Methods and apparatus for enzymatic synthesis of polynucleotides

Abstract

The present invention relates to a method and apparatus for parallel enzymatic synthesis of polynucleotides in an array of reaction chambers using a template-free polymerase with sequence-dependent coupling efficiency. Whenever a sequence causing low-efficiency coupling appears at the 3' end of the growing chain of the synthesized polynucleotide, one or more additional coupling cycles without a deprotection step are inserted into the synthesis schedule to provide additional time to complete the coupling reaction at that position of the polynucleotide.

Description

[0001] There has been a recent increase in interest in enzymatic methods for polynucleotide synthesis, not only because of the increased demand for synthetic polynucleotides in many fields, such as synthetic biology, CRISPR-Cas9 applications, and high-throughput sequencing, but also because of the limitations of chemical methods for polynucleotide synthesis, such as the upper limit on product length and the use and need for disposal of organic solvents, Jensen et al., Biochemistry, 57: 1821-1832 (2018). Enzymatic synthesis is attractive for its specificity and efficiency as well as for its use of mild aqueous reaction conditions that eliminate the need to handle hazardous waste.

[0002] At present, most enzymatic methods use template-free polymerases to repeatedly add 3'-O-blocked nucleoside triphosphates to a single-chain initiator or extended chain connected to a support, and then deblock until a polynucleotide of the desired sequence is obtained. Unfortunately, however, template-free polymerases usually have sequence-specific low efficiency in their coupling yields. That is, under a given set of reaction conditions and time, the sequence at the 3' end of the growing chain where nucleotide coupling occurs may affect the coupling efficiency. Whenever the same reaction time is used for all reaction chambers, this makes it difficult or impossible to obtain consistent product yields in parallel synthesis operations.

[0003] In view of the foregoing, parallel synthesis of polynucleotides using template-free polymerases would be advanced if methods and apparatus were available that could minimize variability in product yields due to sequence-specific coupling inefficiencies. SUMMARY OF THE INVENTION

[0005] The present invention relates to methods and devices for the parallel synthesis of multiple polynucleotides in separate reaction chambers, including microfluidic devices.

[0006] More particularly, the present invention relates to a method for synthesizing a plurality of polynucleotides each having a predetermined sequence using a template-free polymerase, wherein the template-free polymerase has a reduced coupling efficiency at one or more low-efficiency motifs, the method comprising the following steps: (a) providing a reaction chamber for each polynucleotide of the plurality of nucleotides, each reaction chamber having a synthesis support disposed therein with an initiator attached thereto, wherein each initiator has a free 3'-hydroxyl group, and wherein each reaction chamber has an inlet and an outlet and a filter, wherein the filter retains the synthesis support and is operatively associated with the outlet such that a reaction solution exiting the reaction chamber passes through the filter; (b) providing a waste manifold operatively associated with the outlet of the reaction chamber such that whenever a positive pressure differential is generated between the reaction chamber and the waste manifold, the reaction solution is removed from the reaction chamber; and (c) rehydrating each reaction chamber. The invention relates to a method for preparing a reaction chamber comprising: (i) contacting the initiator or the deprotected extension fragment with a 3'-protected nucleoside triphosphate and a template-free polymerase in a coupling solution so that the initiator or the deprotected extension fragment is extended by the 3'-protected nucleoside triphosphate to form a 3'-protected extension fragment, (ii) deprotecting the 3'-protected extension fragment using a deprotection solution, and (iii) applying a pressure differential between the reaction chamber and the waste manifold to remove solution from the reaction chamber; wherein the type of the 3'-protected nucleoside triphosphate contacted in step (i) in the reaction chamber is determined by the sequence of the polynucleotide in the reaction chamber, and wherein, whenever a low-efficiency motif is present at the 3' end of the deprotected extension fragment in such reaction chamber, one or more short cycles of step (i) are performed in the reaction chamber before each cycle. As explained more fully below, the efficiency motif is a sequence segment at the 3' end of the extended segment that causes the template-free polymerase to have a reduced efficiency in coupling nucleotide monomers to the extended segment. In some embodiments, the template-free polymerase includes terminal deoxynucleotidyl transferase (TdT). In some embodiments of TdT disclosed herein, the low efficiency motif includes a 3-mer sequence selected from a set including CCA, CTA, GCA, GTA and CCT. In other embodiments, such a set includes CCA and CTA. In other embodiments, the low efficiency motif of the template-free polymerase is identified, for example, as described below, so that the method and apparatus of the present invention can be implemented.

[0007] The present invention also relates to an apparatus for synthesizing a plurality of polynucleotides each having a predetermined sequence using a template-free polymerase, wherein the template-free polymerase has a reduced coupling efficiency at one or more low-efficiency motifs, the apparatus comprising: (a) a plurality of reaction chambers, each reaction chamber having a synthesis support with an initiator attached thereto disposed therein, wherein each initiator has a free 3'-hydroxyl group, and wherein each reaction chamber has an inlet and an outlet and a filter, wherein the filter retains the synthesis support and is operatively associated with the outlet so that a reaction solution leaving the reaction chamber passes through the filter; (b) a waste manifold operatively associated with the outlet of the reaction chamber so that whenever a positive pressure differential is established between the reaction chamber and the waste manifold, the reaction solution is removed from the reaction chamber and enters the waste manifold; (c) a fluid delivery system for delivering the reaction solution to the reaction chambers of the array; (d) a user interface for receiving nucleotide sequences of the polynucleotides to be synthesized and low-efficiency motif data; and (e) a control system operatively associated with the user interface, the plurality of reaction chambers, the fluid delivery system, and the waste manifold, wherein the control The system determines the number and location of each low-efficiency motif for each polynucleotide and distributes each polynucleotide to a reaction chamber for synthesis, and wherein for each reaction chamber, until its polynucleotide is completed, the control system directs the following steps to be repeated: (i) delivering a coupling solution to the initiator oligonucleotide or the deprotected extension segment under coupling conditions, the coupling solution containing a 3'-protected nucleoside triphosphate and a template-free polymerase to allow the initiator oligonucleotide or the deprotected extension segment to be extended by the 3'-protected nucleoside triphosphate to form a 3'-protected extension segment; , (ii) delivering a deprotection solution to the reaction chamber to deprotect the 3'-protected extension fragment, and (iii) generating a pressure differential between the reaction chamber and the waste manifold to remove the coupling and / or deprotection solution from the reaction chamber; wherein the type of 3'-protected nucleoside triphosphate contacted in step (i) in the reaction chamber is determined by a predetermined sequence of the reaction chamber, and wherein, whenever an inefficient motif is present at the 3' end of the deprotected extension fragment of such reaction chamber, one or more short cycles of step (i) are performed in the reaction chamber before each cycle.

[0008] As above, in some embodiments of the apparatus of the present invention, the template-free polymerase comprises terminal deoxynucleotidyl transferase (TdT). In other embodiments employing TdT disclosed herein, the low efficiency motif comprises a 3-mer sequence selected from a set comprising CCA, CTA, GCA, GTA, and CCT. In other embodiments, such a set comprises CCA and CTA.

[0009] The device of the present invention is particularly suitable for implementing a method for synthesizing a plurality of polynucleotides each having a predetermined sequence using a template-free polymerase, wherein the template-free polymerase has a reduced coupling efficiency at one or more low-efficiency motifs of the present invention. The present invention also relates to a kit for synthesizing a plurality of polynucleotides each having a predetermined sequence using a template-free polymerase, comprising the device of the present invention and 3'-protected nucleoside triphosphates and / or a template-free polymerase and / or a coupling solution and / or a deprotection solution.

[0010] These above-characterized aspects and other aspects of the present invention are exemplified in a number of illustrated embodiments and applications, some of which are shown in the accompanying drawings and characterized in the claims section that follows. However, the above summary is not intended to describe each illustrated embodiment or every implementation of the present invention.

[0011] BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 The basic steps for enzymatic synthesis of polynucleotides are shown.

[0013] Figure 2A An array of reaction wells is shown, for example, each reaction well having an outlet for removing reaction fluid, and each being addressable for the purpose of delivering reagents.

[0014] Figure 2B It shows that it can be Figure 2A A given selection of polynucleotides synthesized in parallel in the reaction wells of , shows the occurrence of inefficient motifs for each polynucleotide, causing difficult coupling for a selected template-free polymerase, such as the TdT variant M27.

[0015] Figure 2C Insertion of a "short" synthesis cycle after the indicated inefficient motifs is shown, which effectively provides additional time for coupling after such inefficient motifs.

[0016] Figure 3A and 3B Data are shown for the relative coupling efficiency for different TdT variants at the 3'-3-mer sequence.

[0017] Figure 4 The apparatus of the invention is shown. DETAILED DESCRIPTION OF THE INVENTION

[0019] The general principles of the present invention are disclosed in more detail herein, particularly by way of example, as shown in the accompanying drawings and described in detail. However, it should be understood that it is not intended to limit the present invention to the specific embodiments described. The present invention is applicable to various modifications and alternative forms, the details of which are shown for several embodiments. It is intended to cover all modifications, equivalents and substitutes that fall within the principles and scope of the present invention.

[0020] Unless otherwise indicated, the practice of the present invention may employ conventional techniques and descriptions of organic chemistry, molecular biology (including recombinant techniques), cell biology, and biochemistry, which are within the technical scope of the art. Such conventional techniques may include, but are not limited to, the preparation and use of synthetic peptides, synthetic polynucleotides, monoclonal antibodies, nucleic acid cloning, amplification, sequencing, and analysis, and related techniques. The schemes of such conventional techniques can be found in product information and standard laboratory manuals from manufacturers, such as Genome Analysis: A Laboratory Manual Series (Volume I-Volume IV); PCR Primer: A Laboratory Manual; and Molecular Cloning: A Laboratory Manual (all from Cold Spring Harbor Laboratory Press); Lutz and Bornscheuer, ed., Protein Engineering Handbook (Wiley-VCH, 2009); Hermanson, Bioconjugate Techniques, Second Edition (Academic Press, 2008); and similar references.

[0021] The present invention relates to methods and apparatus for the parallel enzymatic synthesis of polynucleotides in an array of reaction chambers using a template-free polymerase with sequence-dependent coupling efficiency. Each reaction chamber of the array has an inlet and an outlet, and associated with each outlet is a filter for retaining a solid support, such as beads, in the reaction chamber whenever a solution is removed through the outlet. In some embodiments, the array of reaction chambers is a planar array of microwells operatively associated with a waste manifold that allows simultaneous removal of reaction solutions (e.g., coupling solutions and / or deprotection solutions) from the microwells by applying a pressure differential between the microwells and the manifold.

[0022] A given template-free polymerase has a sequence fragment of a polynucleotide at a reduced coupling efficiency referred to herein as a "low efficiency motif" of the polymerase. Whenever a low efficiency motif appears at the 3' end of a growing chain (or an extended fragment), a reduced coupling efficiency will occur. Such reduced coupling efficiency can be confirmed by an increase in the number of nucleotide deletions next to the 3' end of the low efficiency motif in the completed product compared to the completed product that does not contain the low efficiency motif at the same position when the coupling reaction time is the same for all reactions. Figure 3A-3BThe phenomenon for two terminal deoxynucleotidyl transferase (TdT) variants is shown in . The degree of coupling inefficiency of the template-free polymerase can vary widely, so that the implementation of the present invention depends on this degree and the purity and yield of the desired final product. In some embodiments, the inefficient motif is identified as a 3'-terminal sequence that reduces the coupling efficiency by 10% or more compared to the average coupling efficiency of all 3'-terminal sequences of the same length. In other embodiments, the inefficient motif is identified as a 3'-terminal sequence that reduces the coupling efficiency by 25% or more compared to the average coupling efficiency of all 3'-terminal sequences of the same length. In other embodiments, the inefficient motif is identified as a 3'-terminal sequence that reduces the coupling efficiency by 50% or more compared to the average coupling efficiency of all 3'-terminal sequences of the same length.

[0023] According to the methods and apparatus of the present invention, the inefficiency motif characteristic of the template-free polymerase is located in each of the plurality of polynucleotides to be synthesized. This information is then used to insert so-called "short" coupling cycles into the synthesis plan for each polynucleotide in order to provide additional time for reactions occurring at the inefficiency motifs without affecting the coupling times of reactions not involving the inefficiency motifs. As described more fully below, conventional cycles (e.g., Figure 1 The short cycle may include multiple steps, but the key feature is that it includes a coupling step without any deblocking step, thereby allowing further accumulation of couplings with free 3'-OH groups in the reaction chamber in which the short cycle is implemented.

[0024] The apparatus of the present invention includes a fluid delivery system for delivering reaction solutions to reaction chambers under program control, so that appropriate reaction solutions (e.g., "A", "C", "G" or "T" coupling reagents or solutions, washing solutions, deprotection or deblocking solutions, etc.) are delivered to appropriate reaction chambers at appropriate times to produce polynucleotides of desired sequences. After a plurality of polynucleotide sequences are obtained by, for example, inputting a plurality of polynucleotide sequences through a user interface, a reaction chamber is assigned to each polynucleotide, and the sequence of conventional and short cycles is determined for each polynucleotide. The determination includes analyzing the polynucleotide sequence to identify the number and position of inefficient motifs, and (therefore) the position of inserting one or more short cycles for the synthesis plan of the polynucleotide. The term "synthesis plan" with respect to a polynucleotide means the sequence of conventional and short cycles in a method or apparatus for synthesizing such a polynucleotide. The synthesis plan of a hypothetical polynucleotide (SEQ ID NO: 1) is shown in Figure 2C middle.

[0025] Both conventional and short cycles have the same number of steps with the same duration. After each step in either type of cycle, the reaction solution is removed from all reaction chambers via their outlets. In some embodiments, conventional and short cycles differ only in the activities that occur in the reaction chambers between the reaction solution removals. According to the method and apparatus of the present invention, the reaction solution is removed from the reaction chambers by creating a pressure differential between the reaction chambers and a waste manifold that is operationally associated with the outlet of the reaction chamber array. In some embodiments, this pressure differential is created by connecting the waste manifold to a vacuum source, so that the vacuum pulls the liquid out of the chambers through their filters and outlets and into the chambers of the waste manifold, and ultimately reaches a waste container.

[0026] Typically, the low efficiency motif consists of 1-5 nucleotides, and more typically, the low efficiency motif consists of 3 nucleotides. As explained more fully below, and as Figure 3A and 3B As shown, the inefficiency motifs for a given template-free polymerase can be readily determined by sequencing a sample of polynucleotide products synthesized using such a polymerase in a standard reaction and tabulating the deletions adjacent to subsequences (e.g., 3-mers) in the products. The program also provides a measure of the coupling efficiency at each possible sequence segment of a given length at the 3' end of the growing chain. Once this information is obtained, it is a design choice of one of ordinary skill in the art to select one or more inefficiency motifs (corresponding to inefficient couplings) to implement the methods and apparatus of the present invention; that is, it is a design choice of one of ordinary skill in the art to determine how much reduction in coupling efficiency can be tolerated before improving coupling efficiency by inserting one or more short cycles to provide additional coupling time. As Figure 3A-3B As shown, in some embodiments, for a given template-free polymerase, there may be one or more inefficient motifs. In other embodiments, the number of different inefficient motifs for a given template-free polymerase may range from 1 to 6. In other embodiments, the number of different inefficient motifs for a given template-free polymerase may be multiple; and in some embodiments, the multiple ranges may be 2 to 10; or 2 to 6; or 2 to 4. Although not wishing to be bound by theory or hypothesis, with respect to terminal deoxynucleotidyl transferase (TdT) or related template-free polymerases, it is believed that in some embodiments, the coupling efficiency at certain sequences, especially 3-mer sequences, is proportional to the stability of the connection between the polymerase and the 3' end of the growing chain. That is, the inefficient coupling may be due to the lack of stability of the TdT growing chain complex, so there are fewer such complexes available for capturing monomers to couple with the growing chain.

[0027] Aspects of the present invention are Figure 2A-2C As shown in Figure 2AAs shown, more than one polynucleotide is synthesized in the planar array (200) of reaction chambers (202), which can be labeled as S1 to S2 as shown. n In some embodiments, each polynucleotide is synthesized in a separate reaction chamber. Figure 2B As shown, after user input, the sequence of each polynucleotide is analyzed to determine the identity and position of the inefficient motif (204) (CCA, CTA and GCA in this example) and their nucleotides immediately 3'. This determines the number and position of the synthesis plan for inserting short cycles into each polynucleotide. In some embodiments, one or more short cycles are inserted after each inefficient motif (i.e., 3'). In some embodiments, the number or type of short cycles inserted may depend on the inefficient motif. In some embodiments, a single short cycle is inserted after each inefficient motif. As described above, in some embodiments, both conventional and short cycles include three steps of equal duration, and after each step (or as part of each step), a pressure difference is generated between the reaction chamber and the waste manifold to remove the reaction solution (if any) from the reaction chamber. In some embodiments, this pressure difference is generated by connecting the waste manifold to a conventional vacuum source. A vacuum is typically applied for 10-30 seconds between these steps. A conventional cycle can be represented as follows:

[0028] Steps of a regular cycle

[0029]

[0030] As shown above, the coupling reaction solution (RS) is retained in the reaction chamber, and the deblocking solution is simply added thereto in step (ii). In some embodiments, a washing step to remove the reaction solution may be performed after the coupling step and before the deprotection step. As described above, the short cycle may have various forms shown in the following table (which are not intended to be exhaustive).

[0031] Steps of short cycle (Form 1)

[0032]

[0033] Steps of short cycle (Form 2)

[0034]

[0035] Steps of short cycle (Form 3)

[0036]

[0037] Steps of short cycle (Form 4)

[0038]

[0039] In certain embodiments, further steps may be performed in each cycle or periodically during the synthesis process. For example, from time to time, if enzyme accumulation occurs, for example by adhesion of the template-free polymerase to surfaces such as reaction chamber walls, filter surfaces or pores, resulting in blockages that inhibit fluid transfer, a step of protease treatment may be employed. The reaction times or durations t1, t2 and t3 may vary widely depending on the specific embodiment, wherein, for example, different template-free polymerases, reaction temperatures, reaction buffers, monomers, deprotection solutions, etc. are employed.

[0040] For the hypothetical 21-mer sequence (220) (SEQ ID NO: 1), Figure 2C The above process of inserting short cycles is shown in . Analysis of 21-mer (220) identified two inefficient motifs: CTA (222) and GCA (224). The synthesis plan of the polynucleotide is shown in the following row, where the conventional cycle is represented by "(N)", where N = A, C, G or T, the short cycle is represented by "(n)", where n = a, c, g or t, and the letter represents the type of nucleotide added in the cycle. Due to the inefficient motif, two short cycles (226 and 228) were added in this example, each cycle immediately following the inefficient motif or the 3' insertion of the inefficient motif. The choice of whether to insert one, two, three or more short cycles and the form of the short cycles are design choices of ordinary technicians in the art, which depends at least in part on the specific template-free polymerase used, and in particular on the magnitude of the reduction in coupling efficiency after the inefficient motif. Other factors may include the concentration or activity of the polymerase used.

[0041] A variety of equipment may be constructed or adapted to perform the steps of the method of the invention. Extensive guidance for this purpose is available in the literature on automated chemical synthesis and analysis, for example Miertus et al., eds., Combinatorial Chemistry and Technologies: Methods and Applications, 2nd ed. (CRC Press, 2005); West et al., U.S. Pat. No. 9,103,809; Butendeich et al., J. Laboratory Automation, 18(3):245-250 (2013); Fluent Automated Workstations (Tecan Group); Tisone et al., U.S. Pat. No. 6,063,339; Cathcart et al., U.S. Pat. No. 5,443,791; Ingenhoven et al., U.S. Pat. No. 7,529,598; Glauser et al., U.S. Pat. No. 8,580,197; Sindalar et al., Nucleic Acids Research, 23(6):982-987 (1995); Cheng et al., Nucleic Acids Research, 30(18):e93(2002); Skold et al., U.S. Pat. No. 5,273,718, etc. In some embodiments, the apparatus of the present invention may partially include a conventional fluid delivery robot. In other embodiments, the apparatus of the present invention may partially include an inkjet fluid delivery system.

[0042] In some embodiments, the apparatus of the present invention includes the following elements: (a) a plurality of reaction chambers, each reaction chamber having an inlet and an outlet, wherein the outlet is operatively associated with a filter membrane or layer, the filter membrane or layer allowing a solid support to remain in the reaction chamber when a reaction solution is removed through the outlet, (b) a waste manifold connectable to the plurality of reaction chambers such that when connected, the reaction solution can be removed from all of the reaction chambers whenever a pressure differential is generated between the reaction chambers and the waste manifold, (c) a fluid delivery system for delivering one or more reaction solutions to the plurality of reaction chambers under program control, (d) a user interface for receiving nucleotide sequence and inefficiency motif data of a polynucleotide to be synthesized, and (e) a control system operatively associated with the user interface, the array of reaction chambers, the fluid delivery system, and the waste manifold, for (i) analyzing the input nucleotide sequence and inefficiency motif data and determining a synthesis plan, and assigning a reaction chamber to each polynucleotide, and (ii) for controlling The fluid delivery system, waste manifold and array are designed so that for each reaction chamber, the following steps are repeated until a predetermined sequence thereof is completed: (A) delivering a coupling solution to the initiator oligonucleotide or the deprotected extension fragment under coupling conditions, wherein the coupling solution contains a 3'-protected nucleoside triphosphate and a template-free polymerase to allow the initiator oligonucleotide or the deprotected extension fragment to be extended by the 3'-protected nucleoside triphosphate to form a 3'-protected extension fragment, (B) delivering a deprotection solution to the reaction chamber to deprotect the 3'-protected extension fragment, and (C) generating a pressure differential between the reaction chamber and the waste manifold to remove the deprotection solution from the reaction chamber; wherein the type of 3'-protected nucleoside triphosphate contacted in the reaction chamber in step (A) is determined by the predetermined sequence of the reaction chamber, and wherein one or more short cycles of step (A) are performed in the reaction chamber before each cycle whenever a low efficiency motif is present at the 3' end of the deprotected extension fragment of such reaction chamber.

[0043] In some embodiments, after completing the synthesis of the polynucleotide, further steps may be performed to cut the completed polynucleotide from the solid support and purify them for application. These further steps may be performed in the reaction chamber of the array, or the polynucleotide still connected to the solid support may be transferred to other reaction vessels to perform these further steps. In addition, some cutting methods may result in the product of release, which still needs modification to be converted into an available product. For example, in "endonuclease V-inosine", cutting (as described below) leaves 5'-phosphate, which must be removed for some applications. Therefore, further phosphatase treatment steps may be required.

[0044] Figure 4Elements of one embodiment of the apparatus of the present invention are schematically shown. These elements include: (a) a plurality of reaction chambers (402) in an array (400); (b) a waste manifold (404) operatively associated with the reaction chambers (402) of the array (400); (c) a fluid delivery system (406) for delivering reaction solutions to the reaction chambers (402) under program control; (d) a user interface (408) for inputting nucleotide sequences and inefficient motif data of polynucleotides; and (e) a control system (410) that (i) receives nucleotide sequences and inefficient motif data from the user interface (408), (ii) calculates a synthesis plan for each polynucleotide, (iii) distributes polynucleotides to the reaction chambers (402) for synthesis, and (iv) controls the delivery of reaction solutions to and removal from individual reaction chambers (402) by sending commands to controllers of the fluid delivery system (406), the array (400), and the waste manifold (404). The array (400) of reaction chambers (402) can be a conventional filter plate of 24 wells, 96 wells, 384 wells, 1536 wells or similar forms, such as available from commercial manufacturers such as Pall Corp., Port Washington, NY. The present invention can be used with typical reaction volumes of such filter plates, such as 10-50 μL for a 96 well plate, 3-10 μL for a 384 well plate, and 0.5-3.0 μL for a 1536 well plate. The waste manifold (404) can include a chamber (412) in a body (414) that is sealingly clamped to the array (400) so that when the chamber (412) is connected to a vacuum source (416), a vacuum is transmitted to the outlets of all individual reaction chambers (402) in the array (400), and the waste reaction solution is transferred to a waste container (418). The array (400) can be moved with the waste manifold (404) or separately. For example, the fluid delivery to the reaction chamber can be performed by making the array (400) stationary and the head (420) movable in the x, y and z directions relative to the surface of the array (400), or the array (400) can also be movable in the x and y directions, or the two elements can be movable relative to each other in the x and y directions. The head (420) can be a frame on which a pipette, nozzle or jet port with valves and actuators is installed. The frame can be mounted on a mechanical arm, a gantry rail or other mechanical parts that allow it to be programmably translated on the array (400) to deliver the reaction solution. Fluid delivery can be completed by a variety of methods. In some embodiments, the required synthesis scale is an important factor. For a small amount of a large number of polynucleotides, fluid delivery can be completed by an inkjet method or an inkjet method and an overall flow, such as a combination of overall flows for washing or some deprotection steps.For example, the range is 10 to 2000 or 10 to 1000 or 10 to 384 of a medium number of polynucleotides, fluid delivery can be completed by a liquid transfer volume of 0.5 μ L to 50 μ L or more, and a conventional automated laboratory system (as mentioned above) can be used. In some embodiments, the fluid delivery system (406) includes one or more reagent reservoirs (e.g., 422a-422g) and a removable head (420). In some embodiments, the fluid delivery system can also include a sub-element or even a separate head for aspirating a reaction solution from a reaction chamber (402). For example, in some embodiments, it may be advantageous to transfer the completed polynucleotides connected to a solid support to a separate reaction chamber for cutting and purification. This can be achieved by sucking out such a reaction solution from each reaction chamber where synthesis occurs and depositing the solution in a separate reaction chamber of a separate array. In other embodiments, the polynucleotides completed from the solid support can be cut in a reaction chamber where a cutting solution (422a) is used for synthesis.

[0045] As described, the control system (410) sends signals (411) to the controller, which actuates elements and sub-elements of the fluid delivery system (406), array (400), waste manifold (404), and vacuum source (416) to execute the synthesis plan determined by the control system (410). In some embodiments, the control system (410) can be a general purpose programmable computer running conventional instrument control software (e.g., LabVIEW, National Instruments, Austin, TX).

[0046] Identifying inefficient motifs for template-free polymerases

[0047] The relative efficiency of the nucleotide incorporation of the template-free polymerase as a function of the 3' end sequence of the growing chain can be easily determined by sequencing the samples of the random sequence products synthesized in the standard reaction. Then, according to how many deletions occur in the 3' position next to each other, the 2-mer, 3-mer, 4-mer, etc. of the resulting sequence are tabulated. The 2-mer, 3-mer or 4-mer sequence with a higher number of deletions is a candidate's low efficiency motif. As an exemplary test, 24 52-mer random sequence polynucleotides can be synthesized by template-free polymerase, such as TdT variants (for example, in duplicate or triplicate). 24 52-mers can be selected as follows: each possible 4-mer is represented by approximately its expected frequency, i.e., 1 / 256, or approximately 4 or 5 occurrences in a total of (24x52 effective sequence lengths). (That is, random sequences with a 4-mer tilted representation, such as, for example, those random sequences that just lack many 4-mers are rejected).

[0048] Exemplary synthesis conditions of TdT. In this example, the dNTP of 3'-O blocking is 3'-O-NH2-dNTP, but similar reaction conditions are also applicable to other reversible protecting groups. In TY1x buffer 0.01% Tween 20, prepare respectively the premix containing twice concentrated dNTP-ONH2 (1mM), DMSO (20%) and cofactor (4mM CoCl2) and twice concentrated mutant (32μM). For M59 contrast M27 reaction, add 2x Tween 20 in nucleotide premix. Synthesize 24 sequences (52mer) in duplicate at 37 ℃ with M27 and M59 or M57. The extension time is set to 4min. Synthesize using 500pmol of dI resin (R133) and a filter plate (Pall Laboratory, New York) with wwPTFE membrane. TY1x buffer contains potassium cacodylate 200 mM pH 6.8, MgCl2 5 mM. dI resin R133 is a CNBr agarose resin (GE Healthcare) coupled to 10T-dI-T initial DNA (initiator DNA) via the amino function, and the linker is C12. The complete initiator sequence attached to the resin is 5AmMC12 / TTTTTTTTTT / dI / T.

[0049] Post-synthesis processing. After synthesis, the first wash (1x 100 μL / well H2O, 3x100 μL / well SDS / DTT, 3x100 μL / well H2O) was performed. Immediately after removing the remaining liquid by vacuuming and centrifuging the plate at 2000g for 2min, 3x 50 μL washes were performed with TH1 buffer 1x or WLE1 buffer 1x. The cutting of the synthetic chain was removed directly from the resin afterwards. For dI cutting, the resin was incubated at 42°C, 1250rpm for 30min at 50 μL reaction volume (10mM Tris, pH7.9 containing 173mM NaCl and 50mM MgCl2 or 10mM Tris, pH7.9 containing 50mM KCl and 15mM MgCl2) with 1 μM EndoV, and then centrifuged at 2000g for 2min. The resin was washed with 25 μL of TH1 buffer 1x or WLE1 buffer 1x, incubated at 42°C, 1250 rpm for another 2 min, and centrifuged again at 2000 g for 2 min. If necessary, the recovered filtrate was transferred to a 96-well PCR plate and stored at -20°C. Finally, less than 75 μL was collected from each well. TH1 buffer contained 10 mM Tris, 170 mM NaCl, 50 MgCl2 pH 8. WLE1 buffer contained 10 mM Tris pH 7.9, 50 mM KCl, and 15 mM MgCl2.

[0050] DNA precipitation. Add 0.5 μL glycogen and NaAc (3M) pH 5.2 (7.5 μL) to each well and shake briefly. Add 75 μL isopropanol to each well and centrifuge at 3428g for 1h15 at 4°C. Aspirate the supernatant and only 20 μL is left in each well. Add 180 μL freshly prepared 80% ethanol to each well and centrifuge at 3428g for 30min at 4°C. Remove the supernatant and repeat the wash. After centrifugation, only 10 μL is left in each well and dry in a Speedvac at 35°C for 30min. Resuspend the dried DNA in 15 μL water MB grade and measure the concentration.

[0051] Library preparation: The library was prepared using the Accel-NGS 1S Plus DNA Library Kit (ozymeSW10096) from Illumina 1 96rxns. The library was indexed using the 1S Plus Indexing Kit (ozyme SW16024) from Illumina. The resulting sequence data can be displayed in tabular form or graphically, as shown in Figure 1. Figure 3A-3B As shown, to quickly identify low-efficiency motifs.

[0052] Template-free enzymatic synthesis of oligonucleotides

[0053] Typically, methods for template-free (or equivalently, "template-independent") enzymatic DNA synthesis involve repeated cycles of steps, such as Figure 1 As shown (for so-called "conventional" cycles), in each cycle, a predetermined nucleotide is coupled to an initiator or a growing chain. The general elements of template-free enzymatic synthesis are described in the following references: Ybert et al., International Patent Publication WO / 2015 / 159023; Ybert et al., International Patent Publication WO / 2017 / 216472; Hyman, U.S. Patent 5436143; Hiatt et al., U.S. Patent 5763594; Jensen et al., Biochemistry, 57: 1821-1832 (2018); Mathews et al., Organic & Biomolecular Chemistry, DOI: 0.1039 / c6ob01371f (2016); Schmitz et al., Organic Lett., 1(11): 1729-1731 (1999).

[0054] For example, an initiator polynucleotide (100) having a free 3'-hydroxyl group (130) connected to a solid support (120) is provided. The solid support can be a planar solid surface, a bead, such as a magnetic bead, an agarose bead, etc. Under conditions (140) that effectively enzymatically incorporate a 3'-O protected dNTP into the 3' end of the initiator polynucleotide (100) (or the extended initiator polynucleotide), a 3'-O protected dNTP and a template-free polymerase, such as TdT or a variant thereof (e.g., Ybert et al., WO / 2017 / 216472; Champion et al., WO2019 / 135007) are added to the initiator polynucleotide (100) (or the extended initiator polynucleotide in subsequent cycles). The reaction produces an extended initiator polynucleotide whose 3'-hydroxyl group is protected (160). If the extended sequence is not completed, another addition cycle (180) is implemented. If the extended initiator polynucleotide contains a competing sequence, the 3'-O protecting group can be removed or deprotected, and the desired sequence can be cleaved from the original initiator polynucleotide (182). Such cleavage can be performed using any of a variety of single-strand cleavage techniques, for example, by inserting a cleavable nucleotide at a predetermined position within the original initiator polynucleotide. An exemplary cleavable nucleotide can be a uracil nucleotide that is cleaved by uracil DNA glycosylase. If the extended initiator polynucleotide does not contain the complete sequence, the 3'-O protecting group is removed to expose the free 3'-hydroxyl (130), and the extended initiator polynucleotide is subjected to another cycle of nucleotide addition and deprotection.

[0055] As used herein, "initiator" (or equivalent terms, such as "initiator fragment", "initiator nucleic acid", "initiator oligonucleotide", etc.) generally refers to a short oligonucleotide sequence with a free 3'-hydroxyl group at its end, which can be further extended by a template-free polymerase, such as TdT. In one embodiment, the initiator fragment is a DNA initiator fragment. In an alternative embodiment, the initiator fragment is an RNA initiator fragment. In some embodiments, the initiator fragment has 3-100 nucleotides, particularly 3-20 nucleotides. In some embodiments, the initiator fragment is single-stranded. In an alternative embodiment, the initiator fragment can be double-stranded. In some embodiments, the initiator oligonucleotide can be connected to a synthetic support through its 5' end; and in other embodiments, the initiator oligonucleotide can be indirectly connected to the synthetic support by forming a duplex with a complementary oligonucleotide directly connected to the synthetic support, for example, by a covalent bond. In some embodiments, the synthetic support is a solid support, which can be a discrete area of ​​a solid planar solid, or can be a bead.

[0056] In some embodiments, the initiator can include a non-nucleic acid compound having a free hydroxyl group to which TdT can couple a 3'-O protected dNTP, such as Baiga, U.S. Patent Publications US2019 / 0078065 and US2019 / 0078126.

[0057] After synthesis is complete, the polynucleotide having the desired nucleotide sequence can be released from the initiator and solid support by cleavage.

[0058] A variety of cleavable linkages or cleavable nucleotides can be used for this purpose. In some embodiments, the cleavage of the desired polynucleotide leaves a natural free 5'-hydroxyl on the cleavage strand; however, in alternative embodiments, the cleavage step may leave a portion, such as a 5'-phosphate, which can be removed in a subsequent step, such as by phosphatase treatment. The cleavage step can be performed chemically, thermally, enzymatically, or photochemically. In some embodiments, the cleavable nucleotide can be a nucleotide analog, such as deoxyuridine or 8-oxo-deoxyguanosine, which is recognized by a specific glycosylase (e.g., uracil deoxyglycosylase followed by endonuclease VIII and 8-oxoguanine DNA glycosylase, respectively). In some embodiments, cleavage can be accomplished by providing an initiator having deoxyinosine as the penultimate 3' nucleotide, which can be cleaved by endonuclease V at the 3' end of the initiator, leaving a 5'-phosphate on the released polynucleotide. Other methods for cleaving single-stranded polynucleotides are disclosed in the following references, which are incorporated by reference: U.S. Patent Nos. 5,739,386, 5,700,642, and 5,830,655; and U.S. Patent Publication Nos. 2003 / 0186226 and 2004 / 0106728; and in U.S. Patent No. 5,367,066 to Urdea and Horn.

[0059] Back to Figure 1In some embodiments, in each synthesis step, in the presence of a 3'-O protected dNTP, a template-free polymerase such as TdT is used to couple the nucleotides of the ordered sequence to the initiator nucleic acid. In some embodiments, the method of synthesizing an oligonucleotide comprises the following steps: (a) providing an initiator having a free 3'-hydroxyl group; (b) reacting the initiator or an extension intermediate having a free 3'-hydroxyl group with a template-free polymerase under extension conditions in the presence of a 3'-O protected nucleoside triphosphate to produce a 3'-O protected extension intermediate; (c) deprotecting the extension intermediate to produce an extension intermediate having a free 3'-hydroxyl group; and (d) repeating steps (b) and (c) until a polynucleotide is synthesized. (Sometimes the terms "extension intermediate" or "extension fragment" or "growing chain" are used interchangeably). In some embodiments, the initiator is provided as an oligonucleotide, for example, connected to a solid support by its 5' end. The above method may also include a washing step after the reaction or extension step and after the deprotection step. For example, the reaction step can include a substep of removing unincorporated nucleoside triphosphates, for example by washing, after a predetermined incubation period or reaction time. This predetermined incubation period or reaction time can be a few seconds, for example 30 seconds to a few minutes, for example 30 min.

[0060] When the polynucleotide sequence on the synthetic support includes the reverse complementary subsequence, secondary intramolecular or intermolecular structure can be produced by forming hydrogen bonds between the reverse complementary regions. In some embodiments, the base protection part of the exocyclic amine is selected so that the hydrogen of the protected nitrogen cannot participate in hydrogen bonding, thereby preventing the formation of this type of secondary structure. In other words, the base protection part can be adopted to prevent the formation of hydrogen bonds, such as the hydrogen bonds formed in normal base pairing, for example between nucleosides A and T and between G and C. When the synthesis is finished, the base protection part can be removed and the polynucleotide product can be cut off from the solid support, for example, by cutting it off from its initiator.

[0061] In addition to providing 3'-O blocked dNTP monomers with base protection groups, the extension reaction can also be carried out at a higher temperature using a thermostable template-free polymerase. For example, a thermostable template-free polymerase having activity above 40°C can be used; or, in some embodiments, a thermostable template-free polymerase having activity in the range of 40-85°C can be used; or, in some embodiments, a thermostable template-free polymerase having activity in the range of 40-65°C can be used.

[0062] In some embodiments, the extension condition may include adding a solvent that inhibits hydrogen bonding or base accumulation to the extension reaction mixture. Such solvents include water-miscible solvents with low dielectric constants, such as dimethyl sulfoxide (DMSO), methanol, etc. Similarly, in some embodiments, the extension condition may include providing a chaotropic agent, which includes but is not limited to n-butanol, ethanol, guanidine chloride, lithium perchlorate, lithium acetate, magnesium chloride, phenol, 2-propanol, sodium dodecyl sulfate, thiourea, urea, etc. In some embodiments, the extension condition includes the DMSO with a secondary structure inhibition amount. In some embodiments, the extension condition may include providing a DNA binding protein that inhibits the formation of a secondary structure, wherein such protein includes but is not limited to single-stranded binding protein, helicase, DNA glycolase, etc.

[0063] 3'-O blocked dNTPs without base protection can be purchased from commercial suppliers or synthesized using published techniques, such as U.S. Pat. No. 7,057,026; Guo et al., Proc. Natl. Acad. Sci., 105(27):9145-9150 (2008); Benner, U.S. Pat. Nos. 7,544,794 and 8,212,020; International Patent Publications WO 2004 / 005667, WO 91 / 06678; Canard et al., Gene (cited herein); Metzker et al., Nucleic Acids Research, 22:4259-4267 (1994); Meng et al., J. Org. Chem., 14:3248-3252 (3006); U.S. Pat. No. 2005 / 037991. 3'-O blocked dNTPs with base protection can be synthesized as follows.

[0064] When using base-protected dNTPs, Figure 1 The above process may further comprise the step (e) of removing the base protecting moiety, which in the case of an acyl or amidine protecting group may, for example, comprise treatment with concentrated aqueous ammonia.

[0065] The above method may also include a capping step and a washing step after the reaction or extension step and after the deprotection step. As mentioned above, in some embodiments, a capping step may be included, wherein the unextended free 3'-hydroxyl reacts with a compound that prevents any further extension of the capped chain. In some embodiments, such compounds may be dideoxynucleoside triphosphates. In other embodiments, the non-extended chains with free 3'-hydroxyls can be degraded by treating them with 3'-nuclease activity, such as Exo I. For example, see Hyman, U.S. Patent No. 5436143. Similarly, in some embodiments, the chains that failed to decap can be treated to remove the chains or make them inert to further extension.

[0066] In some embodiments, the reaction conditions of the extension step (also sometimes referred to as the extension step or coupling step) may include the following: 2.0 μM purified TdT; 125-600 μM 3'-O blocked dNTPs (e.g., 3'-O-NH2 blocked dNTPs); about 10 to about 500 mM potassium cacodylate buffer (pH 6.5 to 7.5) and about 0.01 to about 10 mM divalent cations (e.g., CoCl2 or MnCl2), wherein the extension reaction can be carried out in a 50 μL reaction volume at a temperature ranging from RT to 45°C for 3 minutes. In embodiments where the 3'-O blocked dNTP is a 3'-O-NH2 blocked dNTP, the reaction conditions of the deblocking step may include the following: 700 mM NaNO2; 1 M sodium acetate (with acetic acid to adjust the pH value to within the range of 4.8-6.5), wherein the deblocking reaction can be carried out in a 50 μL volume at a temperature ranging from RT to 45°C for 30 seconds to several minutes. Washing can be performed with cacodylate buffer that does not contain coupling reaction components (eg, enzymes, monomers, divalent cations).

[0067] Depending on the specific application, the step of deblocking and / or cutting can include a variety of chemical or physical conditions, such as light, heat, pH, the presence of specific reagents, such as enzymes capable of cutting specific chemical bonds. Guidance for selecting 3'-O blocking groups and corresponding deblocking conditions can be found in the following references, which are incorporated by reference: Benner, U.S. Patents 7544794 and 8212020; U.S. Patent 5808045; U.S. Patent 8808988; International Patent Publication WO91 / 06678; and references cited below. In some embodiments, the cutting agent (sometimes also referred to as a deblocking agent or reagent) is a chemical cutting agent, such as, for example, dithiothreitol (DTT). In alternative embodiments, the cutting agent can be an enzyme cutting agent, such as, for example, a phosphatase, which can cut a 3'-phosphate blocking group. It will be understood by those skilled in the art that the selection of the deblocking agent depends on the type of 3'-nucleotide blocking group used, whether one or more blocking groups are used, whether the initiator is connected to a living cell or organism or to a solid support, etc., which requires gentle handling. For example, phosphines such as tris(2-carboxyethyl)phosphine (TCEP) can be used to cleave the 3'O-azidomethyl group, palladium complexes can be used to cleave the 3'O-allyl group, or sodium nitrite can be used to cleave the 3'O-amino group. In certain embodiments, the cleavage reaction involves TCEP, palladium complexes, or sodium nitrite.

[0068] As mentioned above, in some embodiments, it is desirable to adopt two or more blocking groups, which can be removed using orthogonal blocking conditions. The following exemplary blocking groups are to be used in parallel synthesis embodiments. It should be understood that other blocking groups may be obtained and used in these embodiments of the present invention to or containing more than two groups.

[0069] 3'-O-NH2 3'-O-azidomethyl 3'-O-NH2 3'-O-allyl 3'-O-NH2 3'-O-Phosphate 3'-O-azidomethyl 3'-O-allyl 3'-O-azidomethyl 3'-O-Phosphate 3'-O-allyl 3'-O-Phosphate

[0070] The synthesis of oligonucleotides on living cells requires mild deblocking or deprotection conditions, i.e., conditions that do not damage the cell membrane, do not denature proteins, do not interfere with key cell functions, etc. In some embodiments, the deprotection conditions are within the range of physiological conditions compatible with cell survival. In such embodiments, enzymatic deprotection is desirable because it can be performed under physiological conditions. In some embodiments, specific enzymatically removable blocking groups are associated with specific enzymes for their removal. For example, ester or acyl-based blocking groups can be removed with esterases such as acetylesterase or similar enzymes, and phosphate blocking groups can be removed with 3'phosphatases such as T4 polynucleotide kinases. For example, 3'-O-phosphate can be removed by treatment with a solution of 100mM Tris-HCl (pH 6.5), 10mM MgCl2, 5mM 2-mercaptoethanol and one unit of T4 polynucleotide kinase. The reaction is carried out at a temperature of 37°C for one minute.

[0071] "3'-phosphate blocked" or "3'-phosphate protected" nucleotides refer to nucleotides in which the hydroxyl group at the 3'-position is blocked by the presence of a phosphoric acid-containing moiety. Examples of 3'-phosphate blocked nucleotides according to the present invention are nucleotide-3'-phosphoric acid monoesters / nucleotide-2', 3'-cyclic phosphates, nucleotide-2'-phosphoric acid monoesters and nucleotide-2' or 3'-alkyl phosphodiesters, and nucleotide-2' or 3'-pyrophosphates. Phosphorothioates or other analogs of such compounds may also be used, provided that the substitution does not prevent phosphatase dephosphorylation resulting in a free 3'-OH.

[0072] Other examples of the synthesis and enzymatic deprotection of 3'-O-ester protected dNTPs or 3'-O-phosphate protected dNTPs are described in the following references: Canard et al., Proc. Natl. Acad. Sci., 92: 10859-10863 (1995); Canard et al., Gene, 148: 1-6 (1994); Cameron et al., Biochemistry, 16 (23): 5120-5126 (1977); Rasolonjatovo et al., Nucleosides & Nucleotides, 18 (4 & 5): 1021-1022 (1999); Ferrero et al., Monatshefte fur Chemie, 131: 585-616 (2000); Taunton-Rigby et al., J. Org. Chem., 38(5): 977-985 (1973); Uemura et al., Tetrahedron Lett., 30(29): 3819-3820 (1989); Becker et al., J. Biol. Chem., 242(5): 936-950 (1967); Tsien, International Patent Publication WO1991 / 006678.

[0073] In some embodiments, the modified nucleotide comprises a modified nucleotide or nucleoside molecule comprising a purine or pyrimidine base and a ribose or deoxyribose sugar moiety having a removable 3'-OH blocking group covalently attached thereto such that the 3' carbon atom has attached thereto a set of the structures:

[0074] -OZ

[0075] wherein -Z is any of -C(R')2-OR", -C(R')2-N(R")2, -C(R')2-N(H)R", -C(R')2-SR", and -C(R')2-F, wherein each R" is or is part of a removable protecting group; each R' is independently a hydrogen atom, an alkyl group, a substituted alkyl group, an arylalkyl group, an alkenyl group, an alkynyl group, an aryl group, a heteroaryl group, a heterocyclyl group, an acyl group, a cyano group, an alkoxy group, an aryloxy group, a heteroaryloxy group, or an amido group, or a detectable label attached via a linking group; provided that in some embodiments, such substituents have up to 10 carbon atoms and / or or up to 5 oxygen or nitrogen heteroatoms; or (R')2 represents a set of formula =C(R"')2, wherein each R"' can be the same or different and is selected from hydrogen and halogen atoms and alkyl, provided that in some embodiments, the alkyl group of each R'" has 1-3 carbon atoms; and wherein the molecule can be reacted to produce an intermediate in which each R" is exchanged for H, or, wherein Z is -(R')2-F, F is exchanged for OH, SH or NH2, preferably OH, and the intermediate dissociates under aqueous conditions to give a molecule with a free 3'-OH; provided that wherein Z is -C(R')2-SR", and neither R' group is H. In certain embodiments, R' of the modified nucleotide or nucleoside is an alkyl or substituted alkyl, provided that such alkyl or substituted alkyl has 1-10 carbon atoms and 0-4 oxygen or nitrogen heteroatoms. In certain embodiments, -Z of the modified nucleotide or nucleoside has the formula -C(R')2-N3. In certain embodiments, Z is azidomethyl.

[0076] In some embodiments, Z is a cleavable organic moiety with or without heteroatoms having a molecular weight of 200 or less. In other embodiments, Z is a cleavable organic moiety with or without heteroatoms having a molecular weight of 100 or less. In other embodiments, Z is a cleavable organic moiety with or without heteroatoms having a molecular weight of 50 or less. In some embodiments, Z is an enzymatically cleavable organic moiety with or without heteroatoms having a molecular weight of 200 or less. In other embodiments, Z is an enzymatically cleavable organic moiety with or without heteroatoms having a molecular weight of 100 or less. In other embodiments, Z is an enzymatically cleavable organic moiety with or without heteroatoms having a molecular weight of 50 or less. In other embodiments, Z is an enzymatically cleavable ester group with a molecular weight of 200 or less. In other embodiments, Z is a phosphate group that is removable by 3'-phosphatases. In some embodiments, one or more of the following 3'-phosphatases can be used with the manufacturer's recommended protocol: T4 polynucleotide kinase, calf intestinal alkaline phosphatase, recombinant shrimp alkaline phosphatase (e.g., available from New England Biolabs, Beverly, MA)

[0077] In other embodiments, the 3'-blocked nucleoside triphosphate is blocked with a 3'-O-azidomethyl, 3'-O-NH2, or 3'-O-allyl group.

[0078] In other embodiments, the 3'-O blocking groups of the present invention include 3'-O-methyl, 3'-O-(2-nitrobenzyl), 3'-O-allyl, 3'-O-amine, 3'-O-azidomethyl, 3'-O-tert-butoxyethoxy, 3'-O-(2-cyanoethyl), and 3'-O-propargyl.

[0079] In some embodiments, the 3'-O protecting group is an electrochemically unstable group. That is, the deprotection or cutting of the protecting group is accomplished by changing the electrochemical conditions near the protecting group that causes the cutting. This change in electrochemical conditions can be caused by changing or applying a physical quantity such as a voltage difference or light to activate an auxiliary substance, which in turn causes a change in the electrochemical conditions at the protecting group site, such as an increase or decrease in pH. In some embodiments, the electrochemically unstable group includes, for example, a pH-sensitive protecting group that cuts the pH-sensitive protecting group whenever the pH changes to a predetermined value. In other embodiments, the electrochemically unstable group includes a protecting group that directly cuts the protecting group whenever, for example, the voltage difference at the protecting group site changes the reducing or oxidizing conditions.

[0080] In some embodiments, the enzymatic synthesis method uses a TdT variant that exhibits increased incorporation activity relative to a 3'-O-modified nucleoside triphosphate. For example, such a TdT variant can be produced using the techniques described in Champion et al., U.S. Patent No. 10435676, which is incorporated herein by reference. In some embodiments, a TdT variant is used that has an amino acid sequence at least 60% identical to SEQ ID NO: 2 and a substitution of the first arginine at position 207 and a substitution of the second arginine at position 325, or a functionally equivalent residue thereof. In some embodiments, a terminal deoxynucleotidyl transferase (TdT) variant is employed having an amino acid sequence at least 60% identical to an amino acid sequence selected from SEQ ID NO: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15, having a substitution of arginine (the "first arginine") at position 207 relative to SEQ ID NO 2, 3, 4, 6, 7, 9, 12 and 13, at position 206 relative to SEQ ID NO 5, at position 208 relative to SEQ ID NO 8 and 10, at position 205 relative to SEQ ID NO 11, at position 216 relative to SEQ ID NO 14, and at position 210 relative to SEQ ID NO 15; and a substitution of arginine (the "first arginine") at position 325 relative to SEQ ID NO 2, 9 and 13, at position 324 relative to SEQ ID NO 3 and 4, at position 320 relative to SEQ ID NO 320, at position 331 relative to SEQ ID NO 6 and 8, at position 332 relative to SEQ ID NO 15. 11 at position 323, a substitution of arginine ("second arginine") relative to SEQ ID NOs 12 and 15 at position 328 and relative to SEQ ID NO 14 at position 338; or a functionally equivalent residue thereof; wherein the TdT variant (i) is capable of synthesizing nucleic acid fragments in the absence of a template, and (ii) is capable of incorporating a 3'-O modified nucleotide at the free 3'-hydroxyl group of the nucleic acid fragment. In some embodiments, the above-mentioned percentage of identity value is at least 80% identical to the SEQ ID NO shown; in some embodiments, the above-mentioned percentage of identity value is at least 90% identical to the SEQ ID NO shown; in some embodiments, the above-mentioned percentage of identity value is at least 95% identical to the SEQ ID NO shown; in some embodiments, the above-mentioned percentage of identity value is at least 97% identical; in some embodiments, the above-mentioned percentage of identity value is at least 98% identical; in some embodiments, the above-mentioned percentage of identity value is at least 99% identical.As used herein, the percent identity values ​​used to compare a reference sequence to a variant sequence do not include the explicitly specified amino acid positions containing substitutions in the variant sequence; that is, the percent identity relationship is between the sequence of the reference protein and the sequence of the variant protein outside the explicitly specified positions containing substitutions in the variant. Thus, for example, if the reference sequence and the variant sequence each contain 100 amino acids and the variant sequence has mutations at positions 25 and 81, then the percent homology would be for sequences 1-24, 26-80, and 82-100.

[0081] With respect to (ii), such 3'-O modified nucleotides may comprise 3'-O-NH2-nucleoside triphosphate, 3'-O-azidomethyl-nucleoside triphosphate, 3'-O-allyl-nucleoside triphosphate, 3'-O-(2-nitrobenzyl)-nucleoside triphosphate or 3'-O-propargyl-nucleoside triphosphate.

[0082] In some embodiments, the TdT variants described above have substitutions at the first and second arginines as shown in Table 1.

[0083] Table 1

[0084]

[0085] In some embodiments, other TdT variants for use in the methods and devices of the invention include one or more of further substitutions of methionine, cysteine, or glutamate, as shown in Table 1.

[0086] Other specific TdT variants that can be used in the methods and devices of the present invention are listed in Table 2. Each of the TdT variants DS1001 to DS1018 of Table 2 comprises an amino acid sequence that is at least 60% identical to SEQ ID NO 2 and includes a substitution at the specified position. In some embodiments, TdT variants DS1001 to DS1018 comprise an amino acid sequence at least 80% identical to SEQ ID NO 2 and include substitutions at specified positions; in some embodiments, TdT variants DS1001 to DS1018 comprise an amino acid sequence at least 90% identical to SEQ ID NO 2 and include substitutions at specified positions; in some embodiments, TdT variants DS1001 to DS1018 comprise an amino acid sequence at least 95% identical to SEQ ID NO 2 and include substitutions at specified positions; in some embodiments, TdT variants DS1001 to DS1018 comprise an amino acid sequence at least 97% identical to SEQ ID NO 2 and include substitutions at specified positions; in some embodiments, TdT variants DS1001 to DS1018 comprise an amino acid sequence at least 98% identical to SEQ ID NO 2 and include substitutions at specified positions; in some embodiments, TdT variants DS1001 to DS1018 comprise an amino acid sequence at least 99% identical to SEQ ID NO 2 and include substitutions at specified positions.

[0087] Table 2

[0088] Specific TdT variants for use in the methods of the invention

[0089]

[0090]

[0091] Other specific TdT variants that can be used in the methods and devices of the present invention include amino acid sequences that are at least 90% identical (or at least 95% identical) to SEQ ID NO: 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30, wherein the amino acid sequences of SEQ ID No: 16, 17, 18, 19 and 20 are each substituted at position 4 with a stabilizing mutation selected from E, S, D and N, and wherein the TdT variant (i) is capable of synthesizing nucleic acid fragments in the absence of a template, and (ii) is capable of incorporating a 3'-O protected nucleotide onto a free 3'-hydroxyl group of a polynucleotide. In other embodiments, such a stabilizing mutation at position 4 is E.

[0092] The TdT variants of the present invention as described above each comprise an amino acid sequence having a percentage of sequence identity with a specified SEQ ID NO that is subjected to a specified substitution. In some embodiments, the number and type of sequence differences between the TdT variants of the present invention described in this manner and the specified SEQ ID NO may be due to substitutions, deletions and / or insertions, and the amino acids substituted, deleted and / or inserted may include any amino acid. In some embodiments, such deletions, substitutions and / or insertions include only naturally occurring amino acids. In some embodiments, substitutions include only conservative or synonymous amino acid changes, as described in Grantham, Science, 185: 862-864 (1974). That is, amino acid substitutions can only occur in members of their synonymous amino acid groups. In some embodiments, the synonymous amino acid groups that can be used are listed in Table 3A.

[0093] Table 3A

[0094] Synonymous Group I of Amino Acids

[0095]

[0096]

[0097] In some embodiments, synonymous amino acid groups that may be employed are listed in Table 3B.

[0098] Table 3B

[0099] Synonymous Group II of Amino Acids

[0100]

[0101] definition

[0102] Unless otherwise specifically defined herein, the terms and symbols of nucleic acid chemistry, biochemistry, genetics, and molecular biology used herein follow those of standard treatises and textbooks in the field, such as Kornberg and Baker, DNA Replication, 2nd edition (WH Freeman, New York, 1992); Lehninger, Biochemistry, 2nd edition (Worth Publishers, New York, 1975); Strachan and Read, Human Molecular Genetics, 2nd edition (Wiley-Liss, New York, 1999).

[0103] "Functionally equivalent" with respect to an amino acid position in two or more different TdTs means that (i) the amino acid at each position plays the same functional role in the activity of the TdT, and (ii) the amino acid occurs at a homologous amino acid position in the amino acid sequence of each TdT. Based on sequence alignment and / or molecular modeling, positionally equivalent or homologous amino acid residues in the amino acid sequences of two or more different TdTs can be identified. In some embodiments, the functionally equivalent amino acid positions belong to an inefficient motif that is conserved in the amino acid sequences of TdTs of evolutionarily related species, e.g., a genus, a family, etc. Examples of such conserved inefficient motifs are described in Motea et al., Biochim. Biophys. Acta. 1804 (5): 1151-1166 (2010); Delarue et al., EMBO J., 21: 427-439 (2002) and similar references.

[0104] "Mutant" or "variant" used interchangeably refers to a polypeptide derived from a natural or reference TdT polypeptide described herein and comprising a modification or alteration, i.e., a substitution, insertion and / or deletion, at one or more positions. Variants can be obtained by various techniques well known in the art. In particular, examples of techniques for altering a DNA sequence encoding a wild-type protein include, but are not limited to, site-directed mutagenesis, random mutagenesis, sequence shuffling, and synthetic oligonucleotide construction. The mutagenic activity consists in deleting, inserting, or replacing one or more amino acids in a protein, or in the case of the present invention, a polymerase sequence. The following terms are used to specify substitutions: L238A indicates that the amino acid residue (leucine, L) at position 238 of a reference or wild-type sequence is changed to alanine (A). A132V / I / M indicates that the amino acid residue (alanine, A) at position 132 of the parent sequence is substituted by one of the following amino acids: valine (V), isoleucine (I), or methionine (M). Substitutions can be conservative or non-conservative substitutions. Examples of conservative substitutions are within the following groups: basic amino acids (arginine, lysine, and histidine), acidic amino acids (glutamate and aspartate), polar amino acids (glutamine, asparagine, and threonine), hydrophobic amino acids (methionine, leucine, isoleucine, cysteine, and valine), aromatic amino acids (phenylalanine, tryptophan, and tyrosine), and small amino acids (glycine, alanine, and serine).

[0105] "Polynucleotide" or "oligonucleotide" are used interchangeably and each refers to a linear polymer of a nucleotide monomer or its analog. The monomers constituting polynucleotides and oligonucleotides can specifically bind natural polynucleotides by regular patterns of monomer-monomer interactions, such as base pairing, base stacking, Hoogsteen or reverse Hoogsteen type base pairing, etc. Such monomers and their internucleoside bonds can be naturally occurring or can be analogs thereof, such as naturally occurring or non-naturally occurring analogs. Non-naturally occurring analogs can include PNA, thiophosphate internucleoside bonds, bases containing linking groups that allow connection of markers such as fluorophores or haptens, etc. Whenever the use of oligonucleotides or polynucleotides requires enzymatic processing, such as by polymerase extension, by ligase connection, etc., it will be understood by ordinary technicians that oligonucleotides or polynucleotides will not be included in certain analogs, sugar moieties or bases of internucleoside bonds at any or some positions in those cases. The size range of polynucleotides usually starts from some monomer units, such as 5-40, when they are usually referred to as "oligonucleotides", to several thousand monomer units. Whenever a polynucleotide or oligonucleotide is represented by a sequence of letters (uppercase or lowercase), such as "ATGCCTG", it is understood that the nucleotides are arranged in 5'-3' order from left to right, and "A" represents deoxyadenosine, "C" represents deoxycytidine, "G" represents deoxyguanosine, and "T" represents thymidine, "I" represents deoxyinosine, and "U" represents uridine, unless otherwise specified or obvious from the context. Unless otherwise specified, the terminology and atom numbering conventions will follow those disclosed in Kornberg and Baker, DNA Replication, Second Edition (WH Freeman, 1992) or similar references. Typically polynucleotides contain four natural nucleosides (e.g., deoxyadenosine, deoxycytidine, deoxyguanosine, deoxythymidine for DNA, or their ribose counterparts for RNA) linked by phosphodiester bonds; however, they can also contain non-natural nucleotide analogs, for example, including modified bases, sugars or internucleoside linkages. It is clear to those skilled in the art that enzymes have specific oligonucleotide or polynucleotide substrate requirements for activity, such as single-stranded DNA, RNA / DNA duplexes, etc., and then selecting suitable compositions for oligonucleotide or polynucleotide substrates is entirely within the knowledge of ordinary technicians, especially under the guidance of papers such as Sambrook et al., Molecular Cloning, Second Edition (Cold Spring Harbor Laboratory, New York, 1989) and similar references. Similarly, oligonucleotides and polynucleotides can refer to single-stranded forms or double-stranded forms (i.e., duplexes of oligonucleotides or polynucleotides and their corresponding complements). From the context of the use of the term, ordinary technicians will clearly intend to be which form or whether it is two forms.

[0106] "Sequence identity" refers to the number (or fraction, usually expressed as a percentage) of matches (e.g., identical amino acid residues) between two sequences, such as two polypeptide sequences or two polynucleotide sequences. Sequence identity is determined by comparing sequences when aligned to maximize overlap and identity while minimizing sequence gaps. In particular, sequence identity can be determined using any of a number of mathematical global or local alignment algorithms, depending on the lengths of the two sequences. Sequences of similar length are preferably aligned using a global alignment algorithm (e.g., the Needleman and Wunsch algorithm; Needleman and Wunsch, 1970), which optimally aligns sequences over the entire length, while sequences of substantially different lengths are preferably aligned using a local alignment algorithm (e.g., the Smith and Waterman algorithm (Smith and Waterman, 1981) or the Altschul algorithm (Altschul et al., 1997; Altschul et al., 2005)). Comparison for determining the purpose of amino acid sequence identity percentage can be achieved in various ways within the technical scope of the art, for example, using publicly available computer software that can be obtained on Internet websites such as http: / / blast.ncbi.nlm.nih.gov / or ttp: / / www.ebi.ac.uk / Tools / emboss / . Those skilled in the art can determine the appropriate parameters for measuring the comparison, including any algorithm required for maximum comparison on the full length of the compared sequence. For the purposes of this article, amino acid sequence identity % value refers to the value generated using the paired sequence alignment program EMBOSS Needle, which uses the Needleman-Wunsch algorithm to create the best global comparison of two sequences, wherein all search parameters are set to default values, i.e., scoring matrix=BLOSUM62, gap opening=10, gap extension=0.5, terminal gap penalty=error, terminal gap opening=10 and terminal gap extension=0.5.

[0107] "Substitution" means that one amino acid residue is replaced by another amino acid residue. Preferably, the term "substitution" refers to the replacement of one amino acid residue by another amino acid residue selected from the standard 20 naturally occurring amino acid residues, rare naturally occurring amino acid residues (e.g., hydroxyproline, hydroxylysine, allohydroxylysine, 6-N-methyllysine, N-ethylglycine, N-methylglycine, N-ethylasparagine, alloisoleucine, N-methylisoleucine, N-methylvaline, pyroglutamine, aminobutyric acid, ornithine, norleucine, norvaline) and non-naturally occurring amino acid residues, usually synthetic, (e.g., cyclohexylalanine). Preferably, the term "substitution" refers to the replacement of one amino acid residue by another amino acid residue selected from the standard 20 naturally occurring amino acid residues. The symbol "+" indicates a combination of substitutions. Amino acids are represented herein by their single-letter or three-letter codes according to the following nomenclature: A: alanine (Ala); C: cysteine ​​(Cys); D: aspartic acid (Asp); E: glutamic acid (Glu); F: phenylalanine (Phe); G: glycine (Gly); H: histidine (His); I: isoleucine (Ile); K: lysine (Lys); L: leucine (Leu); M: methionine (Met); N: asparagine (Asn); P: proline (Pro); Q: glutamine (Gln); R: arginine (Arg); S: serine (Ser); T: threonine (Thr); V: valine (Val); W: tryptophan (Trp) and Y: tyrosine (Tyr). Sequence Listing <110> DNA SCRIPT <120> Methods and apparatus for enzymatic synthesis of polynucleotides <130> B3213PC00 <150> EP20159227.6 <151> 2020-02-25 <160> 30 <170> PatentIn version 3.5 <210> 1 <211> twenty one <212> DNA <213> ARTIFICIAL SEQUENCE <220> <223> Nucleotides <400> 1 gtttcctaag ttgcatttgc g 21 <210> 2 <211> 381 <212> PRT <213> Artificial Sequence <220> <223> Truncated Mouse TdT <400> 2 Asn Ser Ser Pro Ser Pro Val Pro Gly Ser Gln Asn Val Pro Ala Pro 1 5 10 15 Ala Val Lys Lys Ile Ser Gln Tyr Ala Cys Gln Arg Arg Thr Thr Leu 20 25 30 Asn Asn Tyr Asn Gln Leu Phe Thr Asp Ala Leu Asp Ile Leu Ala Glu 35 40 45 Asn Asp Glu Leu Arg Glu Asn Glu Gly Ser Cys Leu Ala Phe Met Arg 50 55 60 Ala Ser Ser Val Leu Lys Ser Leu Pro Phe Pro Ile Thr Ser Met Lys 65 70 75 80 Asp Thr Glu Gly Ile Pro Cys Leu Gly Asp Lys Val Lys Ser Ile Ile 85 90 95 Glu Gly Ile Ile Glu Asp Gly Glu Ser Ser Glu Ala Lys Ala Val Leu 100 105 110 Asn Asp Glu Arg Tyr Lys Ser Phe Lys Leu Phe Thr Ser Val Phe Gly 115 120 125 Val Gly Leu Lys Thr Ala Glu Lys Trp Phe Arg Met Gly Phe Arg Thr 130 135 140 Leu Ser Lys Ile Gln Ser Asp Lys Ser Leu Arg Phe Thr Gln Met Gln 145 150 155 160 Lys Ala Gly Phe Leu Tyr Tyr Glu Asp Leu Val Ser Cys Val Asn Arg 165 170 175 Pro Glu Ala Glu Ala Val Ser Met Leu Val Lys Glu Ala Val Val Thr 180 185 190 Phe Leu Pro Asp Ala Leu Val Thr Met Thr Gly Gly Phe Arg Arg Gly 195 200 205 Lys Met Thr Gly His Asp Val Asp Phe Leu Ile Thr Ser Pro Glu Ala 210 215 220 Thr Glu Asp Glu Glu Gln Gln Leu Leu His Lys Val Thr Asp Phe Trp 225 230 235 240 Lys Gln Gln Gly Leu Leu Leu Tyr Cys Asp Ile Leu Glu Ser Thr Phe 245 250 255 Glu Lys Phe Lys Gln Pro Ser Arg Lys Val Asp Ala Leu Asp His Phe 260 265 270 Gln Lys Cys Phe Leu Ile Leu Lys Leu Asp His Gly Arg Val His Ser 275 280 285 Glu Lys Ser Gly Gln Gln Glu Gly Lys Gly Trp Lys Ala Ile Arg Val 290 295 300 Asp Leu Val Met Cys Pro Tyr Asp Arg Arg Ala Phe Ala Leu Leu Gly 305 310 315 320 Trp Thr Gly Ser Arg Gln Phe Glu Arg Asp Leu Arg Arg Tyr Ala Thr 325 330 335 His Glu Arg Lys Met Met Leu Asp Asn His Ala Leu Tyr Asp Arg Thr 340 345 350 Lys Arg Val Phe Leu Glu Ala Glu Ser Glu Glu Glu Ile Phe Ala His 355 360 365 Leu Gly Leu Asp Tyr Ile Glu Pro Trp Glu Arg Asn Ala 370 375 380 <210> 3 <211> 380 <212> PRT <213> Bovine <400> 3 Asp Tyr Ser Ala Thr Pro Asn Pro Gly Phe Gln Lys Thr Pro Pro Leu 1 5 10 15 Ala Val Lys Lys Ile Ser Gln Tyr Ala Cys Gln Arg Lys Thr Thr Leu 20 25 30 Asn Asn Tyr Asn His Ile Phe Thr Asp Ala Phe Glu Ile Leu Ala Glu 35 40 45 Asn Ser Glu Phe Lys Glu Asn Glu Val Ser Tyr Val Thr Phe Met Arg 50 55 60 Ala Ala Ser Val Leu Lys Ser Leu Pro Phe Thr Ile Ile Ser Met Lys 65 70 75 80 Asp Thr Glu Gly Ile Pro Cys Leu Gly Asp Lys Val Lys Cys Ile Ile 85 90 95 Glu Glu Ile Ile Glu Asp Gly Glu Ser Glu Val Lys Ala Val Leu 100 105 110 Asn Asp Glu Arg Tyr Gln Ser Phe Lys Leu Phe Thr Ser Val Phe Gly 115 120 125 Val Gly Leu Lys Thr Ser Glu Lys Trp Phe Arg Met Gly Phe Arg Ser 130 135 140 Leu Ser Lys Ile Met Ser Asp Lys Thr Leu Lys Phe Thr Lys Met Gln 145 150 155 160 Lys Ala Gly Phe Leu Tyr Tyr Glu Asp Leu Val Ser Cys Val Thr Arg 165 170 175 Ala Glu Ala Glu Ala Val Gly Val Leu Val Lys Glu Ala Val Trp Ala 180 185 190 Phe Leu Pro Asp Ala Phe Val Thr Met Thr Gly Gly Phe Arg Arg Gly 195 200 205 Lys Lys Ile Gly His Asp Val Asp Phe Leu Ile Thr Ser Pro Gly Ser 210 215 220 Ala Glu Asp Glu Glu Gln Leu Leu Pro Lys Val Ile Asn Leu Trp Glu 225 230 235 240 Lys Lys Gly Leu Leu Leu Tyr Tyr Asp Leu Val Glu Ser Thr Phe Glu 245 250 255 Lys Phe Lys Leu Pro Ser Arg Gln Val Asp Thr Leu Asp His Phe Gln 260 265 270 Lys Cys Phe Leu Ile Leu Lys Leu His His Gln Arg Val Asp Ser Ser 275 280 285 Lys Ser Asn Gln Gln Glu Gly Lys Thr Trp Lys Ala Ile Arg Val Asp 290 295 300 Leu Val Met Cys Pro Tyr Glu Asn Arg Ala Phe Ala Leu Leu Gly Trp 305 310 315 320 Thr Gly Ser Arg Gln Phe Glu Arg Asp Ile Arg Arg Tyr Ala Thr His 325 330 335 Glu Arg Lys Met Met Leu Asp Asn His Ala Leu Tyr Asp Lys Thr Lys 340 345 350 Arg Val Phe Leu Lys Ala Glu Ser Glu Glu Glu Ile Phe Ala His Leu 355 360 365 Gly Leu Asp Tyr Ile Glu Pro Trp Glu Arg Asn Ala 370 375 380 <210> 4 <211> 380 <212> PRT <213> HUMAN <400> 4 Asp Tyr Ser Asp Ser Thr Asn Pro Gly Pro Pro Lys Thr Pro Pro Ile 1 5 10 15 Ala Val Gln Lys Ile Ser Gln Tyr Ala Cys Gln Arg Arg Thr Thr Leu 20 25 30 Asn Asn Cys Asn Gln Ile Phe Thr Asp Ala Phe Asp Ile Leu Ala Glu 35 40 45 Asn Cys Glu Phe Arg Glu Asn Glu Asp Ser Cys Val Thr Phe Met Arg 50 55 60 Ala Ala Ser Val Leu Lys Ser Leu Pro Phe Thr Ile Ile Ser Met Lys 65 70 75 80 Asp Thr Glu Gly Ile Pro Cys Leu Gly Ser Lys Val Lys Gly Ile Ile 85 90 95 Glu Glu Ile Ile Glu Asp Gly Glu Ser Ser Glu Val Lys Ala Val Leu 100 105 110 Asn Asp Glu Arg Tyr Gln Ser Phe Lys Leu Phe Thr Ser Val Phe Gly 115 120 125 Val Gly Leu Lys Thr Ser Glu Lys Trp Phe Arg Met Gly Phe Arg Thr 130 135 140 Leu Ser Lys Val Arg Ser Asp Lys Ser Leu Lys Phe Thr Arg Met Gln 145 150 155 160 Lys Ala Gly Phe Leu Tyr Tyr Glu Asp Leu Val Ser Cys Val Thr Arg 165 170 175 Ala Glu Ala Glu Ala Val Ser Val Leu Val Lys Glu Ala Val Trp Ala 180 185 190 Phe Leu Pro Asp Ala Phe Val Thr Met Thr Gly Gly Phe Arg Arg Gly 195 200 205 Lys Lys Met Gly His Asp Val Asp Phe Leu Ile Thr Ser Pro Gly Ser 210 215 220 Thr Glu Asp Glu Glu Gln Leu Leu Gln Lys Val Met Asn Leu Trp Glu 225 230 235 240 Lys Lys Gly Leu Leu Leu Tyr Tyr Asp Leu Val Glu Ser Thr Phe Glu 245 250 255 Lys Leu Arg Leu Pro Ser Arg Lys Val Asp Ala Leu Asp His Phe Gln 260 265 270 Lys Cys Phe Leu Ile Phe Lys Leu Pro Arg Gln Arg Val Asp Ser Asp 275 280 285 Gln Ser Ser Trp Gln Glu Gly Lys Thr Trp Lys Ala Ile Arg Val Asp 290 295 300 Leu Val Leu Cys Pro Tyr Glu Arg Arg Ala Phe Ala Leu Leu Gly Trp 305 310 315 320 Thr Gly Ser Arg Gln Phe Glu Arg Asp Leu Arg Arg Tyr Ala Thr His 325 330 335 Glu Arg Lys Met Ile Leu Asp Asn His Ala Leu Tyr Asp Lys Thr Lys 340 345 350 Arg Ile Phe Leu Lys Ala Glu Ser Glu Glu Glu Ile Phe Ala His Leu 355 360 365 Gly Leu Asp Tyr Ile Glu Pro Trp Glu Arg Asn Ala 370 375 380 <210> 5 <211> 376 <212> PRT <213> Chicken <400> 5 Gln Tyr Pro Thr Leu Lys Thr Pro Glu Ser Glu Val Ser Ser Phe Thr 1 5 10 15 Ala Ser Lys Val Ser Gln Tyr Ser Cys Gln Arg Lys Thr Thr Leu Asn 20 25 30 Asn Cys Asn Lys Lys Phe Thr Asp Ala Phe Glu Ile Met Ala Glu Asn 35 40 45 Tyr Glu Phe Lys Glu Asn Glu Ile Phe Cys Leu Glu Phe Leu Arg Ala 50 55 60 Ala Ser Val Leu Lys Ser Leu Pro Phe Pro Val Thr Arg Met Lys Asp 65 70 75 80 Ile Gln Gly Leu Pro Cys Met Gly Asp Arg Val Arg Asp Val Ile Glu 85 90 95 Glu Ile Ile Glu Gly Glu Ser Ser Arg Ala Lys Asp Val Leu Asn 100 105 110 Asp Glu Arg Tyr Lys Ser Phe Lys Glu Phe Thr Ser Val Phe Gly Val 115 120 125 Gly Val Lys Thr Ser Glu Lys Trp Phe Arg Met Gly Leu Arg Thr Val 130 135 140 Glu Glu Val Lys Ala Asp Lys Thr Leu Lys Leu Ser Met Gln Arg 145 150 155 160 Ala Gly Phe Leu Tyr Tyr Glu Asp Leu Will Be Cys Will Be Lys Ala 165 170 175 Glu Ala Asp Ala Val Ser Ile Val Lys Asn Thr Val Cys Thr Phe 180 185 190 Leu Pro Asp Ala Leu Val Thr Ile Thr Gly Gly Phe Arg Arg Gly Lys 195 200 205 Lys Ile Gly His Asp Ile Asp Phe Leu Ile Thr Ser Pro Gly Gln Arg 210 215 220 Glu Asp Asp Glu Leu Leu His Lys Gly Leu Leu Leu Tyr Cys Asp Ile 225 230 235 240 Ile Glu Ser Thr Phe Val Lys Glu Gln Ile Pro Ser Arg His Val Asp 245 250 255 Ala Met Asp His Phe Gln Lys Cys Phe Ala Ile Leu Lys Leu Tyr Gln 260 265 270 Pro Arg Val Asp Asn Ser Ser Tyr Asn Met Ser Lys Lys Cys Asp Met 275 280 285 Ala Glu Val Lys Asp Trp Lys Ala Ile Arg Val Asp Leu Val Ile Thr 290 295 300 Pro Phe Glu Gln Tyr Ala Tyr Ala Leu Leu Gly Trp Thr Gly Ser Arg 305 310 315 320 Gln Phe Gly Arg Asp Leu Arg Arg Tyr Ala Thr His Glu Arg Lys Met 325 330 335 Met Leu Asp Asn His Ala Leu Tyr Asp Lys Arg Lys Arg Val Phe Leu 340 345 350 Lys Ala Gly Ser Glu Glu Glu Ile Phe Ala His Leu Gly Leu Asp Tyr 355 360 365 Val Glu Pro Trp Glu Arg Asn Ala 370 375 <210> 6 <211> 387 <212> PRT <213> Opossum (POSSUM) <400> 6 Ser Ala Asn Pro Asp Pro Thr Ala Gly Thr Leu Asn Ile Leu Pro Pro 1 5 10 15 Thr Thr Lys Thr Ile Ser Gln Tyr Ala Cys Gln Arg Arg Thr Thr Ile 20 25 30 Asn Asn His Asn Gln Arg Phe Thr Asp Ala Phe Glu Ile Leu Ala Lys 35 40 45 Asn Tyr Glu Phe Lys Glu Asn Asp Asp Thr Cys Leu Thr Phe Met Arg 50 55 60 Ala Ile Ser Val Leu Lys Cys Leu Pro Phe Glu Val Val Ser Leu Lys 65 70 75 80 Asp Thr Glu Gly Leu Pro Trp Ile Gly Asp Glu Val Lys Gly Ile Met 85 90 95 Glu Glu Ile Ile Glu Asp Gly Glu Ser Leu Glu Val Gln Ala Val Leu 100 105 110 Asn Asp Glu Arg Tyr Gln Ser Phe Lys Leu Phe Thr Ser Val Phe Gly 115 120 125 Val Gly Leu Lys Thr Ala Asp Lys Trp Tyr Arg Met Gly Phe Arg Thr 130 135 140 Leu Asn Lys Ile Arg Ser Asp Lys Thr Leu Lys Leu Thr Lys Met Gln 145 150 155 160 Lys Ala Gly Leu Cys Tyr Tyr Glu Asp Leu Ile Asp Cys Val Ser Lys 165 170 175 Ala Glu Ala Asp Ala Val Ser Leu Leu Val Gln Asp Ala Val Trp Thr 180 185 190 Phe Leu Pro Asp Ala Leu Val Thr Ile Thr Gly Gly Phe Arg Arg Gly 195 200 205 Lys Glu Phe Gly His Asp Val Asp Phe Leu Ile Thr Ser Pro Gly Ala 210 215 220 Glu Lys Glu Gln Glu Asp Gln Leu Leu Gln Lys Val Thr Asn Leu Trp 225 230 235 240 Lys Lys Gln Gly Leu Leu Leu Tyr Cys Asp Leu Ile Glu Ser Thr Phe 245 250 255 Glu Asp Leu Lys Leu Pro Ser Arg Lys Ile Asp Ala Leu Asp His Phe 260 265 270 Gln Lys Cys Phe Leu Ile Leu Lys Leu Tyr His His Lys Glu Asp Lys 275 280 285 Arg Lys Trp Glu Met Pro Thr Gly Ser Asn Glu Ser Glu Ala Lys Ser 290 295 300 Trp Lys Ala Ile Arg Val Asp Leu Val Val Cys Pro Tyr Asp Arg Tyr 305 310 315 320 Ala Phe Ala Leu Leu Gly Trp Ser Gly Ser Arg Gln Phe Glu Arg Asp 325 330 335 Leu Arg Arg Tyr Ala Thr His Glu Lys Lys Met Met Leu Asp Asn His 340 345 350 Ala Leu Tyr Asp Lys Thr Lys Lys Ile Phe Leu Lys Ala Lys Ser Glu 355 360 365 Glu Glu Ile Phe Ala His Leu Gly Leu Glu Tyr Ile Gln Pro Ser Glu 370 375 380 Arg Asn Ala 385 <210> 7 <211> 381 <212> PRT <213> Shrew <400> 7 Asp Cys Pro Ala Ser His Asp Ser Ser Pro Gln Lys Thr Glu Ser Ala 1 5 10 15 Ala Val Gln Lys Ile Ser Gln Tyr Ala Cys Gln Arg Arg Thr Thr Leu 20 25 30 Asn Asn His Asn His Ile Phe Thr Asp Ala Phe Glu Ile Leu Ala Glu 35 40 45 Asn Cys Glu Phe Arg Glu Asn Glu Gly Ser Tyr Val Thr Tyr Met Arg 50 55 60 Ala Ala Ser Val Leu Lys Ser Leu Pro Phe Ser Ile Ile Ser Met Lys 65 70 75 80 Asp Thr Glu Gly Ile Pro Cys Leu Ala Asp Lys Val Lys Cys Val Ile 85 90 95 Glu Glu Ile Ile Glu Asp Gly Glu Ser Ser Glu Val Lys Ala Val Leu 100 105 110 Asn Asp Glu Arg Tyr Lys Ser Phe Lys Leu Phe Thr Ser Val Phe Gly 115 120 125 Val Gly Leu Lys Thr Ala Glu Lys Trp Phe Arg Leu Gly Phe Arg Thr 130 135 140 Leu Ser Gly Ile Met Asn Asp Lys Thr Leu Lys Leu Thr His Met Gln 145 150 155 160 Lys Ala Gly Phe Leu Tyr Tyr Glu Asp Leu Val Ser Cys Val Thr Arg 165 170 175 Ala Glu Ala Glu Ala Val Gly Val Leu Val Lys Glu Ala Val Trp Ala 180 185 190 Phe Leu Pro Asp Ala Ile Val Thr Met Thr Gly Gly Phe Arg Arg Gly 195 200 205 Lys Lys Val Gly His Asp Val Asp Phe Leu Ile Thr Ser Pro Glu Ala 210 215 220 Thr Glu Glu Gln Glu Gln Gln Leu Leu His Lys Val Ile Thr Phe Trp 225 230 235 240 Glu Lys Glu Gly Leu Leu Leu Tyr Cys Asp Leu Tyr Glu Ser Thr Phe 245 250 255 Glu Lys Leu Lys Met Pro Ser Arg Lys Val Asp Ala Leu Asp His Phe 260 265 270 Gln Lys Cys Phe Leu Ile Leu Lys Leu His Arg Glu Cys Val Asp Asp 275 280 285 Gly Thr Ser Ser Gln Leu Gln Gly Lys Thr Trp Lys Ala Ile Arg Val 290 295 300 Asp Leu Val Val Cys Pro Tyr Glu Cys Arg Ala Phe Ala Leu Leu Gly 305 310 315 320 Trp Thr Gly Ser Pro Gln Phe Glu Arg Asp Leu Arg Arg Tyr Ala Thr 325 330 335 His Glu Arg Lys Met Met Leu Asp Asn His Ala Leu Tyr Asp Lys Thr 340 345 350 Lys Arg Lys Phe Leu Ser Ala Asp Ser Glu Glu Asp Ile Phe Ala His 355 360 365 Leu Gly Leu Asp Tyr Ile Glu Pro Trp Glu Arg Asn Ala 370 375 380 <210> 8 <211> 387 <212> PRT <213> Python <400> 8 Glu Lys Tyr Gln Leu Pro Glu Asp Glu Asp Arg Ser Val Thr Ser Asp 1 5 10 15 Leu Asp Arg Asp Ser Ile Ser Glu Tyr Ala Cys Gln Arg Arg Thr Thr 20 25 30 Leu Lys Asn Tyr Asn Gln Lys Phe Thr Asp Ala Phe Glu Ile Leu Ala 35 40 45 Glu Asn Tyr Glu Phe Asn Glu Asn Lys Gly Phe Cys Thr Ala Phe Arg 50 55 60 Arg Ala Ala Ser Val Leu Lys Cys Leu Pro Phe Thr Ile Val Gln Val 65 70 75 80 His Asp Ile Glu Gly Val Pro Trp Met Gly Lys Gln Val Lys Gly Ile 85 90 95 Ile Glu Asp Ile Ile Glu Glu Gly Glu Ser Ser Lys Val Lys Ala Val 100 105 110 Leu Asp Asn Glu Asn Tyr Arg Ser Val Lys Leu Phe Thr Ser Val Phe 115 120 125 Gly Val Gly Leu Lys Thr Ser Asp Lys Trp Tyr Arg Met Gly Leu Arg 130 135 140 Thr Leu Glu Glu Val Lys Arg Asp Lys Asn Leu Lys Leu Thr Arg Met 145 150 155 160 Gln Lys Ala Gly Phe Leu His Tyr Asp Asp Leu Thr Ser Cys Val Ser 165 170 175 Lys Ala Glu Ala Asp Ala Ala Ser Leu Ile Val Gln Asp Val Val Trp 180 185 190 Lys Ile Val Pro Asn Ala Ile Val Thr Ile Ala Gly Gly Phe Arg Arg 195 200 205 Gly Lys Gln Thr Gly His Asp Val Asp Phe Leu Ile Thr Val Pro Gly 210 215 220 Ser Lys Gln Glu Glu Glu Glu Leu Leu His Thr Val Ile Asp Ile Trp 225 230 235 240 Lys Lys Gln Glu Leu Leu Leu Tyr Tyr Asp Leu Ile Glu Ser Thr Phe 245 250 255 Glu Asp Thr Lys Leu Pro Ser Arg Lys Val Asp Ala Leu Asp His Phe 260 265 270 Gln Lys Cys Phe Ala Ile Leu Lys Val His Lys Glu Arg Glu Asp Lys 275 280 285 Gly Asn Ser Ile Arg Ser Lys Ala Phe Ser Glu Glu Glu Ile Lys Asp 290 295 300 Trp Lys Ala Ile Arg Val Asp Leu Val Val Val Pro Phe Glu Gln Tyr 305 310 315 320 Ala Phe Ala Leu Leu Gly Trp Thr Gly Ser Thr Gln Phe Glu Arg Asp 325 330 335 Leu Arg Arg Tyr Ala Thr His Glu Lys Lys Met Met Leu Asp Asn His 340 345 350 Ala Leu Tyr Asp Lys Thr Lys Lys Ile Phe Leu Asn Ala Ala Ser Glu 355 360 365 Glu Glu Ile Phe Ala His Leu Gly Leu Asp Tyr Leu Glu Pro Trp Glu 370 375 380 Arg Asn Ala 385 <210> 9 <211> 381 <212> PRT <213> Dog <400> 9 Asp Tyr Thr Ala Ser Pro Asn Pro Glu Leu Gln Lys Thr Leu Pro Val 1 5 10 15 Only Lys Lys Is Gln Tyr Only Cys Gln Arg Is Thr Thr Leu 20 25 30 Asn Asn Tyr Asn Asn Val Phe Thr Asp Ala Phe Glu Val Leu Ala Glu 35 40 45 Asn Tyr Glu Phe Arg Glu Asn Glu Val Phe Ser Leu Thr Phe Met Arg 50 55 60 Ala Ala Ser Val Leu Lys Ser Leu Pro Phe Thr Ile Ile Ser Met Lys 65 70 75 80 Asp Thr Glu Gly Ile Pro Cys Leu Gly Asp Gln Val Lys Cys Ile Ile 85 90 95 Glu Glu Isle Isle Glu Asp Gly Ser Ser Glu Val Lys Wing Val Leu 100 105 110 Asn Asp Glu Arg Tyr Gln Ser Phe Lys Leu Phe Thr Ser Val Phe Gly 115 120 125 Val Gly Leu Lys Thr Ser Glu Lys Trp Phe Arg Met Gly Phe Arg Thr 130 135 140 Leu Ser Lys Ile Lys Ser Asp Lys Ser Leu Lys Phe Thr Pro Met Gln 145 150 155 160 Lys Ala Gly Phe Leu Tyr Tyr Glu Asp Leu Val Ser Cys Val Thr Arg 165 170 175 Ala Glu Ala Glu Ala Val Gly Val Leu Val Lys Glu Ala Val Gly Ala 180 185 190 Phe Leu Pro Asp Ala Phe Val Thr Met Thr Gly Gly Phe Arg Arg Gly 195 200 205 Lys Lys Met Gly His Asp Val Asp Phe Leu Ile Thr Ser Pro Gly Ser 210 215 220 Thr Asp Glu Asp Glu Glu Gln Leu Leu Pro Lys Val Ile Asn Leu Trp 225 230 235 240 Glu Arg Lys Gly Leu Leu Leu Tyr Cys Asp Leu Val Glu Ser Thr Phe 245 250 255 Glu Lys Leu Lys Leu Pro Ser Arg Lys Val Asp Ala Leu Asp His Phe 260 265 270 Gln Lys Cys Phe Leu Ile Leu Lys Leu His His Gln Arg Val Asp Gly 275 280 285 Gly Lys Cys Ser Gln Gln Glu Gly Lys Thr Trp Lys Ala Ile Arg Val 290 295 300 Asp Leu Val Met Cys Pro Tyr Glu Arg Arg Ala Phe Ala Leu Leu Gly 305 310 315 320 Trp Thr Gly Ser Arg Gln Phe Glu Arg Asp Leu Arg Arg Tyr Ala Ser 325 330 335 His Glu Arg Lys Met Ile With Asp Asn And His Glu Arg Lys With Tyr Asp Lys Thr 340 345 350 Lys Lys Ile Phe Leu Lys Ala Glu Ser Glu Glu Glu Ile Phe Ala His 355 360 365 Leu Gly Leu Asp Tyr Ile Glu Pro Trp Glu Arg Asn Ala 370 375 380 <210> 10 <211> 382 <212> PRT <213> TRUNC LOST (LOST TRUNC) <400> 10 Gly Asp Cys Pro Ala Ser His Asp Ser Ser Pro Gln Lys Thr Glu Ser 1 5 10 15 Only Gln Lys With Gln Tyr Only Cys Gln Arg Thr Thr 20 25 30 Leu Asn Asn His Asn His Ile Phe Thr Asp Ala Phe Glu Ile Leu Ala 35 40 45 Glu Asn Cys Glu Phe Arg Glu Asn Gly Ser Tyr Val Thr Tyr Met 50 55 60 Arg Ala Ala Ser Val Leu Lys Ser Leu Pro Phe Ser Ile Ile Ser Met 65 70 75 80 Lys Asp Thr Glu Gly Ile Pro Cys Leu Ala Asp Lys Val Lys Cys Val 85 90 95 Ile Glu Glu Ile Ile Glu Asp Gly Glu Ser Ser Glu Val Lys Ala Val 100 105 110 Leu Asn Asp Glu Arg Tyr Lys Ser Phe Lys Leu Phe Thr Ser Val Phe 115 120 125 Gly Val Gly Leu Lys Thr Ala Glu Lys Trp Phe Arg Leu Gly Phe Arg 130 135 140 Thr Leu Ser Gly Ile Met Asn Asp Lys Thr Leu Lys Leu Thr His Met 145 150 155 160 Gln Lys Ala Gly Phe Leu Tyr Tyr Glu Asp Leu Val Ser Cys Val Thr 165 170 175 Arg Ala Glu Ala Glu Ala Val Gly Val Leu Val Lys Glu Ala Val Trp 180 185 190 Ala Phe Leu Pro Asp Ala Ile Val Thr Met Thr Gly Gly Phe Arg Arg 195 200 205 Gly Lys Lys Val Gly His Asp Val Asp Phe Leu Ile Thr Ser Pro Glu 210 215 220 Ala Thr Glu Glu Gln Glu Gln Gln Leu Leu His Lys Val Ile Thr Phe 225 230 235 240 Trp Glu Lys Glu Gly Leu Leu Leu Tyr Cys Asp Leu Tyr Glu Ser Thr 245 250 255 Phe Glu Lys Leu Lys Met Pro Ser Arg Lys Val Asp Ala Leu Asp His 260 265 270 Phe Gln Lys Cys Phe Leu Ile Leu Lys Leu His Arg Glu Cys Val Asp 275 280 285 Asp Gly Thr Ser Ser Gln Leu Gln Gly Lys Thr Trp Lys Ala Ile Arg 290 295 300 Val Asp Leu Val Val Cys Pro Tyr Glu Cys Arg Ala Phe Ala Leu Leu 305 310 315 320 Gly Trp Thr Gly Ser Pro Gln Phe Glu Arg Asp Leu Arg Arg Tyr Ala 325 330 335 Thr His Glu Arg Lys Met Met Leu Asp Asn His Ala Leu Tyr Asp Lys 340 345 350 Thr Lys Arg Lys Phe Leu Ser Ala Asp Ser Glu Glu Asp Ile Phe Ala 355 360 365 His Leu Gly Leu Asp Tyr Ile Glu Pro Trp Glu Arg Asn Ala 370 375 380 <210> 11 <211> 379 <212> PRT <213> PIKA TRUNK <400> 11 Glu Tyr Ser Ala Asn Pro Ser Pro Gly Pro Gln Ala Thr Pro Ala Val 1 5 10 15 Tyr Lys Ile Ser Gln Tyr Ala Cys Gln Arg Arg Thr Thr Leu Asn Asn 20 25 30 His Asn His Ile Phe Thr Asp Ala Phe Glu Ile Leu Ala Glu Asn Tyr 35 40 45 Glu Phe Lys Glu Asn Glu Gly Cys Tyr Val Thr Tyr Met Arg Ala Ala 50 55 60 Ser Val Leu Lys Ser Leu Pro Phe Thr Ile Val Ser Met Lys Asp Thr 65 70 75 80 Glu Gly Ile Pro Cys Leu Glu Asp Lys Val Lys Ser Ile Met Glu Glu 85 90 95 Ile Ile Glu Glu Gly Glu Ser Ser Glu Val Lys Ala Val Leu Ser Asp 100 105 110 Glu Arg Tyr Gln Cys Phe Lys Leu Phe Thr Ser Val Phe Gly Val Gly 115 120 125 Leu Lys Thr Ser Glu Lys Trp Phe Arg Met Gly Phe Arg Ser Leu Ser 130 135 140 Asn Ile Arg Leu Asp Lys Ser Leu Lys Phe Thr Gln Met Gln Lys Ala 145 150 155 160 Gly Phe Arg Tyr Tyr Glu Asp Ile Val Ser Cys Val Thr Arg Ala Glu 165 170 175 Ala Glu Ala Val Asp Val Leu Val Asn Glu Ala Val Arg Ala Phe Leu 180 185 190 Pro Asp Ala Phe Ile Thr Met Thr Gly Gly Phe Arg Arg Gly Lys Lys 195 200 205 Ile Gly His Asp Val Asp Phe Leu Ile Thr Ser Pro Glu Leu Thr Glu 210 215 220 Glu Asp Glu Gln Gln Leu Leu His Lys Val Met Asn Leu Trp Glu Lys 225 230 235 240 Lys Gly Leu Leu Leu Tyr His Asp Leu Val Glu Ser Thr Phe Glu Lys 245 250 255 Leu Lys Gln Pro Ser Arg Lys Val Asp Ala Leu Asp His Phe Gln Lys 260 265 270 Cys Phe Leu Ile Phe Lys Leu Tyr His Glu Arg Val Gly Gly Asp Arg 275 280 285 Cys Arg Gln Pro Glu Gly Lys Asp Trp Lys Ala Ile Arg Val Asp Leu 290 295 300 Val Met Cys Pro Tyr Glu Cys His Ala Phe Ala Leu Leu Gly Trp Thr 305 310 315 320 Gly Ser Arg Gln Phe Glu Arg Asp Leu Arg Arg Tyr Ala Ser His Glu 325 330 335 Arg Lys Met With Asp Asn His Only Tyr Asp Lys Thr Lys Arg 340 345 350 Val Phe Leu Gln Ala Glu Asn Glu Glu Glu Ile Phe Ala His Leu Gly 355 360 365 Leu Asp Tyr With Glu Pro Trp Glu Arg Asn Ala 370,375 <210> 12 <211> 384 <212> PRT <213> TRUNC HEDGEHOG <400> 12 Asp Ala Ser Phe Gly Ser Asn Pro Gly Ser Gln Asn Thr Pro Pro Leu 1 5 10 15 Only Lys Lys Only Has Gln Tyr Only Cys Only Gln Arg Arg Thr Ser Leu 20 25 30 Asn Asn Cys Asn His Process Thr Asp To Leu Asp To Leu To Glu 35 40 45 Asn His Glu Phe Arg Glu Asn Glu Val Ser Cys Val Ala Phe Met Arg 50 55 60 Ala Ala Ser Val Leu Lys Ser Leu Pro Phe Thr Ile Ile Ser Met Lys 65 70 75 80 Asp Thr Lys Gly Ile Pro Cys Leu Gly Asp Lys Ala Lys Cys Val Ile 85 90 95 Glu Glu Ile Ile Glu Asp Gly Glu Ser Ser Glu Val Lys Ala Ile Leu 100 105 110 Asn Asp Glu Arg Tyr Gln Ser Phe Lys Leu Phe Thr Ser Val Phe Gly 115 120 125 Val Gly Leu Lys Thr Ser Glu Lys Trp Phe Arg Met Gly Phe Arg Thr 130 135 140 Leu Asn Lys Ile Met Ser Asp Lys Thr Leu Lys Leu Thr Arg Met Gln 145 150 155 160 Lys Ala Gly Phe Leu Tyr Tyr Glu Asp Leu Val Ser Cys Val Ala Lys 165 170 175 Ala Glu Ala Asp Ala Val Ser Val Leu Val Gln Glu Ala Val Trp Ala 180 185 190 Phe Leu Pro Asp Ala Met Val Thr Met Thr Gly Gly Phe Arg Arg Gly 195 200 205 Lys Lys Leu Gly His Asp Val Asp Phe Leu Ile Thr Ser Pro Gly Ala 210 215 220 Thr Glu Glu Glu Glu Gln Gln Leu Leu Pro Lys Val Ile Asn Phe Trp 225 230 235 240 Glu Arg Lys Gly Leu Leu Leu Tyr His Asp Leu Val Glu Ser Thr Phe 245 250 255 Glu Lys Leu Lys Leu Pro Ser Arg Lys Val Asp Ala Leu Asp His Phe 260 265 270 Gln Lys Cys Phe Leu Ile Leu Lys Leu His Leu Gln His Val Asn Gly 275 280 285 Val Gly Asn Ser Lys Thr Gly Gln Gln Glu Gly Lys Asn Trp Lys Ala 290 295 300 Ile Arg Val Asp Leu Val Met Cys Pro Tyr Glu Arg Arg Ala Phe Ala 305 310 315 320 Leu Leu Gly Trp Thr Gly Ser Arg Gln Phe Glu Arg Asp Leu Arg Arg 325 330 335 Phe Ala Thr His Glu Arg Lys Met Met Leu Asp Asn His Ala Leu Tyr 340 345 350 Asp Lys Thr Lys Arg Ile Phe Leu Lys Ala Glu Ser Glu Glu Glu Ile 355 360 365 Phe Ala His Leu Gly Leu Asp Tyr Ile Asp Pro Trp Glu Arg Asn Ala 370 375 380 <210> 13 <211> 381 <212> PRT <213> Tree Shrew <400> 13 Asp His Ser Thr Ser Pro Ser Pro Gly Pro Gln Lys Thr Pro Ala Leu 1 5 10 15 Ala Val Gln Lys Ile Ser Gln Tyr Ala Cys Gln Arg Arg Thr Thr Leu 20 25 30 Asn Asn Cys Asn Arg Val Phe Thr Asp Ala Phe Glu Thr Leu Ala Glu 35 40 45 Asn Tyr Glu Phe Arg Glu Asn Glu Asp Ser Ser Val Ile Phe Leu Arg 50 55 60 Ala Ala Ser Val Leu Arg Ser Leu Pro Phe Thr Ile Thr Ser Met Arg 65 70 75 80 Asp Thr Glu Gly Leu Pro Cys Leu Gly Asp Lys Val Lys Cys Val Ile 85 90 95 Glu Glu Ile Ile Glu Asp Gly Glu Ser Ser Glu Val Asn Ala Val Leu 100 105 110 Asn Asp Glu Arg Tyr Lys Ser Phe Lys Leu Phe Thr Ser Val Phe Gly 115 120 125 Val Gly Leu Lys Thr Ser Glu Lys Trp Phe Arg Met Gly Phe Arg Thr 130 135 140 Leu Ser Arg Val Arg Ser Asp Lys Ser Leu His Leu Thr Arg Met Gln 145 150 155 160 Gln Ala Gly Phe Leu Tyr Tyr Glu Asp Leu Ala Ser Cys Val Thr Arg 165 170 175 Ala Glu Ala Glu Ala Val Gly Val Leu Val Lys Glu Ala Val Gly Ala 180 185 190 Phe Leu Pro Asp Ala Leu Val Thr Ile Thr Gly Gly Phe Arg Arg Gly 195 200 205 Lys Lys Thr Gly His Asp Val Asp Phe Leu Ile Thr Ser Pro Gly Ser 210 215 220 Thr Glu Glu Lys Glu Glu Glu Leu Leu Gln Lys Val Leu Asn Leu Trp 225 230 235 240 Glu Lys Lys Gly Leu Leu Leu Tyr Tyr Asp Leu Val Glu Ser Thr Phe 245 250 255 Glu Lys Leu Lys Thr Pro Ser Arg Lys Val Asp Ala Leu Asp His Phe 260 265 270 Pro Lys Cys Phe Leu Ile Leu Lys Leu His His Gln Arg Val Asp Gly 275 280 285 Asp Lys Pro Ser Gln Gln Glu Gly Lys Ser Trp Lys Ala Ile Arg Val 290 295 300 Asp Leu Val Met Cys Pro Tyr Glu Arg His Ala Phe Ala Leu Leu Gly 305 310 315 320 Trp Thr Gly Ser Arg Gln Phe Glu Arg Asp Leu Arg Arg Tyr Ala Thr 325 330 335 His Glu Arg Lys Met Met Leu Asp Asn His Ala Leu Tyr Asp Lys Thr 340 345 350 Lys Arg Val Phe Leu Lys Ala Glu Ser Glu Glu Asp Ile Phe Ala His 355 360 365 Leu Gly Leu Asp Tyr Ile Glu Pro Trp Glu Arg Asn Ala 370 375 380 <210> 14 <211> 394 <212> PRT <213> PLATYPUS <400> 14 Leu Thr Asn Ser Ala Pro Ile Asn Cys Met Thr Glu Thr Pro Ser Leu 1 5 10 15 Ala Thr Lys Gln Val Ser Gln Tyr Ala Cys Glu Arg Arg Thr Thr Leu 20 25 30 Asn Asn Cys Asn Gln Lys Phe Thr Asp Ala Phe Glu Ile Leu Ala Lys 35 40 45 Asp Phe Glu Phe Arg Glu Asn Glu Gly Ile Cys Leu Ala Phe Met Arg 50 55 60 Ala Ile Ser Val Leu Lys Cys Leu Pro Phe Thr Ile Val Arg Met Lys 65 70 75 80 Asp Ile Glu Gly Val Pro Trp Leu Gly Asp Gln Val Lys Ser Ile Ile 85 90 95 Glu Glu Ile Ile Glu Asp Gly Glu Ser Ser Ser Val Lys Ala Val Leu 100 105 110 Asn Asp Glu Arg Tyr Arg Ser Phe Gln Leu Phe Asn Ser Val Phe Glu 115 120 125 Val Gly Leu Thr Asp Asn Gly Glu Asn Gly Ile Ala Arg Gly Phe Gln 130 135 140 Thr Leu Asn Glu Val Ile Thr Asp Glu Asn Ile Ser Leu Thr Lys Thr 145 150 155 160 Thr Leu Ser Thr Ser Leu Trp Asn Tyr Leu Pro Gly Phe Leu Tyr Tyr 165 170 175 Glu Asp Leu Val Ser Cys Val Ala Lys Glu Glu Ala Asp Ala Val Tyr 180 185 190 Leu Ile Val Lys Glu Ala Val Arg Ala Phe Leu Pro Glu Ala Leu Val 195 200 205 Thr Leu Thr Gly Gly Phe Arg Arg Gly Lys Lys Ile Gly His Asp Val 210 215 220 Asp Phe Leu Ile Ser Asp Pro Glu Ser Gly Gln Asp Glu Gln Leu Leu 225 230 235 240 Pro Asn Ile Ile Lys Leu Trp Glu Lys Gln Glu Leu Leu Leu Tyr Tyr 245 250 255 Asp Leu Val Glu Ser Thr Phe Glu Lys Thr Lys Ile Pro Ser Arg Lys 260 265 270 Val Asp Ala Met Asp His Phe Gln Lys Cys Phe Leu Ile Leu Lys Leu 275 280 285 His His Gln Lys Val Asp Ser Gly Arg Tyr Lys Pro Pro Pro Glu Ser 290 295 300 Lys Asn His Glu Ala Lys Asn Trp Lys Ala Ile Arg Val Asp Leu Val 305 310 315 320 Met Cys Pro Phe Glu Gln Tyr Ala Tyr Ala Leu Leu Gly Trp Thr Gly 325 330 335 Ser Arg Gln Phe Glu Arg Asp Leu Arg Arg Tyr Ala Thr His Glu Lys 340 345 350 Lys Met Met Leu Asp Asn His Ala Leu Tyr Asp Lys Thr Lys Lys Ile 355 360 365 Phe Leu Lys Ala Glu Ser Glu Glu Asp Ile Phe Thr His Leu Gly Leu 370 375 380 Asp Tyr Ile Glu Pro Trp Glu Arg Asn Ala 385 390 <210> 15 <211> 384 <212> PRT <213> Jerboa <400> 15 Ser Ser Glu Leu Glu Leu Leu Asp Val Ser Trp Leu Ile Glu Cys Met 1 5 10 15 Gly Ala Gly Lys Pro Val Glu Met Thr Gly Arg His Gln Leu Val Lys 20 25 30 Gln Thr Phe Cys Leu Pro Gly Phe Ile Leu Gln Asp Ala Phe Asp Ile 35 40 45 Leu Ala Glu Asn Cys Glu Phe Arg Glu Asn Glu Ala Ser Cys Val Glu 50 55 60 Phe Met Arg Ala Ala Ser Val Leu Lys Ser Leu Pro Phe Pro Ile Ile 65 70 75 80 Ser Val Lys Asp Thr Glu Gly Ile Pro Trp Leu Gly Gly Lys Val Lys 85 90 95 Cys Val Ile Glu Glu Ile Ile Glu Asp Gly Glu Ser Ser Glu Val Lys 100 105 110 Ala Leu Leu Asn Asp Glu Arg Tyr Lys Ser Phe Lys Leu Phe Thr Ser 115 120 125 Val Phe Gly Val Gly Leu Lys Thr Ala Glu Arg Trp Phe Arg Met Gly 130 135 140 Phe Arg Thr Leu Ser Thr Val Lys Leu Asp Lys Ser Leu Thr Phe Thr 145 150 155 160 Arg Met Gln Lys Ala Gly Phe Leu His Tyr Glu Asp Leu Val Ser Cys 165 170 175 Val Thr Arg Ala Glu Ala Glu Ala Val Ser Val Leu Val Gln Gln Ala 180 185 190 Val Val Ala Phe Leu Pro Asp Ala Leu Val Ser Met Thr Gly Gly Phe 195 200 205 Arg Arg Gly Lys Lys Ile Gly His Asp Val Asp Phe Leu Ile Thr Ser 210 215 220 Pro Glu Ala Thr Glu Glu Glu Glu Gln Gln Leu Leu His Lys Val Thr 225 230 235 240 Asn Phe Trp Glu Gln Lys Gly Leu Leu Leu Tyr Cys Asp His Val Glu 245 250 255 Ser Thr Phe Glu Lys Cys Lys Leu Pro Ser Arg Lys Val Asp Ala Leu 260 265 270 Asp His Phe Gln Lys Cys Phe Leu Ile Leu Lys Leu Tyr Arg Glu Arg 275 280 285 Val Asp Ser Val Lys Ser Ser Gln Gln Glu Gly Lys Gly Trp Lys Ala 290 295 300 Ile Arg Val Asp Leu Val Met Cys Pro Tyr Glu Cys Arg Ala Phe Ala 305 310 315 320 Leu Leu Gly Trp Thr Gly Ser Arg Gln Phe Glu Arg Asp Leu Arg Arg 325 330 335 Tyr Ala Thr His Glu Arg Lys Met Arg Leu Asp Asn His Ala Leu Tyr 340 345 350 Asp Lys Thr Lys Arg Val Phe Leu Lys Ala Glu Ser Glu Glu Glu Ile 355 360 365 Phe Ala His Leu Gly Leu Glu Tyr Ile Glu Pro Leu Glu Arg Asn Ala 370 375 380 <210> 16 <211> 362 <212> PRT <213> Truncated mouse <400> 16 Lys Ile Ser Gln Tyr Ala Cys Gln Arg Arg Thr Thr Leu Asn Asn Tyr 1 5 10 15 Asn Glu Leu Phe Thr Arg Ala Leu Asp Ile Leu Ala Glu Asn Asp Glu 20 25 30 Phe Arg Glu Asn Glu Glu Ser Arg Asp Ala Phe Arg Arg Ala Ser Ser 35 40 45 Val Leu Lys Ser Leu Pro Phe Pro Ile Thr Ser Met Lys Asp Thr Glu 50 55 60 Gly Ile Pro Cys Leu Gly Asp Lys Val Lys Arg Ile Ile Glu Glu Ile 65 70 75 80 Ile Glu Asp Gly Glu Ser Ser Glu Val Lys Ala Val Leu Asn Asp Glu 85 90 95 Arg Tyr Lys Ala Phe Lys Leu Phe Thr Ser Val Phe Gly Val Gly Arg 100 105 110 Lys Thr Ala Glu Lys Trp Phe Arg Met Gly Phe Arg Thr Leu Glu Lys 115 120 125 Ile Arg Ser Asp Lys Ser Leu Arg Phe Thr Gln Met Gln Lys Ala Gly 130 135 140 Phe Leu Tyr Tyr Glu Asp Leu Ala Glu Arg Val Asn Arg Pro Glu Ala 145 150 155 160 Glu Ala Ile Arg Met Leu Val Lys Glu Ala Val Val Thr Phe Leu Pro 165 170 175 Asp Ala Leu Val Thr Met Thr Gly Gly Phe Arg Leu Gly Lys Met Thr 180 185 190 Gly His Asp Val Asp Phe Leu Ile Thr Ser Pro Glu Ala Thr Glu Asp 195 200 205 Glu Glu Gln Gln Leu Leu His Lys Val Thr Asp Phe Trp Lys Gln Gln 210 215 220 Gly Leu Leu Leu Tyr Cys Asp Ile Leu Glu Ser Thr Phe Glu Lys Phe 225 230 235 240 Lys Gln Pro Ser Arg Thr Val Asp Ala Leu Asp His Phe Gln Lys Cys 245 250 255 Phe Leu Ile Leu Lys Leu Asp His Pro Arg Val His Ser Val Lys Ser 260 265 270 Gly Gln Gln Glu Gly Lys Gly Trp Lys Ala Ile Arg Val Asp Leu Val 275 280 285 Met Cys Pro Tyr Asp Arg Arg Ala Phe Ala Leu Leu Gly Trp Thr Gly 290 295 300 Ser Pro Gln Phe Asn Arg Asp Leu Arg Arg Tyr Ala Thr His Glu Arg 305 310 315 320 Lys Met Met Leu Asp Asn His Ala Leu Tyr Asp Lys Thr Lys Arg Val 325 330 335 Phe Leu Glu Ala Glu Ser Glu Glu Glu Ile Phe Ala His Leu Gly Leu 340 345 350 Asp Tyr Ile Glu Pro Trp Glu Arg Asn Ala 355 360 <210> 17 <211> 361 <212> PRT <213> ARTIFICIAL SEQUENCE <220> <223> Bovine-Mouse Chimera (M57 without His tag & linker) <400> 17 Lys Ile Ser Gln Tyr Ala Cys Gln Arg Arg Thr Thr Leu Asn Asn Tyr 1 5 10 15 Asn Glu Leu Phe Thr Arg Ala Leu Asp Ile Leu Ala Glu Asn Asp Glu 20 25 30 Phe Arg Glu Asn Glu Glu Ser Arg Asp Ala Phe Arg Arg Ala Ser Ser 35 40 45 Val Leu Lys Ser Leu Pro Phe Pro Ile Thr Ser Met Lys Asp Thr Glu 50 55 60 Gly Ile Pro Cys Leu Gly Asp Lys Val Lys Arg Ile Ile Glu Glu Ile 65 70 75 80 Ile Glu Asp Gly Glu Ser Ser Glu Val Lys Ala Val Leu Asn Asp Glu 85 90 95 Arg Tyr Lys Ala Phe Lys Leu Phe Thr Ser Val Phe Gly Val Gly Arg 100 105 110 Lys Thr Ala Glu Lys Trp Phe Arg Met Gly Phe Arg Thr Leu Glu Lys 115 120 125 Ile Arg Ser Asp Lys Ser Leu Arg Phe Thr Gln Met Gln Lys Ala Gly 130 135 140 Phe Leu Tyr Tyr Glu Asp Leu Val Ser Gly Val Thr Arg Ala Glu Ala 145 150 155 160 Glu Ala Val Gly Val Leu Val Lys Glu Ala Val Trp Ala Phe Leu Pro 165 170 175 Asp Ala Phe Val Thr Met Thr Gly Gly Phe Arg Leu Gly Lys Lys Ile 180 185 190 Gly His Asp Val Asp Phe Leu Ile Thr Ser Pro Gly Ser Ala Glu Asp 195 200 205 Glu Glu Gln Leu Leu Pro Lys Val Ile Asn Leu Trp Glu Lys Lys Gly 210 215 220 Leu Leu Leu Tyr Tyr Asp Leu Val Glu Ser Thr Phe Glu Lys Phe Lys 225 230 235 240 Leu Pro Ser Arg Gln Val Asp Thr Leu Asp His Phe Gln Lys Cys Phe 245 250 255 Leu Ile Leu Lys Leu His His Gln Arg Val Asp Ser Ser Lys Ser Asn 260 265 270 Gln Gln Glu Gly Lys Thr Trp Lys Ala Ile Arg Val Asp Leu Val Met 275 280 285 Cys Pro Tyr Glu Asn Arg Ala Phe Ala Leu Leu Gly Trp Thr Gly Ser 290 295 300 Pro Gln Phe Asn Arg Asp Leu Arg Arg Tyr Ala Thr His Glu Arg Lys 305 310 315 320 Met Met Leu Asp Asn His Ala Leu Tyr Asp Lys Thr Lys Arg Val Phe 325 330 335 Leu Glu Ala Glu Ser Glu Glu Glu Ile Phe Ala His Leu Gly Leu Asp 340 345 350 Tyr Ile Glu Pro Trp Glu Arg Asn Ala 355 360 <210> 18 <211> 361 <212> PRT <213> ARTIFICIAL SEQUENCE <220> <223> Bovine-Mouse Chimera (M59 without His tag and linker) <400> 18 Lys Ile Ser Gln Tyr Ala Cys Gln Arg Arg Thr Thr Leu Asn Asn Tyr 1 5 10 15 Asn Glu Leu Phe Thr Arg Ala Leu Asp Ile Leu Ala Glu Asn Asp Glu 20 25 30 Phe Arg Glu Asn Glu Glu Ser Arg Asp Ala Phe Arg Arg Ala Ser Ser 35 40 45 Val Leu Lys Ser Leu Pro Phe Pro Ile Thr Ser Met Lys Asp Thr Glu 50 55 60 Gly Ile Pro Cys Leu Gly Asp Lys Val Lys Arg Ile Ile Glu Glu Ile 65 70 75 80 Ile Glu Asp Gly Glu Ser Ser Glu Val Lys Ala Val Leu Asn Asp Glu 85 90 95 Arg Tyr Lys Ala Phe Lys Leu Phe Thr Ser Val Phe Gly Val Gly Arg 100 105 110 Lys Thr Ala Glu Lys Trp Phe Arg Met Gly Phe Arg Thr Leu Glu Lys 115 120 125 Ile Arg Ser Asp Lys Ser Leu Arg Phe Thr Gln Met Gln Lys Ala Gly 130 135 140 Phe Leu Tyr Tyr Glu Asp Leu Ala Glu Arg Val Asn Arg Pro Glu Ala 145 150 155 160 Glu Ala Ile Arg Met Leu Val Lys Glu Ala Val Val Thr Phe Leu Pro 165 170 175 Asp Ala Leu Val Thr Met Thr Gly Gly Phe Arg Leu Gly Lys Met Thr 180 185 190 Gly His Asp Val Asp Phe Leu Ile Thr Ser Pro Gly Ser Ala Glu Asp 195 200 205 Glu Glu Gln Leu Leu Pro Lys Val Ile Asn Leu Trp Glu Lys Lys Gly 210 215 220 Leu Leu Leu Tyr Tyr Asp Leu Val Glu Ser Thr Phe Glu Lys Phe Lys 225 230 235 240 Leu Pro Ser Arg Gln Val Asp Thr Leu Asp His Phe Gln Lys Cys Phe 245 250 255 Leu Ile Leu Lys Leu His His Gln Arg Val Asp Ser Ser Lys Ser Asn 260 265 270 Gln Gln Glu Gly Lys Thr Trp Lys Ala Ile Arg Val Asp Leu Val Met 275 280 285 Cys Pro Tyr Glu Asn Arg Ala Phe Ala Leu Leu Gly Trp Thr Gly Ser 290 295 300 Pro Gln Phe Asn Arg Asp Leu Arg Arg Tyr Ala Thr His Glu Arg Lys 305 310 315 320 Met Met Leu Asp Asn His Ala Leu Tyr Asp Lys Thr Lys Arg Val Phe 325 330 335 Leu Glu Ala Glu Ser Glu Glu Glu Ile Phe Ala His Leu Gly Leu Asp 340 345 350 Tyr Ile Glu Pro Trp Glu Arg Asn Ala 355 360 <210> 19 <211> 361 <212> PRT <213> Mouse <400> 19 Lys Ile Ser Gln Tyr Ala Cys Gln Arg Arg Thr Thr Leu Asn Asn Tyr 1 5 10 15 Asn Glu Leu Phe Thr Arg Ala Leu Asp Ile Leu Ala Glu Asn Asp Glu 20 25 30 Phe Arg Glu Asn Glu Glu Ser Arg Asp Ala Phe Arg Arg Ala Ser Ser 35 40 45 Val Leu Lys Ser Leu Pro Phe Pro Ile Thr Ser Met Lys Asp Thr Glu 50 55 60 Gly Ile Pro Cys Leu Gly Asp Lys Val Lys Arg Ile Ile Glu Glu Ile 65 70 75 80 Ile Glu Asp Gly Glu Ser Ser Glu Val Lys Ala Val Leu Asn Asp Glu 85 90 95 Arg Tyr Lys Ala Phe Lys Leu Phe Thr Ser Val Phe Gly Val Gly Arg 100 105 110 Lys Thr Ala Glu Lys Trp Phe Arg Met Gly Phe Arg Thr Leu Glu Lys 115 120 125 Ile Arg Ser Asp Lys Ser Leu Arg Phe Thr Gln Met Gln Lys Ala Gly 130 135 140 Phe Leu Tyr Tyr Glu Asp Leu Ala Glu Arg Val Asn Arg Pro Glu Ala 145 150 155 160 Glu Ala Ile Arg Met Leu Val Lys Glu Ala Val Val Thr Phe Leu Pro 165 170 175 Asp Ala Leu Val Thr Met Thr Gly Gly Phe Arg Leu Gly Lys Met Thr 180 185 190 Gly His Asp Val Asp Phe Leu Ile Thr Ser Pro Gly Ser Ala Glu Asp 195 200 205 Glu Glu Gln Leu Leu Pro Lys Val Ile Asn Leu Trp Glu Lys Lys Gly 210 215 220 Leu Leu Leu Tyr Tyr Asp Leu Val Glu Ser Thr Phe Glu Lys Phe Lys 225 230 235 240 Leu Pro Ser Arg Gln Val Asp Thr Leu Asp His Phe Gln Lys Cys Phe 245 250 255 Leu Ile Leu Lys Leu His His Gln Arg Val Asp Ser Ser Lys Ser Asn 260 265 270 Gln Gln Glu Gly Lys Thr Trp Lys Ala Ile Arg Val Asp Leu Val Met 275 280 285 Cys Pro Tyr Glu Asn Arg Ala Phe Ala Leu Leu Gly Trp Thr Gly Ser 290 295 300 Pro Gln Phe Asn Arg Asp Leu Arg Arg Tyr Ala Thr His Glu Arg Lys 305 310 315 320 Met Met Leu Asp Asn His Ala Leu Tyr Asp Lys Thr Lys Arg Val Phe 325 330 335 Leu Glu Ala Glu Ser Glu Glu Glu Ile Phe Ala His Leu Gly Leu Asp 340 345 350 Tyr Ile Glu Pro Trp Glu Arg Asn Ala 355 360 <210> 20 <211> 361 <212> PRT <213> Artificial Sequence <220> <223> M54 <400> 20 Lys Ile Ser Gln Tyr Ala Cys Gln Arg Lys Thr Thr Leu Asn Asn Tyr 1 5 10 15 Asn Glu Ile Phe Thr Arg Ala Phe Glu Ile Leu Ala Glu Asn Ser Glu 20 25 30 Phe Lys Glu Asn Glu Glu Ser Tyr Val Thr Phe Arg Arg Ala Ala Ser 35 40 45 Val Leu Lys Ser Leu Pro Phe Thr Ile Ile Ser Met Lys Asp Thr Glu 50 55 60 Gly Ile Pro Cys Leu Gly Asp Lys Val Lys Arg Ile Ile Glu Glu Ile 65 70 75 80 Ile Glu Asp Gly Glu Ser Ser Glu Val Lys Ala Val Leu Asn Asp Glu 85 90 95 Arg Tyr Gln Ala Phe Lys Leu Phe Thr Ser Val Phe Gly Val Gly Arg 100 105 110 Lys Thr Ser Glu Lys Trp Phe Arg Met Gly Phe Arg Ser Leu Glu Lys 115 120 125 Ile Arg Ser Asp Lys Thr Leu Lys Phe Thr Lys Met Gln Lys Ala Gly 130 135 140 Phe Leu Tyr Tyr Glu Asp Leu Val Ser Gly Val Thr Arg Ala Glu Ala 145 150 155 160 Glu Ala Val Gly Val Leu Val Lys Glu Ala Val Trp Ala Phe Leu Pro 165 170 175 Asp Ala Phe Val Thr Met Thr Gly Gly Phe Arg Leu Gly Lys Lys Ile 180 185 190 Gly His Asp Val Asp Phe Leu Ile Thr Ser Pro Gly Ser Ala Glu Asp 195 200 205 Glu Glu Gln Leu Leu Pro Lys Val Ile Asn Leu Trp Glu Lys Lys Gly 210 215 220 Leu Leu Leu Tyr Tyr Asp Leu Val Glu Ser Thr Phe Glu Lys Phe Lys 225 230 235 240 Leu Pro Ser Arg Gln Val Asp Thr Leu Asp His Phe Gln Lys Cys Phe 245 250 255 Leu Ile Leu Lys Leu His His Gln Arg Val Asp Ser Ser Lys Ser Asn 260 265 270 Gln Gln Glu Gly Lys Thr Trp Lys Ala Ile Arg Val Asp Leu Val Met 275 280 285 Cys Pro Tyr Glu Asn Arg Ala Phe Ala Leu Leu Gly Trp Thr Gly Ser 290 295 300 Ala Phe Phe Asn Arg Asp Ile Arg Arg Tyr Ala Thr His Glu Arg Lys 305 310 315 320 Met Met Leu Asp Asn His Ala Leu Tyr Asp Lys Thr Lys Arg Val Phe 325 330 335 Leu Lys Ala Glu Ser Glu Glu Glu Ile Phe Ala His Leu Gly Leu Asp 340 345 350 Tyr Ile Glu Pro Arg Glu Arg Asn Ala 355 360 <210> 21 <211> 362 <212> PRT <213> Artificial Sequence <220> M77 without His tag <400> 21 Lys Lys Ile Ser Glu Tyr Ala Cys Gln Arg Arg Thr Thr Leu Asn Asn 1 5 10 15 Tyr Asn Glu Leu Phe Thr Arg Ala Leu Asp Ile Leu Ala Glu Asn Asp 20 25 30 Glu Phe Arg Glu Asn Glu Glu Ser Arg Asp Ala Phe Arg Arg Ala Ser 35 40 45 Ser Val Leu Lys Ser Leu Pro Phe Pro Ile Thr Ser Met Lys Asp Thr 50 55 60 Glu Gly Ile Pro Cys Leu Gly Asp Lys Val Lys Arg Ile Ile Glu Glu 65 70 75 80 Ile Ile Glu Asp Gly Glu Ser Ser Glu Val Lys Ala Val Leu Asn Asp 85 90 95 Glu Arg Tyr Lys Ala Phe Lys Leu Phe Thr Ser Val Phe Gly Val Gly 100 105 110 Arg Lys Thr Ala Glu Lys Trp Phe Arg Met Gly Phe Arg Thr Leu Glu 115 120 125 Lys Ile Arg Ser Asp Lys Ser Leu Arg Phe Thr Gln Met Gln Lys Ala 130 135 140 Gly Phe Leu Tyr Tyr Glu Asp Leu Val Ser Gly Val Thr Arg Ala Glu 145 150 155 160 Ala Glu Ala Val Gly Val Leu Val Lys Glu Ala Val Trp Ala Phe Leu 165 170 175 Pro Asp Ala Phe Val Thr Met Thr Gly Gly Phe Arg Leu Gly Lys Lys 180 185 190 Ile Gly His Asp Val Asp Phe Leu Ile Thr Ser Pro Gly Ser Ala Glu 195 200 205 Asp Glu Glu Gln Leu Leu Pro Lys Val Ile Asn Leu Trp Glu Lys Lys 210 215 220 Gly Leu Leu Leu Tyr Tyr Asp Leu Val Glu Ser Thr Phe Glu Lys Phe 225 230 235 240 Lys Leu Pro Ser Arg Gln Val Asp Thr Leu Asp His Phe Gln Lys Cys 245 250 255 Phe Leu Ile Leu Lys Leu His His Gln Arg Val Asp Ser Ser Lys Ser 260 265 270 Asn Gln Gln Glu Gly Lys Thr Trp Lys Ala Ile Arg Val Asp Leu Val 275 280 285 Met Cys Pro Tyr Glu Asn Arg Ala Phe Ala Leu Leu Gly Trp Thr Gly 290 295 300 Ser Pro Gln Phe Asn Arg Asp Leu Arg Arg Tyr Ala Thr His Glu Arg 305 310 315 320 Lys Met Met Leu Asp Asn His Ala Leu Tyr Asp Lys Thr Lys Arg Val 325 330 335 Phe Leu Glu Ala Glu Ser Glu Glu Glu Ile Phe Ala His Leu Gly Leu 340 345 350 Asp Tyr Ile Glu Pro Trp Glu Arg Asn Ala 355 360 <210> 22 <211> 377 <212> PRT <213> Artificial Sequence <220> <223> M77 with His-tag <400> 22 Met Ala Ser Ser His His His His His His Ser Ser Gly Ser Glu Lys 1 5 10 15 Lys Ile Ser Glu Tyr Ala Cys Gln Arg Arg Thr Thr Leu Asn Asn Tyr 20 25 30 Asn Glu Leu Phe Thr Arg Ala Leu Asp Ile Leu Ala Glu Asn Asp Glu 35 40 45 Phe Arg Glu Asn Glu Glu Ser Arg Asp Ala Phe Arg Arg Ala Ser Ser 50 55 60 Val Leu Lys Ser Leu Pro Phe Pro Ile Thr Ser Met Lys Asp Thr Glu 65 70 75 80 Gly Ile Pro Cys Leu Gly Asp Lys Val Lys Arg Ile Ile Glu Glu Ile 85 90 95 Ile Glu Asp Gly Glu Ser Ser Glu Val Lys Ala Val Leu Asn Asp Glu 100 105 110 Arg Tyr Lys Ala Phe Lys Leu Phe Thr Ser Val Phe Gly Val Gly Arg 115 120 125 Lys Thr Ala Glu Lys Trp Phe Arg Met Gly Phe Arg Thr Leu Glu Lys 130 135 140 Ile Arg Ser Asp Lys Ser Leu Arg Phe Thr Gln Met Gln Lys Ala Gly 145 150 155 160 Phe Leu Tyr Tyr Glu Asp Leu Val Ser Gly Val Thr Arg Ala Glu Ala 165 170 175 Glu Ala Val Gly Val Leu Val Lys Glu Ala Val Trp Ala Phe Leu Pro 180 185 190 Asp Ala Phe Val Thr Met Thr Gly Gly Phe Arg Leu Gly Lys Lys Ile 195 200 205 Gly His Asp Val Asp Phe Leu Ile Thr Ser Pro Gly Ser Ala Glu Asp 210 215 220 Glu Glu Gln Leu Leu Pro Lys Val Ile Asn Leu Trp Glu Lys Lys Gly 225 230 235 240 Leu Leu Leu Tyr Tyr Asp Leu Val Glu Ser Thr Phe Glu Lys Phe Lys 245 250 255 Leu Pro Ser Arg Gln Val Asp Thr Leu Asp His Phe Gln Lys Cys Phe 260 265 270 Leu Ile Leu Lys Leu His His Gln Arg Val Asp Ser Ser Lys Ser Asn 275 280 285 Gln Gln Glu Gly Lys Thr Trp Lys Ala Ile Arg Val Asp Leu Val Met 290 295 300 Cys Pro Tyr Glu Asn Arg Ala Phe Ala Leu Leu Gly Trp Thr Gly Ser 305 310 315 320 Pro Gln Phe Asn Arg Asp Leu Arg Arg Tyr Ala Thr His Glu Arg Lys 325 330 335 Met Met Leu Asp Asn His Ala Leu Tyr Asp Lys Thr Lys Arg Val Phe 340 345 350 Leu Glu Ala Glu Ser Glu Glu Glu Ile Phe Ala His Leu Gly Leu Asp 355 360 365 Tyr Ile Glu Pro Trp Glu Arg Asn Ala 370 375 <210> 23 <211> 361 <212> PRT <213> Artificial Sequence <220> <223> C1-40 without His tag <400> 23 Lys Ile Ser Glu Tyr Ala Cys Gln Arg Arg Thr Thr Leu Asn Asn Tyr 1 5 10 15 Asn Glu Leu Phe Thr Arg Ala Leu Asp Ile Leu Ala Glu Asn Asp Glu 20 25 30 Phe Arg Glu Asn Glu Glu Ser Arg Asp Ala Phe Arg Arg Ala Ser Ser 35 40 45 Val Leu Lys Ser Leu Pro Phe Pro Ile Thr Ser Met Lys Asp Thr Glu 50 55 60 Gly Ile Pro Cys Leu Gly Asp Lys Val Lys Arg Ile Ile Glu Glu Ile 65 70 75 80 Ile Glu Asp Gly Glu Ser Ser Glu Val Lys Ala Val Leu Asn Asp Glu 85 90 95 Arg Tyr Lys Ala Phe Lys Leu Phe Thr Ser Val Phe Gly Val Gly Arg 100 105 110 Lys Thr Ala Glu Lys Trp Phe Arg Met Gly Phe Arg Thr Leu Glu Lys 115 120 125 Ile Arg Ser Asp Lys Ser Leu Arg Phe Thr Gln Met Gln Lys Ala Gly 130 135 140 Phe Leu Tyr Tyr Glu Asp Leu Val Ser Gly Val Thr Arg Ala Glu Ala 145 150 155 160 Glu Ala Val Gly Val Leu Val Lys Glu Ala Val Trp Ala Phe Leu Pro 165 170 175 Asp Ala Phe Val Thr Met Thr Gly Gly Phe Arg Leu Gly Lys Lys Ile 180 185 190 Gly His Asp Val Asp Phe Leu Ile Thr Ser Pro Gly Ser Ala Glu Asp 195 200 205 Glu Glu Gln Leu Leu Pro Lys Val Ile Asn Leu Trp Glu Lys Lys Gly 210 215 220 Leu Leu Leu Tyr Tyr Asp Leu Val Glu Ser Thr Phe Glu Lys Phe Lys 225 230 235 240 Leu Pro Ser Arg Gln Val Asp Thr Leu Asp His Phe Gln Lys Cys Phe 245 250 255 Leu Ile Leu Lys Leu His His Gln Arg Val Asp Ser Ser Lys Ser Asn 260 265 270 Gln Gln Glu Gly Lys Thr Trp Lys Ala Ile Arg Val Asp Leu Val Met 275 280 285 Cys Pro Tyr Glu Asn Arg Ala Phe Ala Leu Leu Gly Trp Thr Gly Ser 290 295 300 Pro Gln Phe Asn Arg Asp Leu Arg Arg Tyr Ala Thr His Glu Arg Lys 305 310 315 320 Met Met Leu Asp Asn His Ala Leu Tyr Asp Lys Thr Lys Arg Val Phe 325 330 335 Leu Glu Ala Glu Ser Glu Glu Glu Ile Phe Ala His Leu Gly Leu Asp 340 345 350 Tyr Ile Glu Pro Trp Glu Arg Asn Ala 355 360 <210> 24 <211> 376 <212> PRT <213> Artificial Sequence <220> <223> C1-40 with His tag <400> 24 Met Ala Ser Ser His His His His His His Ser Ser Gly Ser Glu Lys 1 5 10 15 Ile Ser Glu Tyr Ala Cys Gln Arg Arg Thr Thr Leu Asn Asn Tyr Asn 20 25 30 Glu Leu Phe Thr Arg Ala Leu Asp Ile Leu Ala Glu Asn Asp Glu Phe 35 40 45 Arg Glu Asn Glu Glu Ser Arg Asp Ala Phe Arg Arg Ala Ser Ser Val 50 55 60 Leu Lys Ser Leu Pro Phe Pro Ile Thr Ser Met Lys Asp Thr Glu Gly 65 70 75 80 Ile Pro Cys Leu Gly Asp Lys Val Lys Arg Ile Ile Glu Glu Ile Ile 85 90 95 Glu Asp Gly Glu Ser Ser Glu Val Lys Ala Val Leu Asn Asp Glu Arg 100 105 110 Tyr Lys Ala Phe Lys Leu Phe Thr Ser Val Phe Gly Val Gly Arg Lys 115 120 125 Thr Ala Glu Lys Trp Phe Arg Met Gly Phe Arg Thr Leu Glu Lys Ile 130 135 140 Arg Ser Asp Lys Ser Leu Arg Phe Thr Gln Met Gln Lys Ala Gly Phe 145 150 155 160 Leu Tyr Tyr Glu Asp Leu Val Ser Gly Val Thr Arg Ala Glu Ala Glu 165 170 175 Ala Val Gly Val Leu Val Lys Glu Ala Val Trp Ala Phe Leu Pro Asp 180 185 190 Ala Phe Val Thr Met Thr Gly Gly Phe Arg Leu Gly Lys Lys Ile Gly 195 200 205 His Asp Val Asp Phe Leu Ile Thr Ser Pro Gly Ser Ala Glu Asp Glu 210 215 220 Glu Gln Leu Leu Pro Lys Val Ile Asn Leu Trp Glu Lys Lys Gly Leu 225 230 235 240 Leu Leu Tyr Tyr Asp Leu Val Glu Ser Thr Phe Glu Lys Phe Lys Leu 245 250 255 Pro Ser Arg Gln Val Asp Thr Leu Asp His Phe Gln Lys Cys Phe Leu 260 265 270 Ile Leu Lys Leu His His Gln Arg Val Asp Ser Ser Lys Ser Asn Gln 275 280 285 Gln Glu Gly Lys Thr Trp Lys Ala Ile Arg Val Asp Leu Val Met Cys 290 295 300 Pro Tyr Glu Asn Arg Ala Phe Ala Leu Leu Gly Trp Thr Gly Ser Pro 305 310 315 320 Gln Phe Asn Arg Asp Leu Arg Arg Tyr Ala Thr His Glu Arg Lys Met 325 330 335 Met Leu Asp Asn His Ala Leu Tyr Asp Lys Thr Lys Arg Val Phe Leu 340 345 350 Glu Ala Glu Ser Glu Glu Glu Ile Phe Ala His Leu Gly Leu Asp Tyr 355 360 365 Ile Glu Pro Trp Glu Arg Asn Ala 370 375 <210> 25 <211> 363 <212> PRT <213> Artificial Sequence <220> <223> A2-20 without His tag <400> 25 Val Thr Thr Val Ser Glu Tyr Ala Cys Gln Arg Arg Thr Thr Leu Asn 1 5 10 15 Asn Tyr Asn Glu Leu Phe Thr Arg Ala Leu Asp Ile Leu Ala Glu Asn 20 25 30 Asp Glu Phe Arg Glu Asn Glu Glu Ser Arg Asp Ala Phe Arg Arg Ala 35 40 45 Ser Ser Val Leu Lys Ser Leu Pro Phe Pro Ile Thr Ser Met Lys Asp 50 55 60 Thr Glu Gly Ile Pro Cys Leu Gly Asp Lys Val Lys Arg Ile Ile Glu 65 70 75 80 Glu Ile Ile Glu Asp Gly Glu Ser Ser Glu Val Lys Ala Val Leu Asn 85 90 95 Asp Glu Arg Tyr Lys Ala Phe Lys Leu Phe Thr Ser Val Phe Gly Val 100 105 110 Gly Arg Lys Thr Ala Glu Lys Trp Phe Arg Met Gly Phe Arg Thr Leu 115 120 125 Glu Lys Ile Arg Ser Asp Lys Ser Leu Arg Phe Thr Gln Met Gln Lys 130 135 140 Ala Gly Phe Leu Tyr Tyr Glu Asp Leu Val Ser Gly Val Thr Arg Ala 145 150 155 160 Glu Ala Glu Ala Val Gly Val Leu Val Lys Glu Ala Val Trp Ala Phe 165 170 175 Leu Pro Asp Ala Phe Val Thr Met Thr Gly Gly Phe Arg Leu Gly Lys 180 185 190 Lys Ile Gly His Asp Val Asp Phe Leu Ile Thr Ser Pro Gly Ser Ala 195 200 205 Glu Asp Glu Glu Gln Leu Leu Pro Lys Val Ile Asn Leu Trp Glu Lys 210 215 220 Lys Gly Leu Leu Leu Tyr Tyr Asp Leu Val Glu Ser Thr Phe Glu Lys 225 230 235 240 Phe Lys Leu Pro Ser Arg Gln Val Asp Thr Leu Asp His Phe Gln Lys 245 250 255 Cys Phe Leu Ile Leu Lys Leu His His Gln Arg Val Asp Ser Ser Lys 260 265 270 Ser Asn Gln Gln Glu Gly Lys Thr Trp Lys Ala Ile Arg Val Asp Leu 275 280 285 Val Met Cys Pro Tyr Glu Asn Arg Ala Phe Ala Leu Leu Gly Trp Thr 290 295 300 Gly Ser Pro Gln Phe Asn Arg Asp Leu Arg Arg Tyr Ala Thr His Glu 305 310 315 320 Arg Lys Met Met Leu Asp Asn His Ala Leu Tyr Asp Lys Thr Lys Arg 325 330 335 Val Phe Leu Glu Ala Glu Ser Glu Glu Glu Ile Phe Ala His Leu Gly 340 345 350 Leu Asp Tyr Ile Glu Pro Trp Glu Arg Asn Ala 355 360 <210> 26 <211> 379 <212> PRT <213> Artificial Sequence <220> <223> A2-20 with His-tag <400> 26 Met Ala Ser Ser His His His His His His Ser Ser Gly Ser Glu Asn 1 5 10 15 Val Thr Thr Val Ser Glu Tyr Ala Cys Gln Arg Arg Thr Thr Leu Asn 20 25 30 Asn Tyr Asn Glu Leu Phe Thr Arg Ala Leu Asp Ile Leu Ala Glu Asn 35 40 45 Asp Glu Phe Arg Glu Asn Glu Glu Ser Arg Asp Ala Phe Arg Arg Ala 50 55 60 Ser Ser Val Leu Lys Ser Leu Pro Phe Pro Ile Thr Ser Met Lys Asp 65 70 75 80 Thr Glu Gly Ile Pro Cys Leu Gly Asp Lys Val Lys Arg Ile Ile Glu 85 90 95 Glu Ile Ile Glu Asp Gly Glu Ser Ser Glu Val Lys Ala Val Leu Asn 100 105 110 Asp Glu Arg Tyr Lys Ala Phe Lys Leu Phe Thr Ser Val Phe Gly Val 115 120 125 Gly Arg Lys Thr Ala Glu Lys Trp Phe Arg Met Gly Phe Arg Thr Leu 130 135 140 Glu Lys Ile Arg Ser Asp Lys Ser Leu Arg Phe Thr Gln Met Gln Lys 145 150 155 160 Ala Gly Phe Leu Tyr Tyr Glu Asp Leu Val Ser Gly Val Thr Arg Ala 165 170 175 Glu Ala Glu Ala Val Gly Val Leu Val Lys Glu Ala Val Trp Ala Phe 180 185 190 Leu Pro Asp Ala Phe Val Thr Met Thr Gly Gly Phe Arg Leu Gly Lys 195 200 205 Lys Ile Gly His Asp Val Asp Phe Leu Ile Thr Ser Pro Gly Ser Ala 210 215 220 Glu Asp Glu Glu Gln Leu Leu Pro Lys Val Ile Asn Leu Trp Glu Lys 225 230 235 240 Lys Gly Leu Leu Leu Tyr Tyr Asp Leu Val Glu Ser Thr Phe Glu Lys 245 250 255 Phe Lys Leu Pro Ser Arg Gln Val Asp Thr Leu Asp His Phe Gln Lys 260 265 270 Cys Phe Leu Ile Leu Lys Leu His His Gln Arg Val Asp Ser Ser Lys 275 280 285 Ser Asn Gln Gln Glu Gly Lys Thr Trp Lys Ala Ile Arg Val Asp Leu 290 295 300 Val Met Cys Pro Tyr Glu Asn Arg Ala Phe Ala Leu Leu Gly Trp Thr 305 310 315 320 Gly Ser Pro Gln Phe Asn Arg Asp Leu Arg Arg Tyr Ala Thr His Glu 325 330 335 Arg Lys Met Met Leu Asp Asn His Ala Leu Tyr Asp Lys Thr Lys Arg 340 345 350 Val Phe Leu Glu Ala Glu Ser Glu Glu Glu Ile Phe Ala His Leu Gly 355 360 365 Leu Asp Tyr Ile Glu Pro Trp Glu Arg Asn Ala 370 375 <210> 27 <211> 363 <212> PRT <213> Artificial Sequence <220> <223> ii-ell-40 without His tag <400> 27 Val Ala Thr Val Ser Glu Tyr Ala Cys Gln Arg Arg Thr Thr Leu Asn 1 5 10 15 Asn Tyr Asn Glu Leu Phe Thr Arg Ala Leu Asp Ile Leu Ala Glu Asn 20 25 30 Asp Glu Phe Arg Glu Asn Glu Glu Ser Arg Asp Ala Phe Arg Arg Ala 35 40 45 Ser Ser Val Leu Lys Ser Leu Pro Phe Pro Ile Thr Ser Met Lys Asp 50 55 60 Thr Glu Gly Ile Pro Cys Leu Gly Asp Lys Val Lys Arg Ile Ile Glu 65 70 75 80 Glu Ile Ile Glu Asp Gly Glu Ser Ser Glu Val Lys Ala Val Leu Asn 85 90 95 Asp Glu Arg Tyr Lys Ala Phe Lys Leu Phe Thr Ser Val Phe Gly Val 100 105 110 Gly Arg Lys Thr Ala Glu Lys Trp Phe Arg Met Gly Phe Arg Thr Leu 115 120 125 Glu Lys Ile Arg Ser Asp Lys Ser Leu Arg Phe Thr Gln Met Gln Lys 130 135 140 Ala Gly Phe Leu Tyr Tyr Glu Asp Leu Val Ser Gly Val Thr Arg Ala 145 150 155 160 Glu Ala Glu Ala Val Gly Val Leu Val Lys Glu Ala Val Trp Ala Phe 165 170 175 Leu Pro Asp Ala Phe Val Thr Met Thr Gly Gly Phe Arg Leu Gly Lys 180 185 190 Lys Ile Gly His Asp Val Asp Phe Leu Ile Thr Ser Pro Gly Ser Ala 195 200 205 Glu Asp Glu Glu Gln Leu Leu Pro Lys Val Ile Asn Leu Trp Glu Lys 210 215 220 Lys Gly Leu Leu Leu Tyr Tyr Asp Leu Val Glu Ser Thr Phe Glu Lys 225 230 235 240 Phe Lys Leu Pro Ser Arg Gln Val Asp Thr Leu Asp His Phe Gln Lys 245 250 255 Cys Phe Leu Ile Leu Lys Leu His His Gln Arg Val Asp Ser Ser Lys 260 265 270 Ser Asn Gln Gln Glu Gly Lys Thr Trp Lys Ala Ile Arg Val Asp Leu 275 280 285 Val Met Cys Pro Tyr Glu Asn Arg Ala Phe Ala Leu Leu Gly Trp Thr 290 295 300 Gly Ser Pro Gln Phe Asn Arg Asp Leu Arg Arg Tyr Ala Thr His Glu 305 310 315 320 Arg Lys Met Met Leu Asp Asn His Ala Leu Tyr Asp Lys Thr Lys Arg 325 330 335 Val Phe Leu Glu Ala Glu Ser Glu Glu Glu Ile Phe Ala His Leu Gly 340 345 350 Leu Asp Tyr Ile Glu Pro Trp Glu Arg Asn Ala 355 360 <210> 28 <211> 379 <212> PRT <213> ARTIFICIAL SEQUENCE <220> <223> ii-ell-40 with His tag <400> 28 Met Ala Ser Ser His His His His His His Ser Ser Gly Ser Glu Asn 1 5 10 15 Will There Be Glu Tyr Ala Cys Gln Arg Thr Thr Leu Asn 20 25 30 Asn Tyr Asn Glu To Phe Thr Arg To Asp And To Glu Asn 35 40 45 Asp Glu Phe Arg Glu Asn Glu Glu Ser Arg Asp Ala Phe Arg Arg Ala 50 55 60 Ser Ser Val Leu Lys Ser Leu Pro Phe Pro Ile Thr Ser Met Lys Asp 65 70 75 80 Thr Glu Gly Ile Pro Cys Leu Gly Asp Lys Val Lys Arg Ile Ile Glu 85 90 95 Glu Ile Ile Glu Asp Gly Glu Ser Ser Glu Val Lys Ala Val Leu Asn 100 105 110 Asp Glu Arg Tyr Lys Ala Phe Lys Leu Phe Thr Ser Val Phe Gly Val 115 120 125 Gly Arg Lys Thr Ala Glu Lys Trp Phe Arg Met Gly Phe Arg Thr Leu 130 135 140 Glu Lys With Arg Server Asp Lys Serve Leu Arg Phe Thr Gln Met Gln Lys 145 150 155 160 Ala Gly Phe Leu Tyr Tyr Glu Asp Leu Val Ser Gly Val Thr Arg Ala 165 170 175 Glu Ala Glu Ala Val Gly Val Leu Val Lys Glu Ala Val Trp Ala Phe 180 185 190 Leu Pro Asp Ala Phe Val Thr Met Thr Gly Gly Phe Arg Leu Gly Lys 195 200 205 Lys Ile Gly His Asp Val Asp Phe Leu Ile Thr Ser Pro Gly Ser Ala 210 215 220 Glu Asp Glu Glu Gln Leu Leu Pro Lys Val Ile Asn Leu Trp Glu Lys 225 230 235 240 Lys Gly Leu Leu Leu Tyr Tyr Asp Leu Val Glu Ser Thr Phe Glu Lys 245 250 255 Phe Lys Leu Pro Ser Arg Gln Val Asp Thr Leu Asp His Phe Gln Lys 260 265 270 Cys Phe Leu Ile Leu Lys Leu His His Gln Arg Val Asp Ser Ser Lys 275 280 285 Ser Asn Gln Gln Glu Gly Lys Thr Trp Lys Ala Ile Arg Val Asp Leu 290 295 300 Val Met Cys Pro Tyr Glu Asn Arg Ala Phe Ala Leu Leu Gly Trp Thr 305 310 315 320 Gly Ser Pro Gln Phe Asn Arg Asp Leu Arg Arg Tyr Ala Thr His Glu 325 330 335 Arg Lys Met Met Leu Asp Asn His Ala Leu Tyr Asp Lys Thr Lys Arg 340 345 350 Val Phe Leu Glu Ala Glu Ser Glu Glu Glu Ile Phe Ala His Leu Gly 355 360 365 Leu Asp Tyr Ile Glu Pro Trp Glu Arg Asn Ala 370 375 <210> 29 <211> 363 <212> PRT <213> ARTIFICIAL SEQUENCE <220> <223> 9-40 <400> 29 Val Ala Thr Val Ser Glu Tyr Ala Cys Gln Arg Arg Thr Thr Leu Asn 1 5 10 15 Asn Tyr Asn Glu Leu Phe Thr Arg Ala Leu Asp Ile Leu Ala Glu Asn 20 25 30 Asp Glu Phe Arg Glu Asn Glu Glu Ser Arg Asp Ala Phe Arg Arg Ala 35 40 45 Ser Ser Val Leu Lys Ser Leu Pro Phe Pro Ile Thr Ser Met Lys Asp 50 55 60 Thr Glu Gly Ile Pro Cys Leu Gly Asp Lys Val Lys Arg Ile Ile Glu 65 70 75 80 Glu Ile Ile Glu Asp Gly Glu Ser Ser Glu Val Lys Ala Val Leu Asn 85 90 95 Asp Glu Arg Tyr Lys Ala Phe Lys Leu Phe Thr Ser Val Phe Gly Val 100 105 110 Gly Arg Lys Thr Ala Glu Lys Trp Phe Arg Met Gly Phe Arg Thr Leu 115 120 125 Glu Lys Ile Arg Ser Asp Lys Ser Leu Arg Phe Thr Gln Met Gln Lys 130 135 140 Ala Gly Phe Leu Tyr Tyr Glu Asp Leu Val Ser Gly Val Thr Arg Ala 145 150 155 160 Glu Ala Glu Ala Val Gly Val Leu Val Lys Glu Ala Val Trp Ala Phe 165 170 175 Leu Pro Asp Ala Phe Val Thr Met Thr Gly Gly Phe Arg Leu Gly Lys 180 185 190 Lys Ile Gly His Asp Val Asp Phe Leu Ile Thr Ser Pro Gly Ser Ala 195 200 205 Glu Asp Glu Glu Gln Leu Leu Pro Lys Val Ile Asn Leu Trp Glu Lys 210 215 220 Lys Gly Leu Leu Leu Tyr Tyr Asp Leu Val Glu Ser Thr Phe Glu Lys 225 230 235 240 Phe Lys Leu Pro Ser Arg Gln Val Asp Thr Leu Asp His Phe Gln Lys 245 250 255 Cys Phe Leu Ile Leu Lys Leu His His Gln Arg Val Asp Ser Ser Lys 260 265 270 Ser Asn Gln Gln Glu Gly Lys Thr Trp Lys Ala Ile Arg Val Asp Leu 275 280 285 Val Met Cys Pro Tyr Glu Asn Arg Ala Phe Ala Leu Leu Gly Trp Thr 290 295 300 Gly Ser Pro Gln Phe Asn Arg Asp Leu Arg Arg Tyr Ala Thr His Glu 305 310 315 320 Arg Lys Met Met Leu Asp Asn His Ala Leu Tyr Asp Lys Thr Lys Arg 325 330 335 Val Phe Leu Glu Ala Glu Ser Glu Glu Glu Ile Phe Ala His Leu Gly 340 345 350 Leu Asp Tyr Ile Glu Pro Trp Glu Arg Asn Ala 355 360 <210> 30 <211> 379 <212> PRT <213> Artificial Sequence <220> <223> 9 - 40 with His - tag <400> 30 Met Ala Ser Ser His His His His His His Ser Ser Gly Ser Glu Asn 1 5 10 15 Val Ala Thr Val Ser Glu Tyr Ala Cys Gln Arg Arg Thr Thr Leu Asn 20 25 30 Asn Tyr Asn Glu Leu Phe Thr Arg Ala Leu Asp Ile Leu Ala Glu Asn 35 40 45 Asp Glu Phe Arg Glu Asn Glu Glu Ser Arg Asp Ala Phe Arg Arg Ala 50 55 60 Ser Ser Val Leu Lys Ser Leu Pro Phe Pro Ile Thr Ser Met Lys Asp 65 70 75 80 Thr Glu Gly Ile Pro Cys Leu Gly Asp Lys Val Lys Arg Ile Ile Glu 85 90 95 Glu Ile Ile Glu Asp Gly Glu Ser Ser Glu Val Lys Ala Val Leu Asn 100 105 110 Asp Glu Arg Tyr Lys Ala Phe Lys Leu Phe Thr Ser Val Phe Gly Val 115 120 125 Gly Arg Lys Thr Ala Glu Lys Trp Phe Arg Met Gly Phe Arg Thr Leu 130 135 140 Glu Lys Ile Arg Ser Asp Lys Ser Leu Arg Phe Thr Gln Met Gln Lys 145 150 155 160 Ala Gly Phe Leu Tyr Tyr Glu Asp Leu Val Ser Gly Val Thr Arg Ala 165 170 175 Glu Ala Glu Ala Val Gly Val Leu Val Lys Glu Ala Val Trp Ala Phe 180 185 190 Leu Pro Asp Ala Phe Val Thr Met Thr Gly Gly Phe Arg Leu Gly Lys 195 200 205 Lys Ile Gly His Asp Val Asp Phe Leu Ile Thr Ser Pro Gly Ser Ala 210 215 220 Glu Asp Glu Glu Gln Leu Leu Pro Lys Val Ile Asn Leu Trp Glu Lys 225 230 235 240 Lys Gly Leu Leu Leu Tyr Tyr Asp Leu Val Glu Ser Thr Phe Glu Lys 245 250 255 Phe Lys Leu Pro Ser Arg Gln Val Asp Thr Leu Asp His Phe Gln Lys 260 265 270 Cys Phe Leu Ile Leu Lys Leu His His Gln Arg Val Asp Ser Ser Lys 275 280 285 Ser Asn Gln Gln Glu Gly Lys Thr Trp Lys Ala Ile Arg Val Asp Leu 290 295 300 Val Met Cys Pro Tyr Glu Asn Arg Ala Phe Ala Leu Leu Gly Trp Thr 305 310 315 320 Gly Ser Pro Gln Phe Asn Arg Asp Leu Arg Arg Tyr Ala Thr His Glu 325 330 335 Arg Lys Met Met Leu Asp Asn His Ala Leu Tyr Asp Lys Thr Lys Arg 340 345 350 Val Phe Leu Glu Ala Glu Ser Glu Glu Glu Ile Phe Ala His Leu Gly 355 360 365 Leu Asp Tyr Ile Glu Pro Trp Glu Arg Asn Ala 370 375

Claims

1. A method for synthesizing a plurality of polynucleotides each having a predetermined sequence using a template-free polymerase, wherein the template-free polymerase has a reduced coupling efficiency at one or more low-efficiency motifs, the method comprising the following steps: (a) providing a reaction chamber for each polynucleotide of the plurality of nucleotides, each reaction chamber having a synthesis support disposed therein to which an initiator is attached, wherein each initiator has a free 3'-hydroxyl group, and wherein each reaction chamber has an inlet and an outlet and a filter, the filter retaining the synthesis support and operatively associated with the outlet such that a reaction solution exiting the reaction chamber passes through the filter; (b) providing a waste manifold operatively associated with an outlet of the reaction chamber such that a reaction solution is removed from the reaction chamber whenever a positive pressure differential is created between the reaction chamber and the waste manifold; (c) repeating a cycle of the following reaction steps for each reaction chamber until the polynucleotide in such reaction chamber is complete: (i) contacting the initiator or deprotected extension segment with a 3'-protected nucleoside triphosphate and a template-free polymerase in a coupling solution such that the initiator or deprotected extension segment is extended by the 3'-protected nucleoside triphosphate to form a 3'-protected extension segment, (ii) deprotecting the 3'-protected extension segment using a deprotection solution, and (iii) applying a pressure differential between the reaction chamber and the waste manifold to remove the reaction solution from the reaction chamber; wherein the type of 3'-protected nucleoside triphosphate contacted in step (i) in the reaction chamber is determined by the polynucleotide sequence of the reaction chamber, and Wherein, whenever a low efficiency motif is present at the 3' end of the deprotected extension segment of such reaction chamber, prior to each cycle, one or more short cycles of step (i) are performed in the reaction chamber.

2. The method of claim 1, wherein the cycle and the short cycle have the same duration.

3. The method of claim 1 or 2, further comprising the step of cleaving the polynucleotide from the synthetic support.

4. The method of claim 1, wherein the template-free polymerase is terminal deoxynucleotidyl transferase (TdT).

5. The method of claim 4, wherein the low efficiency motif is CCA, CTA, GCA, GTA or CCT.

6. The method of claim 4 or 5, wherein the TdT is a TdT variant having an amino acid sequence that is at least 90% identical to SEQ ID NO: 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30, wherein the amino acid sequences of SEQ ID No: 16, 17, 18, 19 and 20 each have the substitution Q4E / S / D / N, and wherein the TdT variant (i) is capable of synthesizing nucleic acid fragments in the absence of a template, and (ii) is capable of incorporating 3'-O protected nucleotides onto free 3'-hydroxyl groups of polynucleotides.

7. An apparatus for synthesizing a plurality of polynucleotides each having a predetermined sequence using a template-free polymerase, wherein the template-free polymerase has a reduced coupling efficiency at one or more low-efficiency motifs, the apparatus comprising: (a) a plurality of reaction chambers, each reaction chamber having a synthesis support disposed therein with an initiator attached thereto, wherein each initiator has a free 3'-hydroxyl group, and wherein each reaction chamber has an inlet and an outlet and a filter, the filter retaining the synthesis support and operatively associated with the outlet such that a reaction solution exiting the reaction chamber passes through the filter; (b) a waste manifold operatively associated with the outlet of the reaction chamber such that whenever a positive pressure differential is established between the reaction chamber and the waste manifold, reaction solution is removed from the reaction chamber and enters the waste manifold; (c) a fluid delivery system for delivering a reaction solution to the reaction chambers of the array; (d) a user interface for receiving the nucleotide sequence of the polynucleotide to be synthesized and low efficiency motif data; (e) a control system operatively associated with the user interface, the array of reaction chambers, the fluid delivery system, and the waste manifold, wherein the control system determines the number and position of each inefficient motif for each polynucleotide and distributes each polynucleotide to a reaction chamber for synthesis, and wherein for each reaction chamber, until its polynucleotide is complete, the control system directs the following repeated steps: (i) delivering a coupling solution to the initiator oligonucleotide or deprotected extension segment under coupling conditions, the coupling solution containing a 3'-protected nucleoside triphosphate and a template-free polymerase to allow extension of the initiator oligonucleotide or deprotected extension segment by the 3'-protected nucleoside triphosphate to form a 3'-protected extension segment, (ii) delivering a deprotection solution to the reaction chamber to deprotect the 3'-protected extension segment, and (iii) creating a pressure differential between the reaction chamber and the waste manifold to remove the deprotection solution from the reaction chamber; wherein the type of 3'-protected nucleoside triphosphate contacted in step (i) in the reaction chamber is determined by the predetermined sequence of the reaction chamber, and Wherein, whenever a low efficiency motif is present at the 3' end of the deprotected extension segment of such reaction chamber, prior to each cycle, one or more short cycles of step (i) are performed in the reaction chamber.

8. The apparatus of claim 7, wherein the template-free polymerase is terminal deoxynucleotidyl transferase (TdT).

9. The device of claim 7, wherein the low efficiency motif is CCA, CTA, GCA, GTA or CCT.

10. Use of the device of claim 7 for implementing the method of synthesizing a plurality of polynucleotides each having a predetermined sequence using a template-free polymerase according to any one of claims 1 to 6.

11. A kit for synthesizing a plurality of polynucleotides each having a predetermined sequence using a template-free polymerase, the kit comprising the device of claim 7 and 3'-protected nucleoside triphosphates and / or one or more template-free polymerases and / or a coupling solution and / or a deprotection solution.

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