 308Y / 314I, 322E, 328A, 389V, 390L, 390M, 394N, 394S, 404I / 409D, 404M / 409D, 405A / 409D, 405V / 409D, 461A, and 484W, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12 to the carboxy terminus of SEQ ID NO: 1504, or to the reference sequence corresponding to SEQ ID NO: 1504.

29. The engineered terminal nucleotidyl transferase of any one of Claims 1, 3, and 7, wherein the amino acid sequence of the engineered terminal nucleotidyl transferase comprises (a) at least an amino acid residue difference at an amino acid positions(s) 52, 175, 182, 193, 195,Docket No. CX10-267WO3 196, 201, or 278, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12 to the carboxy terminus of SEQ ID NO: 1522, or to the reference sequence corresponding to SEQ ID NO: 1522; or (b) at least an amino acid residue difference(s) 133E / 197Q / 203E, 192H, 197R / 203E, 261G, 261R, 261V, 263K, 263R, 281G, 281S / 440D, 295T / 297G, 295T / 297P, 295T / 301V / 308Y, 308L, 308Y, 308Y / 314I, 322E, 328A, 389V, 390L, 390M, 394N, 394S, 404I / 409D, 404M / 409D, 405A / 409D, 405V / 409D, 461A, or 484W, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12 to the carboxy terminus of SEQ ID NO: 1522, or to the reference sequence corresponding to SEQ ID NO: 1522; or (c) at least an amino acid residue difference at an amino acid positions(s) 52, 175, 182, 193, 195, 196, 201, or 278, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12 to the carboxy terminus of SEQ ID NO: 1522, or to the reference sequence corresponding to SEQ ID NO: 1522; or (d) at least an amino acid residue difference(s) 52G, 175E, 175S, 175T, 182V, 193A, 193G, 193V, 195R, 195T, 196V, 201S, or 278R, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12 to the carboxy terminus of SEQ ID NO: 1522, or to the reference sequence corresponding to SEQ ID NO: 1522; or (e) at least an amino acid residue difference at an amino acid positions(s) 173, 193, 195, 196, 201, 272, 275, 278, 282, 297, 299, 301, 314, 412, 418, 461, 462, 479, or 494, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12 to the carboxy terminus of SEQ ID NO: 1522, or to the reference sequence corresponding to SEQ ID NO: 1522; or (f) at least an amino acid residue difference(s) 173P, 193R, 195F, 195W, 196R, 201P, 201R, 272R, 275R, 275S, 278R, 282N, 297A, 297P, 297T, 297V, 297W, 299G, 299H, 299P, 301A, 314A, 412S, 412V, 418S, 418T, 461V, 462F, 479M, 479T, 494L, 494M, 494Q, or 494V, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12 to the carboxy terminus of SEQ ID NO: 1522, or to the reference sequence corresponding to SEQ ID NO: 1522; or (g) at least an amino acid residue difference at an amino acid positions(s) 174 / 194 / 195 / 197 / 319 / 361 / 420, 174 / 194 / 197 / 260 / 263 / 319 / 353 / 409 / 420, 174 / 194 / 197 / 260 / 263 / 353 / 409 / 420, 194 / 195 / 197 / 319, 194 / 195 / 319 / 420, 194 / 195 / 361, 194 / 197, 194 / 197 / 319 / 361 / 420, 194 / 319, 194 / 319 / 361 / 420, 194 / 319 / 420, 195 / 319 / 361 / 420, 195 / 319 / 420, 197 / 420, 300 / 325 / 406, 300 / 353 / 420 / 525, 300 / 406 / 409, 319 / 420, 325 / 406, or 361 / 420, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12 to the carboxy terminus of SEQ ID NO: 1522, or to the reference sequence corresponding to SEQ ID NO: 1522; or (h) at least an amino acid residue difference(s) 174L / 194L / 195Q / 197M / 319V / 361V / 420V, 174L / 194L / 197M / 260Q / 263H / 319V / 353V / 409D / 420V, 174L / 194L / 197M / 260Q / 263H / 353V / 409D / 420V, 194L / 195Q / 197M / 319V, 194L / 195Q / 319V / 420V, 194L / 195Q / 361V, 194L / 197M, 194L / 197M / 319V / 361V / 420V, 194L / 319V, 194L / 319V / 361V / 420V, 194L / 319V / 420V, 195Q / 319V / 361V / 420V, 195Q / 319V / 420V, 197M / 420V, 300N / 325V / 406D, 300N / 353V / 420V / 525T, 300N / 406D / 409D, 319V / 420V, 325V / 406D, or 361V / 420V, or combinations thereof, wherein the amino acidDocket No. CX10-267WO3 positions are relative to the reference sequence corresponding to amino acid residues 12 to the carboxy terminus of SEQ ID NO: 1522, or to the reference sequence corresponding to SEQ ID NO: 1522.

30. The engineered terminal nucleotidyl transferase of any one of Claims 1, 3, and 7, wherein the amino acid sequence of the engineered terminal nucleotidyl transferase comprises (a) at least an amino acid residue difference at an amino acid positions(s) 261 / 409, 297, 300, and 409, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12 to the carboxy terminus of SEQ ID NO: 1702, or to the reference sequence corresponding to SEQ ID NO: 1702; or (b) at least an amino acid residue difference(s) 261V / 409D, 297P, 300N, and 409D, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12 to the carboxy terminus of SEQ ID NO: 1702, or to the reference sequence corresponding to SEQ ID NO: 1702.

31. The engineered terminal nucleotidyl transferase of any one of Claims 1, 3, and 7, wherein the amino acid sequence of the engineered terminal nucleotidyl transferase comprises (a) at least an amino acid residue difference at an amino acid positions(s) 52 / 201 / 390, 175 / 193 / 297 / 300, 175 / 297 / 300, 175 / 297 / 300 / 462, 175 / 300, 175 / 300 / 314, 175 / 300 / 442, 193 / 195 / 297 / 300 / 314, 193 / 297 / 300 / 314 / 442, 195 / 297 / 300 / 462, 221 / 297 / 300, or 297 / 300, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12 to the carboxy terminus of SEQ ID NO: 1742, or to the reference sequence corresponding to SEQ ID NO: 1742; or (b) at least an amino acid residue difference(s) 52G / 201S / 390M, 175S / 193G / 297V / 300N, 175S / 297V / 300N, 175S / 297V / 300N / 462F, 175S / 300N, 175S / 300N / 314A, 175S / 300N / 442R, 193G / 195T / 297V / 300N / 314A, 193G / 297V / 300N / 314A / 442R, 195T / 297A / 300N / 462F, 195T / 297V / 300N / 462F, 221Q / 297A / 300N, 297A / 300N, or 297V / 300N, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12 to the carboxy terminus of SEQ ID NO: 1742, or to the reference sequence corresponding to SEQ ID NO: 1742; or (c) at least an amino acid residue difference at an amino acid positions(s) 152 / 281, 182, 196, 197, 261, 281, 297, 303, 307, 319, 397, 404 / 409, 405 / 409, 406 / 409, 409, 463, 474, or 477, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12 to the carboxy terminus of SEQ ID NO: 1742, or to the reference sequence corresponding to SEQ ID NO: 1742; or (d) at least an amino acid residue difference(s) 152H / 281S, 182V, 196D, 196L, 196S, 197I, 261T, 281S, 297T, 303R, 307N, 319P, 397S, 404E / 409E, 404G / 409E, 405S / 409E, 406A / 409E, 406P / 409E, 409E, 463P, 474L, or 477S, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12 to the carboxy terminus of SEQ ID NO: 1742, or to the reference sequence corresponding to SEQ ID NO: 1742; or (e) at least an amino acid residue difference at an amino acid positions(s) 152 / 281, 190, 197, 208, 274, 281, 297, 299, 303, 307, 308, 313, 319, 321, 360, 404 / 409, 406 / 409, 417, 460, 461, 477, 479, 492, or 495, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12 to the carboxy terminus of SEQ ID NO: 1742, or to the referenceDocket No. CX10-267WO3 sequence corresponding to SEQ ID NO: 1742; or (f) at least an amino acid residue difference(s) 152H / 281S, 190R, 197I, 208E, 208T, 274S, 281A, 281G, 281H, 281S, 297T, 299N, 303R, 303T, 307D, 307E, 307M, 308Y, 313A, 319G, 319L, 319M, 319P, 321T, 360V, 404E / 409E, 404M / 409E, 404V / 409E, 406A / 409E, 406M / 409E, 406P / 409E, 417G, 460D, 461A, 477E, 479M, 479T, 492G, or 495A, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12 to the carboxy terminus of SEQ ID NO: 1742, or to the reference sequence corresponding to SEQ ID NO: 1742.

32. The engineered terminal nucleotidyl transferase of any one of Claims 1, 3, and 7, wherein the amino acid sequence of the engineered terminal nucleotidyl transferase comprises (a) at least an amino acid residue difference at an amino acid positions(s) 161 / 251 / 408, 183 / 522, 196 / 231 / 522, 231 / 416 / 474, 273, 273 / 274 / 325 / 416 / 522, 416, or 416 / 474, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12 to the carboxy terminus of SEQ ID NO: 1928, or to the reference sequence corresponding to SEQ ID NO: 1928; or (b) at least an amino acid residue difference(s) 161G / 251M / 408A, 183T / 522V, 196Q / 231M / 522V, 231M / 416S / 474L, 273M, 273M / 274R / 325V / 416G / 522V, 416S, or 416S / 474L, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12 to the carboxy terminus of SEQ ID NO: 1928, or to the reference sequence corresponding to SEQ ID NO: 1928.

33. The engineered terminal nucleotidyl transferase of any one of Claims 1, 3, and 7, wherein the amino acid sequence of the engineered terminal nucleotidyl transferase comprises (a) at least an amino acid residue difference at an amino acid positions(s) 231 / 408, or 406 / 463, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12 to the carboxy terminus of SEQ ID NO: 1944, or to the reference sequence corresponding to SEQ ID NO: 1944; or (b) at least an amino acid residue difference(s) 231M / 408A, and 406P / 463P, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12 to the carboxy terminus of SEQ ID NO: 1944, or to the reference sequence corresponding to SEQ ID NO: 1944; or (c) at least an amino acid residue difference at an amino acid positions(s) 231, 273, 367, 407, 424, 427, 429, 430, or 499, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12 to the carboxy terminus of SEQ ID NO: 1944, or to the reference sequence corresponding to SEQ ID NO: 1944; or (d) at least an amino acid residue difference(s) 231N, 273I, 273V, 367N, 367Q, 407D, 424P, 427M, 429S, 430G, 499N, or 499R, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12 to the carboxy terminus of SEQ ID NO: 1944, or to the reference sequence corresponding to SEQ ID NO: 1944.

34. The engineered terminal nucleotidyl transferase of any one of Claims 1, 3, and 7, wherein the amino acid sequence of the engineered terminal nucleotidyl transferase comprisesDocket No. CX10-267WO3 (a) at least an amino acid residue difference at an amino acid positions(s) 196, 196 / 273, 196 / 273 / 325, 196 / 273 / 325 / 335, 196 / 273 / 325 / 335 / 367, 196 / 273 / 430, 196 / 335, 196 / 335 / 367, 196 / 335 / 367 / 430, 196 / 367 / 479, 196 / 430, 273, 273 / 274, 273 / 274 / 367 / 430, 273 / 325 / 335 / 367, 273 / 325 / 430, 273 / 335 / 430, 273 / 367, 273 / 430, 273 / 430 / 479, 274, 274 / 325 / 335 / 479, 274 / 430, 325 / 335, 325 / 335 / 417, 325 / 335 / 417 / 430 / 479, 325 / 367, 335, 335 / 367, 335 / 367 / 479, 335 / 430, 335 / 479, 367, 367 / 430, 367 / 479, 417, 417 / 430 / 479, 430, and 479, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12 to the carboxy terminus of SEQ ID NO: 1946, or to the reference sequence corresponding to SEQ ID NO: 1946; or (b) at least an amino acid residue difference(s) 196Q, 196Q / 273I / 325V, 196Q / 273I / 325V / 335V, 196Q / 273I / 430G, 196Q / 335V, 196Q / 335V / 367R / 430G, 196Q / 367R / 479M, 196Q / 430G, 196S / 273I, 196S / 273I / 325V / 335V / 367Q, 196S / 335V / 367R, 196S / 367R / 479M, 273I, 273I / 274R, 273I / 274R / 367R / 430G, 273I / 325V / 335V / 367Q, 273I / 325V / 430G, 273I / 335V / 430G, 273I / 367R, 273I / 430G, 273I / 430G / 479M, 274R, 274R / 325V / 335V / 479M, 274R / 430G, 325V / 335V, 325V / 335V / 417S, 325V / 335V / 417S / 430G / 479M, 325V / 367Q, 325V / 367R, 335V, 335V / 367Q, 335V / 367R / 479M, 335V / 430G, 335V / 479M, 367Q, 367Q / 479M, 367R, 367R / 430G, 417S, 417S / 430G / 479M, 430G, or 479M, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12 to the carboxy terminus of SEQ ID NO: 1946, or to the reference sequence corresponding to SEQ ID NO: 1946.

35. The engineered terminal nucleotidyl transferase of any one of Claims 1, 3, and 7, wherein the amino acid sequence of the engineered terminal nucleotidyl transferase comprises (a) at least an amino acid residue difference at an amino acid positions(s) 231, 231 / 273, 231 / 273 / 380 / 417 / 500, 231 / 273 / 506, 231 / 380 / 417 / 501, 231 / 500 / 501 / 506, or 231 / 501, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12 to the carboxy terminus of SEQ ID NO: 1984, or to the reference sequence corresponding to SEQ ID NO: 1984; or (b) at least an amino acid residue difference(s) 231N, 231N / 273K, 231N / 273K / 380K / 417S / 500N, 231N / 273K / 506K, 231N / 273M, 231N / 380K / 417S / 501K, 231N / 500N / 501K / 506K, or 231N / 501K, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12 to the carboxy terminus of SEQ ID NO: 1984, or to the reference sequence corresponding to SEQ ID NO: 1984; or (c) at least an amino acid residue difference at an amino acid positions(s) 187, 190, 259, 260, 263, 273, 296, 300, 314, 343, 361, 367, 376, 384, 389, 390, 393, 395, 416, 417, 428, or 446, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12 to the carboxy terminus of SEQ ID NO: 1984, or to the reference sequence corresponding to SEQ ID NO: 1984; or (d) at least an amino acid residue difference(s) 187L, 187T, 187V, 187Y, 190Q, 190V, 259F, 260K, 260S, 260T, 263R, 273M, 296L, 300A, 300K, 314E, 343M, 343V, 361I, 367E, 376E, 384S, 389V, 390S, 390T, 393T, 395F, 395S, 416G, 417A, 428L, 446D, 446G, 446H, 446S, or 446T, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residuesDocket No. CX10-267WO3 12 to the carboxy terminus of SEQ ID NO: 1984, or to the reference sequence corresponding to SEQ ID NO: 1984.

36. The engineered terminal nucleotidyl transferase of any one of Claims 1, 3, and 7, wherein the amino acid sequence of the engineered terminal nucleotidyl transferase comprises (a) at least an amino acid residue difference at an amino acid positions(s) 161, 162, 174, 201, 219, 223, 244, 246, 252, 254, 318, 433, 437, 503, or 525, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12 to the carboxy terminus of SEQ ID NO: 2064, or to the reference sequence corresponding to SEQ ID NO: 2064; or (b) at least an amino acid residue difference(s) 161L, 162P, 174W, 201Q, 219D, 223H, 244R, 246L, 252L, 254N, 254Q, 254R, 318K, 433A, 437H, 503V, or 525T, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12 to the carboxy terminus of SEQ ID NO: 2064, or to the reference sequence corresponding to SEQ ID NO: 2064.

37. The engineered terminal nucleotidyl transferase of any one of Claims 1, 3, and 7, wherein the amino acid sequence of the engineered terminal nucleotidyl transferase comprises (a) at least an amino acid residue difference at an amino acid positions(s) 174 / 199 / 389 / 390, 174 / 231, 174 / 231 / 260 / 273 / 389 / 390, 174 / 231 / 263 / 273 / 343, 174 / 231 / 263 / 389, 174 / 231 / 389 / 390 / 446, 174 / 231 / 390, 174 / 259 / 260 / 263 / 273 / 361 / 389 / 390 / 446, 174 / 259 / 273 / 343 / 361 / 389, 174 / 263 / 389 / 390, 174 / 273 / 343 / 389 / 390 / 446, 187 / 190 / 231 / 252 / 273 / 296 / 300 / 353 / 417, 187 / 190 / 252 / 273 / 300 / 417, 187 / 190 / 273 / 353, 187 / 231 / 252 / 273 / 296 / 300 / 353, 187 / 231 / 273 / 296 / 300 / 353, 187 / 231 / 296 / 300 / 353, 187 / 231 / 300 / 353, 187 / 273 / 353, 190 / 231 / 273, 196 / 199 / 231 / 263 / 273 / 389, 199 / 231 / 260 / 263 / 361 / 389 / 446, 231, 231 / 259 / 263 / 273, 231 / 273, 259 / 263 / 273 / 389 / 390, 260 / 273 / 446, 263 / 273 / 343 / 446, 273, 273 / 353, 273 / 389 / 446, or 273 / 390, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12 to the carboxy terminus of SEQ ID NO: 2104, or to the reference sequence corresponding to SEQ ID NO: 2104; or (b) at least an amino acid residue difference(s) 174L / 199V / 389V / 390T, 174L / 231N, 174L / 231N / 260K / 273K / 389V / 390T, 174L / 231N / 263R / 389V, 174L / 231N / 263T / 273K / 343V, 174L / 231N / 263T / 389V, 174L / 231N / 389V / 390T / 446S, 174L / 231N / 390T, 174L / 259F / 260K / 263R / 273K / 361I / 389V / 390T / 446S, 174L / 259F / 273K / 343V / 361I / 389V, 174L / 263R / 389V / 390T, 174L / 273K / 343V / 389V / 390T / 446S, 187V / 190Q / 252M / 273K / 300K / 417S, 187V / 190Q / 273K / 353V, 187V / 231N / 252E / 273K / 296L / 300K / 353R, 187V / 231N / 300K / 353V, 187V / 273K / 353V, 187Y / 190Q / 231N / 252M / 273K / 296L / 300K / 353V / 417S, 187Y / 231N / 273K / 296L / 300K / 353R, 187Y / 231N / 296L / 300K / 353R, 190Q / 231N / 273K, 196Q / 199V / 231N / 263T / 273K / 389V, 199V / 231N / 260K / 263T / 361I / 389V / 446S, 231N, 231N / 259F / 263T / 273K, 231N / 273K, 259F / 263R / 273K / 389V / 390T, 260K / 273K / 446S, 263T / 273K / 343V / 446S, 273K, 273K / 353V, 273K / 389V / 446S, or 273K / 390T, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12 to the carboxy terminus of SEQ ID NO: 2104, or to the reference sequence corresponding to SEQ ID NO: 2104.Docket No. CX10-267WO3 38. The engineered terminal nucleotidyl transferase of any one of Claims 1, 3, and 7, wherein the amino acid sequence of the engineered terminal nucleotidyl transferase comprises (a) at least an amino acid residue difference at an amino acid positions(s) 161 / 162 / 254 / 417, 161 / 162 / 318 / 446, 174 / 244 / 273 / 390 / 437 / 503, 174 / 244 / 437 / 525, 174 / 273 / 503, 201 / 376, 433, or 525, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12 to the carboxy terminus of SEQ ID NO: 2210, or to the reference sequence corresponding to SEQ ID NO: 2210; or (b) at least an amino acid residue difference(s) 161L / 162P / 254R / 417A, 161L / 162P / 318K / 446S, 174W / 244G / 437H / 525T, 174W / 244R / 273K / 390T / 437H / 503L, 174W / 273K / 503L, 201Q / 376E, 433P, or 525T, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12 to the carboxy terminus of SEQ ID NO: 2210, or to the reference sequence corresponding to SEQ ID NO: 2210; or (c) at least an amino acid residue difference at an amino acid positions(s) 303, 397, or 466, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12 to the carboxy terminus of SEQ ID NO: 2210, or to the reference sequence corresponding to SEQ ID NO: 2210; or (d) at least an amino acid residue difference(s) 303E, 397S, or 466I, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12 to the carboxy terminus of SEQ ID NO: 2210, or to the reference sequence corresponding to SEQ ID NO: 2210; or (e) at least an amino acid residue difference at an amino acid positions(s) 278, 290, 297, 303, 397, or 525, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12 to the carboxy terminus of SEQ ID NO: 2210, or to the reference sequence corresponding to SEQ ID NO: 2210; or (f) at least an amino acid residue difference 278S, 290L, 297L, 297Y, 303E, 397G, 397S, or 525G, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12 to the carboxy terminus of SEQ ID NO: 2210, or to the reference sequence corresponding to SEQ ID NO: 2210.

39. The engineered terminal nucleotidyl transferase of any one of Claims 1, 3, and 7, wherein the amino acid sequence of the engineered terminal nucleotidyl transferase comprises (a) at least an amino acid residue difference at an amino acid positions(s) 22, 22 / 162 / 201 / 244 / 254 / 318 / 376 / 417 / 433 / 437 / 525, 22 / 201 / 376, 22 / 244, 22 / 244 / 254, 22 / 244 / 433 / 525, 22 / 318 / 376 / 433 / 525, 22 / 318 / 376 / 446, 22 / 376 / 417 / 437 / 446 / 525, 161 / 162 / 201 / 244 / 318 / 376 / 417 / 437, 161 / 162 / 201 / 244 / 376, 162, 162 / 201 / 318 / 376 / 417 / 433 / 525, 162 / 201 / 318 / 376 / 433 / 437 / 446, 162 / 244 / 254 / 433, 162 / 244 / 318 / 376 / 417 / 433 / 437, 162 / 254, 162 / 254 / 318 / 376 / 417 / 433, 162 / 254 / 376 / 417 / 433, 162 / 376 / 525, 162 / 433, 201 / 244 / 318 / 376, 201 / 244 / 376 / 433 / 525, 201 / 318 / 376 / 446, 244 / 318 / 376 / 417, 244 / 318 / 376 / 417 / 433 / 437 / 525, 318 / 376, 318 / 376 / 433, 433, or 437, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12 to the carboxy terminus of SEQ ID NO: 2256, or to the reference sequence corresponding to SEQ ID NO: 2256; orDocket No. CX10-267WO3 (b) at least an amino acid residue difference(s) 22P, 22P / 162P / 201Q / 244G / 254R / 318K / 376E / 417A / 433P / 437H / 525T, 22P / 201Q / 376E, 22P / 244G, 22P / 244G / 254R, 22P / 244G / 433P / 525T, 22P / 318K / 376E / 433P / 525T, 22P / 318K / 376E / 446S, 22P / 376E / 417A / 437H / 446S / 525T, 161L / 162P / 201Q / 244G / 318K / 376E / 417A / 437H, 161L / 162P / 201Q / 244G / 376E, 162P, 162P / 201Q / 318K / 376E / 417A / 433P / 525T, 162P / 201Q / 318K / 376E / 433P / 437H / 446S, 162P / 244G / 254R / 433P, 162P / 244G / 318K / 376E / 417A / 433P / 437H, 162P / 254R, 162P / 254R / 318K / 376E / 417A / 433P, 162P / 254R / 376E / 417A / 433P, 162P / 376E / 525T, 162P / 433P, 201Q / 244G / 318K / 376E, 201Q / 244G / 376E / 433P / 525T, 201Q / 318K / 376E / 446S, 244G / 318K / 376E / 417A, 244G / 318K / 376E / 417A / 433P / 437H / 525T, 318K / 376E, 318K / 376E / 433P, 433P, or 437H, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12 to the carboxy terminus of SEQ ID NO: 2256, or to the reference sequence corresponding to SEQ ID NO: 2256.

40. The engineered terminal nucleotidyl transferase of any one of Claims 1, 3, and 7, wherein the amino acid sequence of the engineered terminal nucleotidyl transferase comprises (a) at least an amino acid residue difference at an amino acid positions(s) 22 / 27 / 437 / 466, 22 / 34 / 278 / 318 / 437 / 466, 22 / 466, 41 / 42 / 43 / 397, 43 / 244 / 397, 45 / 397, or 397, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12 to the carboxy terminus of SEQ ID NO: 2280, or to the reference sequence corresponding to SEQ ID NO: 2280; or (b) at least an amino acid residue difference(s) 22P / 27P / 437H / 466I, 22P / 34Y / 278S / 318K / 437H / 466I, 22P / 466I, 41T / 42T / 43- / 397S, 43T / 244G / 397S, 45T / 397S, or 397S, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12 to the carboxy terminus of SEQ ID NO: 2280, or to the reference sequence corresponding to SEQ ID NO: 2280; or (c) at least an amino acid residue difference at an amino acid positions(s) 173, 175, 176, 179, 182, 196, 197, 208, 263, 273, 274, 277, 308, 310, 319, 328, 341, 376, 388, 405, 408, 411, 462, 477, or 480, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12 to the carboxy terminus of SEQ ID NO: 2280, or to the reference sequence corresponding to SEQ ID NO: 2280; or (d) at least an amino acid residue difference(s) 173P, 175L, 175R, 176H, 179C, 179K, 179R, 182I, 196D, 197L, 208T, 263R, 263T, 273M, 274L, 277D, 308Y, 310S, 319A, 328A, 341T, 376T, 388V, 405L, 408G, 411T, 462Y, 477L, 477W, or 480K, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12 to the carboxy terminus of SEQ ID NO: 2280, or to the reference sequence corresponding to SEQ ID NO: 2280; or (e) at least an amino acid residue difference at an amino acid position 195, 203, 273, 274, 277, 278, 291, 299, 300, 324, or 477, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12 to the carboxy terminus of SEQ ID NO: 2280, or to the reference sequence corresponding to SEQ ID NO: 2280; or [[****Table 70.2]] (f) at least an amino acid residue difference 195T, 203Q, 273E, 273G, 273M, 273N, 273Q, 274L, 277D, 278D, 291L, 299G, 299H, 300S, 324T, or 477W, or combinations thereof, wherein the amino acidDocket No. CX10-267WO3 positions are relative to the reference sequence corresponding to amino acid residues 12 to the carboxy terminus of SEQ ID NO: 2280, or to the reference sequence corresponding to SEQ ID NO: 2280.

41. The engineered terminal nucleotidyl transferase of any one of Claims 1, 3, and 7, wherein the amino acid sequence of the engineered terminal nucleotidyl transferase comprises (a) at least an amino acid residue difference at an amino acid positions(s) 34 / 273 / 300 / 477 / 480, 34 / 396, or 300 / 396 / 477 / 480, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12 to the carboxy terminus of SEQ ID NO: 2342, or to the reference sequence corresponding to SEQ ID NO: 234; or (b) at least an amino acid residue difference(s) 34D / 273M / 300S / 477L / 480K, 34D / 396T, or 300S / 396T / 477L / 480K, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12 to the carboxy terminus of SEQ ID NO: 2342, or to the reference sequence corresponding to SEQ ID NO: 2342.

42. The engineered terminal nucleotidyl transferase of any one of Claims 1, 3, and 7, wherein the amino acid sequence of the engineered terminal nucleotidyl transferase comprises (a) at least an amino acid residue difference at an amino acid positions(s) 27 / 34 / 173 / 176 / 179 / 195 / 277, 27 / 173, 27 / 173 / 176, 27 / 173 / 176 / 179 / 273 / 277, 27 / 173 / 176 / 179 / 277, 27 / 173 / 179 / 273, 27 / 173 / 179 / 273 / 277, 27 / 173 / 179 / 277, 27 / 176 / 179 / 273, 27 / 179, 27 / 179 / 273 / 277, 27 / 179 / 277, 34, 34 / 173 / 176 / 197 / 277, 105 / 173 / 176 / 197 / 277, 173 / 176 / 179, 173 / 176 / 179 / 197, 173 / 176 / 179 / 277, 173 / 179, 173 / 179 / 273, 173 / 179 / 273 / 277, 173 / 277, 176 / 179, 176 / 179 / 197, 176 / 179 / 273, 176 / 179 / 273 / 277, 179, 179 / 197, 179 / 273, or 179 / 273 / 277, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12 to the carboxy terminus of SEQ ID NO: 2438, or to the reference sequence corresponding to SEQ ID NO: 2438; or (b) at least an amino acid residue difference(s) 27P / 34D / 173P / 176H / 179K / 195T / 277D, 27P / 173P, 27P / 173P / 176H, 27P / 173P / 176H / 179K / 273M / 277D, 27P / 173P / 176H / 179R / 277D, 27P / 173P / 179K / 273M, 27P / 173P / 179K / 273M / 277D, 27P / 173P / 179K / 277D, 27P / 176H / 179R / 273M, 27P / 179K, 27P / 179K / 273M / 277D, 27P / 179R / 277D, 34D, 34D / 173P / 176H / 197L / 277D, 105G / 173P / 176H / 197L / 277D, 173P / 176H / 179K / 277D, 173P / 176H / 179R, 173P / 176H / 179R / 197L, 173P / 179K / 273M / 277D, 173P / 179R, 173P / 179R / 273M, 173P / 179R / 273M / 277D, 173P / 277D, 176H / 179K / 273M, 176H / 179K / 273M / 277D, 176H / 179R, 176H / 179R / 197L, 176H / 179R / 273M / 277D, 179K, 179K / 197L, 179K / 273M, 179K / 273M / 277D, 179R, or 179R / 197L, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12 to the carboxy terminus of SEQ ID NO: 2438, or to the reference sequence corresponding to SEQ ID NO: 2438; or (c) at least an amino acid residue difference at an amino acid positions(s) 177, 199, 202, 229, 292, 339, 342, 352, 363, 376, 406, 407, 430, 487, 508, or 525, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12 to the carboxy terminus of SEQ ID NO: 2438, or to the reference sequence corresponding to SEQ ID NO: 2438; or (d) at least an amino acid residue difference(s) 177C, 199E, 202V, 229L, 292A, 339T, 342T, 352A, 352T, 363G, 376H, 406D, 406P, 407N, 430G, 430S, 487R, 508C, or 525T, or combinations thereof, whereinDocket No. CX10-267WO3 the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12 to the carboxy terminus of SEQ ID NO: 2438, or to the reference sequence corresponding to SEQ ID NO: 2438.

43. The engineered terminal nucleotidyl transferase of any one of Claims 1, 3, and 7, wherein the amino acid sequence of the engineered terminal nucleotidyl transferase comprises (a) at least an amino acid residue difference at an amino acid positions(s) 176 / 183, 176 / 183 / 205, 183, 183 / 205, 183 / 205 / 408, or 205, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12 to the carboxy terminus of SEQ ID NO: 2452, or to the reference sequence corresponding to SEQ ID NO: 2452; or (b) at least an amino acid residue difference(s) 176N / 183R, 176N / 183R / 205L, 183G, 183G / 205L, 183R, 183R / 205L, 183R / 205L / 408V, or 205L, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12 to the carboxy terminus of SEQ ID NO: 2452, or to the reference sequence corresponding to SEQ ID NO: 2452.

44. The engineered terminal nucleotidyl transferase of any one of Claims 1, 3, and 7, wherein the amino acid sequence of the engineered terminal nucleotidyl transferase comprises (a) at least an amino acid residue difference at an amino acid positions(s) 177, 177 / 292 / 339 / 342 / 406, 177 / 292 / 339 / 406, 177 / 292 / 342 / 363 / 376 / 406, 177 / 292 / 342 / 406, 177 / 339 / 342, 177 / 339 / 342 / 376, 177 / 339 / 342 / 406, 177 / 339 / 342 / 406 / 487, 177 / 342 / 376 / 407, 177 / 342 / 406 / 430, 177 / 407, 177 / 487, 292 / 339 / 342 / 376 / 406 / 487, 292 / 342, 292 / 342 / 406, 292 / 406, 339 / 342, 339 / 342 / 407, 339 / 342 / 407 / 487, or 406, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12 to the carboxy terminus of SEQ ID NO: 2562, or to the reference sequence corresponding to SEQ ID NO: 2562; or (b) at least an amino acid residue difference(s) 177K, 177K / 292A / 339T / 342T / 406D, 177K / 292A / 339T / 406D, 177K / 292A / 339T / 406P, 177K / 292A / 342T / 363G / 376H / 406D, 177K / 292A / 342T / 406P, 177K / 339T / 342T, 177K / 339T / 342T / 376H, 177K / 339T / 342T / 406D, 177K / 339T / 342T / 406P / 487R, 177K / 342T / 376H / 407N, 177K / 342T / 406P / 430G, 177K / 407N, 177K / 487R, 292A / 339T / 342T / 376H / 406P / 487R, 292A / 342T, 292A / 342T / 406D, 292A / 406P, 339T / 342T, 339T / 342T / 407N, 339T / 342T / 407N / 487R, or 406P, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12 to the carboxy terminus of SEQ ID NO: 2562, or to the reference sequence corresponding to SEQ ID NO: 2562; or (c) at least an amino acid residue difference at an amino acid positions(s) 174, 187, 190, 198, 267, 274, 277, 307, 316, 343, 359, 379, 402 / 407, 407, 409, 412, 414, 481, 494, or 523, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12 to the carboxy terminus of SEQ ID NO: 2562, or to the reference sequence corresponding to SEQ ID NO: 2562; or (d) at least an amino acid residue difference(s) 174I, 187T, 190G, 198P, 198S, 198T, 267G, 274V, 277S, 307K, 316L, 343R, 359F, 379M, 402P / 407A, 407S, 409S, 412P, 414Q, 481H, 494T, 494Y, 523A, or 523H, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12 to the carboxy terminus of SEQ ID NO: 2562, or to the reference sequence corresponding to SEQ ID NO: 2562.Docket No. CX10-267WO3 45. The engineered terminal nucleotidyl transferase of any one of Claims 1, 3, and 7, wherein the amino acid sequence of the engineered terminal nucleotidyl transferase comprises (a) at least an amino acid residue difference at an amino acid positions(s) 202, 208, 221, 224, 235, 236, 238, 239, 242, 249, 251, 252, 259, 263, 405 / 406, 406, 477, 484 / 487, or 487, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12 to the carboxy terminus of SEQ ID NO: 2576, or to the reference sequence corresponding to SEQ ID NO: 2576; or (b) at least an amino acid residue difference(s) 202M, 208M, 221I, 224V, 235R, 236R, 238I, 238L, 239G, 239L, 239R, 239S, 242L, 249L, 249Q, 251A, 252R, 259F, 263R, 405G / 406G, 405V / 406G, 406E, 406M, 406T, 477I, 484L / 487K, 484Y / 487K, or 487K, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12 to the carboxy terminus of SEQ ID NO: 2576, or to the reference sequence corresponding to SEQ ID NO: 2576; or (c) at least an amino acid residue difference at an amino acid positions(s) 174 / 190, 174 / 190 / 202 / 204 / 254, 174 / 190 / 202 / 254, 174 / 190 / 204, 174 / 190 / 236, 174 / 202 / 204 / 254, 174 / 202 / 204 / 254 / 353, 174 / 204 / 236 / 254 / 353, 187, 187 / 224, 187 / 224 / 321 / 436, 187 / 224 / 379 / 436, 187 / 249, 187 / 249 / 321 / 379 / 436, 204 / 254 / 353, 249 / 379, 359 / 407, 359 / 407 / 412, 379, or 436, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12 to the carboxy terminus of SEQ ID NO: 2576, or to the reference sequence corresponding to SEQ ID NO: 2576; or (d) at least an amino acid residue difference(s) 174I / 190G, 174I / 190G / 202H / 204M / 254I, 174I / 190G / 202H / 254I, 174I / 190G / 204M, 174I / 190G / 236R, 174I / 202H / 204M / 254I, 174I / 202H / 204M / 254I / 353V, 174I / 204M / 236R / 254I / 353V, 187T, 187T / 224V, 187T / 224V / 321R / 436S, 187T / 224V / 379M / 436S, 187T / 249M, 187T / 249M / 321R / 379M / 436S, 204V / 254I / 353V, 249M / 379M, 359F / 407A, 359F / 407S / 412P, 379M, or 436S, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12 to the carboxy terminus of SEQ ID NO: 2576, or to the reference sequence corresponding to SEQ ID NO: 2576; or (e) at least an amino acid residue difference at an amino acid positions(s) 219, 293, 300, 310, 313, 318, 340, 364, or 392, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12 to the carboxy terminus of SEQ ID NO: 2576, or to the reference sequence corresponding to SEQ ID NO: 2576; or (f) at least an amino acid residue difference(s) 219Q, 293R, 300R, 310D, 313Q, 318P, 340L, 340T, 364S, 392Q, or 392S, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12 to the carboxy terminus of SEQ ID NO: 2576, or to the reference sequence corresponding to SEQ ID NO: 2576; or (g) at least an amino acid residue difference at an amino acid positions(s) 160, 161, 173, 175, 222, 244, 278, 293, 300, 301, 310, 318, 319, 324, 334, 339, 341, 342, 366, 367, 368, 370, 376, or 378, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12 to the carboxy terminus of SEQ ID NO: 2576, or to the reference sequence corresponding to SEQ ID NO: 2576; or (h) at least an amino acid residue difference(s) 160A, 160D, 160T, 160W, 161A, 161L, 173H, 175H,Docket No. CX10-267WO3 175P, 222N, 244I, 244S, 278A, 278L, 293R, 300R, 301F, 310D, 318P, 318R, 318S, 319P, 324A, 334S, 339A, 339S, 341L, 342S, 366F, 366H, 366P, 367V, 368R, 370L, 370R, 376A, 376G, 376R, 376S, 376V, or 378M, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12 to the carboxy terminus of SEQ ID NO: 2576, or to the reference sequence corresponding to SEQ ID NO: 2576.

46. The engineered terminal nucleotidyl transferase of any one of Claims 1, 3, and 7, wherein the amino acid sequence of the engineered terminal nucleotidyl transferase comprises (a) at least an amino acid residue difference at an amino acid positions(s) 187, 187 / 224, 187 / 224 / 263, 187 / 224 / 407, 187 / 224 / 407 / 412, 187 / 224 / 412, 187 / 254 / 263 / 359 / 379 / 407, 187 / 263, 187 / 359, 187 / 379, 224 / 359 / 407, 249 / 263, 249 / 263 / 407, 263, or 263 / 407 / 412, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12 to the carboxy terminus of SEQ ID NO: 2714, or to the reference sequence corresponding to SEQ ID NO: 2714: or (b) at least an amino acid residue difference(s) 187T, 187T / 224V, 187T / 224V / 263R, 187T / 224V / 407A, 187T / 224V / 407A / 412P, 187T / 224V / 412P, 187T / 254R / 263R / 359F / 379M / 407S, 187T / 263R, 187T / 359F, 187T / 379M, 224V / 359F / 407A, 249M / 263R, 249M / 263R / 407S, 263R, or 263R / 407S / 412P, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12 to the carboxy terminus of SEQ ID NO: 2714, or to the reference sequence corresponding to SEQ ID NO: 2714.

47. The engineered terminal nucleotidyl transferase of any one of Claims 1, 3, and 7, wherein the amino acid sequence of the engineered terminal nucleotidyl transferase comprises (a) at least an amino acid residue difference at an amino acid positions(s) 160 / 263 / 318 / 339, 175 / 244 / 293 / 364 / 407, 219 / 310, 219 / 310 / 340, 244 / 293 / 313, 244 / 293 / 334, 244 / 313, 244 / 313 / 334 / 364 / 407, 263 / 310 / 318 / 366, 263 / 318, 293 / 313 / 334 / 364, 293 / 334 / 364 / 407, 310 / 318 / 340, 318, or 334 / 364, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12 to the carboxy terminus of SEQ ID NO: 2876, or to the reference sequence corresponding to SEQ ID NO: 2876; or (b) at least an amino acid residue difference(s) 60T / 263R / 318P / 339S, 175H / 244I / 293R / 364S / 407P, 219S / 310D, 219S / 310D / 340T, 244I / 293R / 313Q, 244I / 313Q, 244I / 313Q / 334S / 364S / 407S, 244S / 293R / 334S, 263R / 310D / 318S / 366H, 263R / 318P, 293R / 313Q / 334S / 364S, 293R / 334S / 364S / 407P, 310D / 318P / 340T, 318P, or 334S / 364S, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12 to the carboxy terminus of SEQ ID NO: 2876, or to the reference sequence corresponding to SEQ ID NO: 2876.

48. The engineered terminal nucleotidyl transferase of any one of Claims 1, 3, and 7, wherein the amino acid sequence of the engineered terminal nucleotidyl transferase comprises (a) at least an amino acid residue difference at an amino acid positions(s) 254 / 293 / 310, 254 / 293 / 310 / 334 / 340 / 407, 310 / 334, 334, 334 / 340, 334 / 407, 340, or 407, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12 to the carboxy terminus of SEQ ID NO: 2884, or to the reference sequence corresponding to SEQ ID NO: 2884; orDocket No. CX10-267WO3 (b) at least an amino acid residue difference(s) 254R / 293R / 310D, 254R / 293R / 310D / 334S / 340T / 407P, 310D / 334S, 334S, 334S / 340T, 334S / 407P, 340T, or 407P, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12 to the carboxy terminus of SEQ ID NO: 2884, or to the reference sequence corresponding to SEQ ID NO: 2884; or (c) at least an amino acid residue difference at an amino acid positions(s) 183, 190, 203, 232, 252, 259 / 263, 261 / 263, 397, 408, or 413, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12 to the carboxy terminus of SEQ ID NO: 2884, or to the reference sequence corresponding to SEQ ID NO: 2884; or (d) at least an amino acid residue difference(s) 183V, 190N, 203L, 232G, 252H, 259F / 263S, 261S / 263S, 397Q, 408D, or 413T, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12 to the carboxy terminus of SEQ ID NO: 2884, or to the reference sequence corresponding to SEQ ID NO: 2884; or at least an amino acid residue difference at an amino acid positions(s) 179, 200, 201, 208, 218, 223, 227, 229, 230, 235, 297, or 408, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12 to the carboxy terminus of SEQ ID NO: 2884, or to the reference sequence corresponding to SEQ ID NO: 2884; or (e) at least an amino acid residue difference 179V, 200Q, 201R, 208G, 218I, 223L, 227G, 227S, 229G, 229S, 230V, 235N, 297Q, or 408D, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12 to the carboxy terminus of SEQ ID NO: 2884, or to the reference sequence corresponding to SEQ ID NO: 2884.

49. The engineered terminal nucleotidyl transferase of any one of Claims 1, 3, and 7, wherein the amino acid sequence of the engineered terminal nucleotidyl transferase comprises (a) at least an amino acid residue difference at an amino acid positions(s) 307 / 429 / 474, or 508, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 2910; or (b) at least an amino acid residue difference(s) 307K / 429A / 474L, or 508Q, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12 to the carboxy terminus of SEQ ID NO: 2910, or to the reference sequence corresponding to SEQ ID NO: 2910.

50. The engineered terminal nucleotidyl transferase of any one of Claims 1, 3, and 7, wherein the amino acid sequence of the engineered terminal nucleotidyl transferase comprises (a) at least an amino acid residue difference at an amino acid positions(s) 190, 190 / 203 / 205 / 508, 205 / 208, 205 / 223, 205 / 230, 205 / 239 / 403 / 508, 205 / 297 / 508, 205 / 370 / 403, 205 / 403, 205 / 413, 205 / 508, or 218 / 319 / 508, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12 to the carboxy terminus of SEQ ID NO: 2974, or to the reference sequence corresponding to SEQ ID NO: 2974; or (b) at least an amino acid residue difference(s) 190N, 190N / 203F / 205R / 508K, 205R / 208G, 205R / 223L, 205R / 230V, 205R / 239S / 403F / 508K, 205R / 297Q / 508K, 205R / 370R / 403F, 205R / 403F,Docket No. CX10-267WO3 205R / 413T, 205R / 508K, or 218I / 319M / 508K, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12 to the carboxy terminus of SEQ ID NO: 2974, or to the reference sequence corresponding to SEQ ID NO: 2974.

51. The engineered terminal nucleotidyl transferase of any one of Claims 1, 3, and 7, wherein the amino acid sequence of the engineered terminal nucleotidyl transferase comprises (a) at least an amino residue difference at an amino acid position(s) 230 / 251 / 352 / 357, 238 / 297 / 403, 238 / 297 / 403 / 466, 297, or 403 / 466, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12 to the carboxy terminus of SEQ ID NO: 2998, or to the Ference sequence corresponding to SEQ ID NO: 2998; or (b) at least an amino acid residue difference(s) 230I / 251A / 352T / 357I, 238L / 297V / 403F, 238L / 297V / 403F / 466L, 297V, or 403F / 466L, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12 to the carboxy terminus of SEQ ID NO: 2998, or to the reference sequence corresponding to SEQ ID NO: 2998.

52. The engineered terminal nucleotidyl transferase of Claim 1, comprising an amino acid sequence comprising amino acid residues 12 to the carboxy terminus of an even-numbered SEQ ID NO. of SEQ ID NOs: 4-1056, 1130-1880, and 1928-3008, or an amino acid sequence comprising an even-numbered SEQ ID NO. of SEQ ID NOs: 4-1056, 1130-1880, and 1928-3008.

53. The engineered terminal nucleotidyl transferase of any of Claims 1-52, wherein the polypeptide sequence further comprises an N-terminal truncation of 1-156 amino acids.

54. The engineered terminal nucleotidyl transferase of any of Claims 1-53, wherein the engineered terminal nucleotidyl transferase polypeptide is fused with a second polypeptide; optionally, wherein the second polypeptide has inorganic pyrophosphatase (IPP) activity.

55. The engineered terminal nucleotidyl transferase of any of Claims 1-54, wherein said engineered terminal nucleotidyl transferase is capable of template-independent synthesis.

56. The engineered terminal nucleotidyl transferase of any of Claims 1-55, having at least one improved property, as compared to a wild-type or reference terminal nucleotidyl transferase or template- independent polymerase.

57. The engineered terminal nucleotidyl transferase of Claim 56, wherein said improved property is selected from increased thermostability, increased activity at elevated temperatures, increased soluble expression or isolated protein yield, decreased by-product formation, increased specific activity on one or more NTP-3’-O-RBG or natural or modified NTP substrates, increased incorporation efficiency in extension of oligo acceptor substrates, increased activity on one or more oligo acceptor substrates, and increased activity on oligo acceptor substrates of a length of three to seven nucleotides.

58. The engineered terminal nucleotidyl transferase of Claim 56, wherein said engineered terminal nucleotidyl transferase comprises increased soluble expression or isolated protein yield as compared to a wild-type or reference terminal nucleotidyl transferase or template-independent polymerase.Docket No. CX10-267WO3 59. The engineered terminal nucleotidyl transferase of Claim 56, wherein said engineered terminal nucleotidyl transferase comprises increased thermal stability as compared to a wild-type or reference terminal nucleotidyl transferase or template-independent polymerase.

60. The engineered terminal nucleotidyl transferase of Claim 59, wherein said engineered terminal nucleotidyl transferase comprises increased activity after incubation at a temperature of 40 °C, 45 °C, 50 °C, 55 °C, 60 °C, 65 °C, 68 °C, 68.5 °C, 69 °C, 70 °C, or 72 °C as compared to a wild-type or reference terminal nucleotidyl transferase.

61. The engineered terminal nucleotidyl transferase of Claim 56, wherein said engineered terminal nucleotidyl transferase comprises increased activity on one or more NTP-3’-O-RBG or natural or modified NTP substrates or increased activity on one or more oligo acceptor substrates, or both, as compared to a wild-type or reference terminal nucleotidyl transferase or template-independent polymerase.

62. The engineered terminal nucleotidyl transferase of Claim 61, wherein said engineered terminal nucleotidyl transferase comprises conversion of at least one oligo acceptor substrate of 4 to 7 basepairs to the oligo acceptor substrate extended by one nucleotide with at least 1.3 fold, 1.4 fold, or 1.5 fold the activity relative to SEQ ID NO: 148 or 256.

63. The engineered terminal nucleotidyl transferase of Claim 62, wherein said engineered terminal nucleotidyl transferase comprises an amino acid sequence with at least 80% sequence identity to SEQ ID NO:

682.

64. The engineered terminal nucleotidyl transferase of Claim 61, wherein said engineered terminal nucleotidyl transferase has activity in the conversion of a variety of oligo acceptor substrate of 4 to 7 basepairs to the oligo acceptor substrate extended by one nucleotide.

65. The engineered terminal nucleotidyl transferase of Claim 64, wherein said engineered terminal nucleotidyl transferase comprises an amino acid sequence with at least 80% sequence identity to SEQ ID NO: 148, 256, or 682.

66. The engineered terminal nucleotidyl transferase of Claim 61, wherein said engineered terminal nucleotidyl transferase comprises at least 31% or 91% conversion an oligo acceptor substrate of 3 to 4 basepairs to the oligo acceptor substrate extended by one nucleotide.

67. The engineered terminal nucleotidyl transferase of Claim 66, wherein said engineered terminal nucleotidyl transferase comprises an amino acid sequence with at least 80% sequence identity to SEQ ID NO:

932.

68. The engineered terminal nucleotidyl transferase of Claim 61, wherein said engineered terminal nucleotidyl transferase comprises at least 75% conversion of an oligo acceptor substrate to the oligo acceptor substrate extended by a glycol nucleic acid nucleotide ((S)-GNA) or a locked nucleic acid nucleotide (LNA).Docket No. CX10-267WO3 69. The engineered terminal nucleotidyl transferase of Claim 68, wherein said engineered terminal nucleotidyl transferase comprises an amino acid sequence with at least 80% sequence identity to SEQ ID NO:

992.

70. The engineered terminal nucleotidyl transferase of Claim 61, wherein said engineered terminal nucleotidyl transferase comprises increased substrate promiscuity for NTP-3’-O-RBG substrates of 2'-methoxyadenosine, 2'-methoxycytidine, 2'-methoxyguanosine, 2'-methoxyuridine, 2'-fluoroadenosine, 2'- fluorocytidine, 2'-fluoroguanosine, 2'-fluorouridine, adenosine ribonucleotide, cytidine ribonucleotide, guanosine ribonucleotide, uridine ribonucleotide, phosphorothioate linkage, 5'-phosphorylation modification, adenosine (S)-glycol nucleic acid, cytidine (S)-glycol nucleic acid, guanosine (S)-glycol nucleic acid, thymidine (S)-glycol nucleic acid, uridine (S)-glycol nucleic acid, C2'-endo locked adenosine ribonucleotide, C2'-endo locked cytidine ribonucleotide, C2'-endo locked guanosine ribonucleotide, C2'-endo locked thymidine ribonucleotide, C2'-endo locked uridine ribonucleotide, 2'-O-(2-methoxyethyl)adenosine, 2'-O-(2- methoxyethyl)cytidine, 2'-O-(2-methoxyethyl)guanosine, 2'-O-(2-methoxyethyl)uridine, 2',3'- dideoxyadenosine, 2',3'-dideoxycytidine, 2',3'-dideoxyguanosine, 2',3'-dideoxythymidine, 2',3'-dideoxyuridine vinylphosphonate 2'O-methoxy uridine, 1-((2S))-1-(2,3-dihydroxypropyl))thymine, and 2'O-hexadecyl cytidine, as compared to a wild-type or engineered reference TnT.

71. The engineered terminal nucleotidyl transferase of Claim 70, wherein said engineered terminal nucleotidyl transferase comprises an amino acid sequence selected from the even-numbered sequences in SEQ ID NO: 4-1056, 1130-1880, and 1928-3008.

72. The engineered terminal nucleotidyl transferase of Claim 70, wherein said engineered terminal nucleotidyl transferase comprises an amino acid sequence with at least 80% sequence identity to SEQ ID NO:

992.

73. The engineered terminal nucleotidyl transferase of Claim 61, wherein said engineered terminal nucleotidyl transferase comprises increased substrate promiscuity for oligo acceptor substrates of 5'- 6-FAM-T10mCmCmUfA, 5'-6-FAM-T31mUfCmAfU, 5'-6-FAM-T41mAfUfCfC, 5'-6-FAM- T51mU*fA*fAfG, 5'-6-FAM-T16mC*mA*mGmA, 5'-6-FAM-T31mAmAmAfG, 5'-6-FAM- T36mC*mU*mAmC, 5'-6-FAM-T46mAfGmUfG, 5'-6-FAM-T26mUfGmUfC, 5'-6-FAM-T36fCmAmUmC, 5'-6-FAM-T11mU*fA*fA, 5'-6-FAM-T17*fA*fAfG, 5'-6-FAM-T27fGmAfU, 5'-6-FAM-T41*fC*mAfA, 5'- 6-FAM-T21mGfUfAfC, 5'-6-FAM-T36fCmAfUfC, 5'-6-FAM-T46fAmUmUfG, 5'-6-FAM- T56mA*fC*mAfA, 5'-6-FAM-T26mCfCmCfG, 5'-6-FAM-T36mCfCmGfG, 5'-6-FAM-T41mGfUmGfG, 5'- 6-FAM-T16mGmUmC*mC, 5'-6-FAM-T51fGmUfGmU, 5'-6-FAM-T11AmC*mA*mG, 5'-6-FAM- T15mAmUmCmU, 5'-6-FAM-T46mUmCmUmU, 5'-6-FAM-T41mAmUmCmU, T8mUfGmUfC, T8fCmAmUmC, T8mCfCmCfG, T8mCfCmGfG, mAmAmAmAmUmCmU, mAmAmAmUmCmU, mAmAmUmCmU, mAmUmCmU, mUmCmU, 5'P-mUmCmU, mC*mA*mGmA, mAfGmUfG, mGmUmC*mC, 5'-6-FAM-T9mAmAmAmUmCmU, 5'-6-FAM-T9mAmAmAmUmCmU(sgna)A, 5'-6-FAM- T9mAmAmAmUmCmU+A, 5'-6-FAM-T9mAmAmAmUmCmU+G, 5'-6-FAM-T9mAmAmAmUmCmU+C, 5'-6-FAM-T9mAmAmAmUmCmU+U, 5'-6-FAM-T9mAmAmAmUmCmU+A+A, 5'-6-FAM- T9mAmAmAmUmCmU+G+G, 5'-6-FAM-T9mAmAmAmUmCmU+U+U, 5'-6-FAM-T16mAfGmAfG, 5'-6-Docket No. CX10-267WO3 FAM-T26fCfAfUmG, 5'-6-FAM-T10AmC*mA*mG, 5'-6-FAM-T16*fC*mAfAfA, 5'-6-FAM- T16*fA*mAmGfAfG, 5'-6-FAM-T11fUfUfUfC, 5'-6-FAM-T21mUmAmG*mA, 5'-6-FAM- T20mAmUfUmGfG, 5'-6-FAM-T25mAmGfUfGfG, 5'-6-FAM-T20mAmGfAmGfG, 5'-6-FAM- T16*fU*fUfAfU, 5'-6-FAM-T31mUfCmAmU, 5'-6-FAM-T35fAfGfUmGfG, 5'-6-FAM-T51mAmUmCfC, 5'-6-FAM-T56fGmUfCmU, T8AmC*mA*mG, T8mCfCmGfG, T8*fC*mAfAfA, T8*fA*mAmGfAfG, T8mGfUfAfC, T8fUfUfUfC, T8mAfUfCfC, T8mAmUfUmGfG, T8mAmGfUfGfG, T8mAmGfAmGfG, T8*fU*fUfAfU, T8mGfUmGfG, T8fAmUmUfG, T8mU*fA*fAfG, T8fAfGfUmGfG, T8fGmUfCmU, and T8mA*fC*mAfA, as compared to a wild-type or engineered reference terminal nucleotidyl transferase.

74. The engineered terminal nucleotidyl transferase of any of Claims 1-73, wherein said terminal nucleotidyl transferase is purified.

75. The engineered terminal nucleotidyl transferase of any of Claims 1-74, wherein said terminal nucleotidyl transferase is immobilized.

76. A polynucleotide comprising a polynucleotide sequence encoding at least one engineered terminal nucleotidyl transferase of any of Claims 1-73.

77. The polynucleotide of Claim 76, comprising a polynucleotide sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference polynucleotide sequence corresponding to nucleotide residue 34 to the 3’-terminus of SEQ ID NO: 1, 7, 69, 147, 183, 233, 255, 477, 667, 681, 729, 733, 741, 931, 991, 1143, 1211, 1235, 1263, 1413, 1503, 1521, 1701, 1741, 1927, 1943, 1945, 1983, 2063, 2103, 2209, 2255, 2279, 2341, 2437, 2451, 2561, 2575, 2713, 2875, 2883, 2909, 2973, 2997, or 3001, or to a reference polynucleotide sequence comprising SEQ ID NO: 1, 7, 69, 147, 183, 233, 255, 477, 667, 681, 729, 733, 741, 931, 991, 1143, 1211, 1235, 1263, 1413, 1503, 1521, 1701, 1741, 1927, 1943, 1945, 1983, 2063, 2103, 2209, 2255, 2279, 2341, 2437, 2451, 2561, 2575, 2713, 2875, 2883, 2909, 2973, 2997, or 3001, wherein the polynucleotide sequence encodes a terminal nucleotidyl transferase.

78. The polynucleotide of Claim 76, comprising a polynucleotide sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference polynucleotide sequence corresponding to nucleotide residues 34 to 3’-terminus of an odd numbered SEQ ID NO. of SEQ ID NOs: 3-1055, 1129-1879, and 1927-3007, or to the reference polynucleotide sequence corresponding to an odd numbered SEQ ID NO. of SEQ ID NOs: 3-1055, 1129-1879, and 1927-3007.

79. The polynucleotide of any one of Claims 76-78, wherein the polynucleotide sequence is codon-optimized.

80. The polynucleotide of Claim 76, comprising a polynucleotide sequence comprising an odd numbered SEQ ID NO. of SEQ ID NOs: 3-1055, 1129-1879, and 1927-3007.

81. An expression vector comprising at least one polynucleotide of any one of Claims 76-80.Docket No. CX10-267WO3 82. The polynucleotide sequence of Claims 81, wherein said polynucleotide sequence is operably linked to a control sequence.

83. A host cell comprising at least one expression vector of any one of Claims 76-82.

84. A method of producing an engineered terminal nucleotidyl transferase polypeptide in a host cell comprising culturing a host cell of Claim 83, under suitable culture conditions, such that at least one engineered terminal nucleotidyl transferase is produced.

85. The method of Claim 84, further comprising recovering at least one engineered terminal nucleotidyl transferase from the culture and / or host cells.

86. The method of Claim 84 or 85, further comprising the step of purifying said at least one engineered terminal nucleotidyl transferase.

87. A composition comprising at least one engineered terminal nucleotidyl transferase of any of Claims 1-75.

88. A method of template independent synthesis of an oligonucleotide, comprising contacting an oligonucleotide acceptor substrate with a terminal nucleotidyl transferase of any one of Claims 1-75 in presence of one or more nucleotide triphosphates under reaction conditions sufficient for addition of the nucleotide to the 3’-end of the oligonucleotide acceptor substrate.

89. The method of Claim 88, wherein the nucleotide triphosphate comprises a modified nucleotide triphosphate.

90. The method of Claim 89, wherein the modified nucleotide triphosphate comprises a modified sugar moiety, modified nucleobase, and / or modified phosphate group.

91. The method of Claim 90, wherein the modified nucleotide triphosphate comprises a 3’- reversible blocking group (RGB).

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