Engineered luciferases and luciferin substrates

Abstract

The present disclosure provides a protein having luciferase activity comprising a secondary structural arrangement H1-L1-H2-L2-B1-L3-B2-L4-H3-L5-B3-L6-L4-L7-L5-L8-B6 wherein (i) "H" is a helical domain, "L" is a loop domain, and "B" is a beta chain domain wherein the length of the B5 domain is at least 11, 12, 13, or 14 amino acids, and wherein "L" is a cyclic domain, and "B" is a beta chain domain. And residue 11 of the B5 domain is F, Y or L; and the length of the B4 domain is at least 12 amino acids, and the residue 10 of the B4 domain is F, Y, L, I, K or M; and / or residue 12 of the B4 domain is F, L, R, D, M, Q or V; (ii) the H1 domain is at least 18 or 19 amino acids in length; the residue 9 of the H1 domain is T, S, H, R, C, L, D, V, A, Q, G, E, K, I, N or M; (iii) the length of the B3 domain is at least 6, 7, 8, 9 or 10 amino acids, and the residue 1 of the B3 domain is W or H; (iv) the length of the B4 domain is at least 12 amino acids, and the residue 10 of the B4 domain is F, Y, L, I, K or M; or (v) the length of the B4 domain is at least 12 amino acids, and the residue 12 of the B4 domain is L, R, D, M, Q, or V. Additional proteins disclosed herein are illustrated in the claims. Also provided herein are luciferase substrates, assay buffers, and kits comprising one or more of a protein having luciferase activity or a nucleic acid encoding the protein, an assay buffer, and a luciferase substrate.

Description

Cross Reference to Related Applications

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 625,901, filed January 26, 2024, U.S. Provisional Patent Application No. 63 / 505,939, filed June 2, 2023, and U.S. Provisional Patent Application No. 63 / 498,236, filed April 25, 2023, which are incorporated by reference herein in their entirety. XML Listing Incorporated by Reference

[0002] The sequences are provided in the sequence listing XML “MOBI-010WO_SEQLIST_4-23-24.XML” which was created on April 23, 2024 and has a size of 2,879,287 bytes. The contents of the text file are incorporated by reference herein in their entirety. INTRODUCTION

[0003] Bioluminescence produced by oxidation of a luciferin substrate by the enzymatic activity of luciferase has been used in biological assays in cell-free systems, in vitro, and in vivo. Because excitation is not required to induce emission, luminescence occurs in the dark, providing a significant advantage over fluorescence, including lower background signal and no need for excitation that can cause phototoxicity in tissues.

[0004] Work has been done to engineer natural luciferases to improve their use as molecular probes. However, a satisfactory luciferase based on a natural luciferase has not been produced. The Beck Lab at the University of Washington developed a synthetic luciferase called LuxSit (Nature 614, 774-780 (2023)).

[0005] D-luciferin and coelenterazine, and their respective luciferases, are well-known luciferin / luciferase pairs and are commonly used in most applications of bioluminescence, such as genetic assays, detection of protein-protein interactions, high-throughput screening (HTS) in drug discovery, sanitation management, pollution analysis in ecosystems, and in vivo imaging of small mammals (Syed et al., Chem. Soc. Rev., 2021, 50, 5668).

[0006] Much work has been done in the field of synthetic chemistry to develop two luciferins with beneficial properties. Synthetic luciferin analogs are known to have more persistent and sustained bioluminescent signals compared to the bioluminescent signal of D-luciferin. Synthetic coelenterazine analogs are reported to have higher brightness and better solubility than coelenterazine (Syed et al., Chem. Soc. Rev., 2021, 50, 5668). There remains a need to develop new luciferins with improved properties. SUMMARY

[0007] The present disclosure provides a protein having luciferase activity, the protein comprising the secondary structure arrangement H1-L1-H2-L2-B1-L3-B2-L4-H3-L5-B3-L6-B4-L7-B5-L8-B6, wherein (i) "H" is a helix domain, "L" is a loop domain, and "B" is a beta strand domain, wherein the B5 domain is at least 11, 12, 13, or 14 amino acids in length and residue 11 of the B5 domain is F, Y, or L; and the B4 domain is at least 12 amino acids in length and residue 10 of the B4 domain is F, Y, L, I, K, or M; and / or residue 12 of the B4 domain is F, L, R, D, M, Q, or V; (ii) the H1 domain is at least 18 or 19 amino acids in length; residue 9 of the H1 domain is T, S, H, R, C, L, D, V, A, Q, G, E, K, I, N, or M; (iii) the B3 domain is at least 6, 7, 8, 9, or 10 amino acids in length and residue 1 of the B3 domain is W, L, or H; (iv) the B4 domain is at least 12 amino acids in length and residue 10 of the B4 domain is F, Y, L, I, K, or M; or (v) the B4 domain is at least 12 amino acids in length and residue 12 of the B4 domain is L, R, D, M, Q, or V. Split versions of these proteins are also provided, wherein the protein is split into two or three components that are self-complementary and have luciferase activity when non-covalently associated. Circularly permuted polypeptides having luciferase activity are also disclosed. An assay solution for measuring luciferase activity of a protein is also provided, the assay solution comprising 1 mM-1000 mM imidazole.

[0008] Luciferin substrates are also provided. These luciferin substrates can be used to measure the activity of luciferases, such as the luciferase activity of the proteins disclosed herein. BRIEF DESCRIPTION OF DRAWINGS

[0009] Figure 1. The secondary structure arrangement H1-L1-H2-L2-B1-L3-B2-L4-H3-L5-B3-L6-B4-L7-B5-L8-B6 is mapped onto the amino acid sequence of LuxSit-i (SEQ ID NO: 1).

[0010] Figure 2. Single mutants of LuxSit-i proteins enriched for luciferase activity.

[0011] Figure 3. Graphical representation of mutation frequency at positions found to improve luciferase activity of a protein in single saturation mutagenesis (SSM).

[0012] Figure 4A. Comparison of luciferase activity of MBIO-148, MBIO-158, and LuxSit-i.

[0013] Figure 4B. Comparison of luciferase activity of MBIO-148, MBIO-301, MBIO-302, and LuxSit-i.

[0014] Figure 5A. Single mutants of LuxSit-i protein enriched for stability.

[0015] Figure 5B. Comparison of stability of LuxSit-i protein and LuxSit-i protein variants MBIO-301, MBIO-3073, and MBIO-4039.

[0016] Figure 5C. Static light scattering (SLS) at 266 nm used to detect formation of early aggregates during thermal denaturation.

[0017] Figure 6A. Kinetic curves of ring-substituted LuxSit-i variants over one hour.

[0018] Figure 6B. Initial relative light unit (RLU) values of ring-substituted LuxSit variants.

[0019] Figure 7. Luminescent activity of high-affinity two-component luciferase variants fused to the rapamycin-inducible FRB:FKBP system.

[0020] Figure 8. Luminescent activity of low-affinity two-component luciferase variants fused to the rapamycin-inducible FRB:FKBP system.

[0021] Figures 9A and 9B. Improvement of luciferase activity in buffers with high imidazole concentrations.

[0022] Figure 10 shows bioluminescent emission spectra of synthetic luciferin substrates la, lb, lc, Id, lk, ln, and lp incubated with MBIO-301 enzyme.

[0023] Figure 11 shows bioluminescent emission spectra of synthetic luciferin substrates 2a, 2b, 2c, 2d, 2f, 2h, and 2p incubated with MBIO-301 enzyme.

[0024] Figure 12 shows bioluminescent emission spectra of synthetic luciferin substrates 3b and 3i incubated with MBIO-301 enzyme.

[0025] Figure 13A shows luminescence obtained after incubation of synthetic luciferin substrate with MBIO-301 enzyme. Figure 13B shows luminescence obtained after incubation of synthetic luciferin substrate with MBIO-4039 enzyme. Figure 13C shows luminescence obtained after incubation of synthetic luciferin substrate with MBIO-4040 enzyme.

[0026] Figure 14 compares bioluminescence emission of synthetic luciferin substrate lc and DTZ after incubation with MBIO-301 under two assay conditions: 100% assay buffer and 20% human serum + 80% assay buffer.

[0027] Figure 15 shows luminescence obtained after incubation of synthetic luciferin substrate with MBIO-301 -derived split enzymes.

[0028] Figure 16A, Figure 16B, and Figure 16C show mass spectrometry of compound Ip_2p according to embodiments of the present disclosure.

[0029] Figure 17A, Figure 17B, and Figure 17C show mass spectrometry of compound Ip according to embodiments of the present disclosure.

[0030] Figure 18A, Figure 18B, and Figure 18C show mass spectrometry of compound 2a according to embodiments of the present disclosure.

[0031] Figure 19A, Figure 19B, and Figure 19C show mass spectrometry of compound 2al u according to embodiments of the present disclosure.

[0032] Figure 20A, Figure 20B, and Figure 20C show mass spectrometry of compound 2al v according to embodiments of the present disclosure.

[0033] Figure 21A, Figure 21B, and Figure 21C show mass spectrometry of compound 2al w according to embodiments of the present disclosure.

[0034] Figure 22A, Figure 22B, and Figure 22C show mass spectrometry of compound 2al x according to embodiments of the present disclosure.

[0035] Figure 23A, Figure 23B, and Figure 23C show mass spectrometry of compound 2k according to embodiments of the present disclosure.

[0036] Figure 24A, Figure 24B, and Figure 24C show mass spectrometry of compound 2o according to embodiments of the present disclosure.

[0037] Figure 25A, Figure 25B, and Figure 25C show mass spectrometry of compound 2p according to embodiments of the present disclosure.

[0038] FIG. 26A, FIG. 26B, and FIG. 26C show mass spectrometry of compound 2plu according to an embodiment of the disclosure.

[0039] FIG. 27A, FIG. 27B, and FIG. 27C show mass spectrometry of compound 2plv according to an embodiment of the disclosure.

[0040] FIG. 28A, FIG. 28B, and FIG. 28C show mass spectrometry of compound 2plw according to an embodiment of the disclosure.

[0041] FIG. 29A, FIG. 29B, and FIG. 29C show mass spectrometry of compound 2plx according to an embodiment of the disclosure.

[0042] FIG. 30A, FIG. 30B, and FIG. 30C show mass spectrometry of compound In2a according to an embodiment of the disclosure.

[0043] FIG. 31A, FIG. 31B, and FIG. 31C show mass spectrometry of compound In2p according to an embodiment of the disclosure.

[0044] FIG. 32A, FIG. 32B, and FIG. 32C show mass spectrometry of compound Ip2a according to an embodiment of the disclosure.

[0045] FIG. 33A, FIG. 33B, and FIG. 33C show mass spectrometry of compound Ip2p according to an embodiment of the disclosure.

[0046] FIG. 34A, FIG. 34B, and FIG. 34C show mass spectrometry of compound 1w according to an embodiment of the disclosure.

[0047] FIG. 35A, FIG. 35B, and FIG. 35C show mass spectrometry of compound 1x according to an embodiment of the disclosure.

[0048] FIG. 36A-FIG. 36F show enzyme activity of LuxSit-i variants in a functional complementation assay.

[0049] FIG. 37A-FIG. 37H show enzyme activity of LuxSit-i variants.

[0050] FIG. 38 shows a comparison of enzyme activity of LuxSit-i variant MBIO-4039 to enzyme activity of LuxSit-i.

[0051] FIG. 39 shows enzyme activity of LuxSit-i variant Mbio-3073 measured in different buffers, tris buffered saline (TBS), OB2.0, and OB3.0.

[0052] FIG. 40. LuxSit-i variant MBIO-4039 has improved yield compared to LuxSit-i.

[0053] Figure 41. Schematic of constructs used to test LuxSit Split.

[0054] Figures 42A-42G show the enzyme activity of LuxSit-i variants measured in different buffers: phosphate buffered saline (PBS), OPT 1.0, OPT 2.0, and OPT 3.0.

[0055] Figures 43A-43D show the enzyme activity of LuxSit-i variants measured using substrate 1c and buffers PBS, OPT 1.0, OPT 2.0, and OPT 3.0.

[0056] Figure 44. Schematic of constructs used to measure the stability of LgLux produced by error-prone PCR and to identify LgLux with improved protease resistance.

[0057] Figure 45. Schematic of constructs used to measure the stability of computationally produced LgLux and to identify LgLux with improved protease resistance.

[0058] Figures 46A-46C. MBIO-4517 and MBIO-4039 were split into large and small fragments and conjugated to proteins that bind to a rapamycin molecule (FKBP and FRB). Luciferase activity before and after addition of rapamycin is shown in Figure 46A. Plots comparing the luciferase activity of split luciferase versions of MBIO-4517 and MBIO-4039 to the full-length versions are shown in Figures 46B and 46C, respectively. DETAILED DESCRIPTION

[0059] The present disclosure provides a protein having luciferase activity, the protein comprising a secondary structure arrangement H1-L1-H2-L2-B1-L3-B2-L4-H3-L5-B3-L6-B4-L7-B5-L8-B6, wherein (i) "H" is a helix domain, "L" is a loop domain, and "B" is a beta strand domain, wherein the B5 domain is at least 11, 12, 13, or 14 amino acids in length, and residue 11 of the B5 domain is F, Y, or L; and the B4 domain is at least 12 amino acids in length, and residue 10 of the B4 domain is F, Y, L, I, K, or M; and / or residue 12 of the B4 domain is F, L, R, D, M, Q, or V; (ii) the H1 domain is at least 18 or 19 amino acids in length; residue 9 of the H1 domain is T, S, H, R, C, L, D, V, A, Q, G, E, K, I, N, or M; (iii) the B3 domain is at least 6, 7, 8, 9, or 10 amino acids in length, and residue 1 of the B3 domain is W, L, or H; (iv) the B4 domain is at least 12 amino acids in length, and residue 10 of the B4 domain is F, Y, L, I, K, or M; or (v) the B4 domain is at least 12 amino acids in length, and residue 12 of the B4 domain is L, R, D, M, Q, or V. Split versions of these proteins are also provided, wherein the protein is split into two or three components that are self-complementary. When the components are not physically associated with one another, they lack or have significantly reduced luciferase activity, and when the components are physically associated, they have luciferase activity. The physical association can be non-covalent or covalent. Examples of non-covalent association include association mediated via one or more moieties (e.g., proteins or small molecules) that bind to the individual components and are physically close enough to enable functional complementation. Examples of covalent association include linkers (e.g., peptides or polypeptides) that link two components (or more components) to form a polypeptide having luciferase activity. The linker can be cleavable, e.g., include a cleavage site. Upon cleavage of the linker, the two components (or more components) are physically separated, resulting in a loss or significant reduction in luciferase activity.

[0060] Also disclosed are circularly permuted polypeptides having luciferase activity.

[0061] Also provided are assay solutions (e.g., buffers) for measuring luciferase activity of a protein or protein complex, the assay solution comprising 1 mM-1000 mM imidazole. In certain experiments, the assay solution can have a basic pH.

[0062] Before the present application is described in detail, it is to be understood that this application is not limited to the particular formulations described as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting, as the scope of the present application will be limited only by the appended claims.

[0063] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the application. The upper and lower limits of these smaller ranges can independently be included in the smaller ranges, and are also encompassed within the application, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the application.

[0064] Certain ranges are presented herein with numerical values being presented with certain notations. The use of "about" in the herein indicates that the precise numerical value that it follows is an approximation which can vary by a small amount, as understood by those of ordinary skill in the art. In determining whether a numerically-approximate value is close enough to the recited value at issue, the unrecited value can be a number that is substantially equivalent in its context to the recited value.

[0065] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as those commonly understood by one of ordinary skill in the art to which this application belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present application, representative illustrative methods and materials are now described.

[0066] All publications and patents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference and are incorporated herein by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. The citation of any publication is for its disclosure prior to the filing date of this application. Nothing herein is to be construed as an admission that the present application is not entitled to antedate such publication by virtue of prior application. Further, the dates of publication provided can be different from the dates that can appear on the publications that were published by the U.S. Patent and Trademark Office.

[0067] It should be noted that, as used in this document and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. It should also be noted that the claims can be drafted to exclude any optional element. Thus, this statement is intended to serve as antecedent basis for use of such exclusive terminology in the description and / or claims to designate one or more elements or features as "essential," "sole," "only," or the like.

[0068] As will be apparent to those of ordinary skill in the art in the context of the present disclosure, each of the individual embodiments described and illustrated herein has constituent parts and features that can be readily separated from or combined with features of any other number of embodiments without departing from the scope or spirit of the present application. Any method recited can be carried out in the order of events recited or in any other order that is logically possible.

[0069] While the apparatus and method have been or will be described in the context of functional explanations for grammatical flow, it should be expressly understood that the claims should not be necessarily limited in any way by construction of "means" or "steps" limitations unless expressly recited in the claims under 35 U.S.C. § 112, and should be accorded the full scope of the meaning of the claims provided and the full scope of equivalents under the judicial doctrine of equivalents, and where the claims are expressly recited under 35 U.S.C. § 112, the claims should be accorded the full statutory equivalents under 35 U.S.C. § 112. Definitions

[0070] In the context of an amino acid sequence or polynucleotide sequence, "derived from" is intended to mean that the polypeptide or nucleic acid has a sequence based on the sequence of the reference polypeptide or nucleic acid, and is not intended to limit the source or method in which the protein or nucleic acid is made.

[0071] The terms "polypeptide" and "protein" are used interchangeably herein to refer to a linear series of amino acid residues connected to one another by peptide bonds between the amino groups of adjacent residues. The amino acid residues are typically in the "L" isomeric form. However, residues in the "D" isomeric form can be substituted for any L-amino acid residue, as long as the desired functional properties are retained by the polypeptide. In addition, in addition to the 20 "standard" amino acids, amino acids include modified amino acids and unusual amino acids, including but not limited to those listed in 37 CFR (§ 1.822(b)(4)). Furthermore, it is noted that a dash at the beginning or end of an amino acid residue sequence indicates a peptide bond to a another sequence of one or more amino acid residues or to a covalent bond to a carboxyl or hydroxyl terminal group. However, the absence of a dash does not indicate that such a peptide bond or covalent bond to a carboxyl or hydroxyl terminal group is absent, as it is conventional to omit such a dash in the representation of an amino acid sequence. The term "peptide" also refers to a linear series of amino acid residues connected to one another by peptide bonds between the amino groups of adjacent residues, but is typically shorter than a protein or polypeptide, e.g., less than 50 amino acids in length, e.g., 2-50 amino acids in length. The terms protein, polypeptide, and peptide can be used interchangeably.

[0072] As used herein, the term "bind" refers to a type of non-covalent interaction that occurs between two molecules. The strength or affinity of a binding interaction can be expressed as a dissociation constant (K D ) for the interaction, where a smaller K D indicates a greater affinity. The binding properties of a selected polypeptide can be quantified using methods well known in the art.

[0073] "Isolated" refers to the environment in which the subject entity is found as distinct from the environment in which the entity can naturally occur or be initially produced. An "isolated" compound (e.g., an "isolated" polypeptide) is separated from all or some of the components with which it is accompanied when produced or initially recovered by the hand of man. It can be significantly enriched, e.g., purified, such that the compound is at least about 70% pure, at least about 80% pure, at least about 90% pure, at least about 95% pure, at least about 98% pure, at least about 99% or greater than 99% pure, or free of impurities, contaminants, and / or components other than the compound. "Isolated" also refers to the state of a compound that is separated from all or some of the components with which it is accompanied during the manufacturing process (e.g., chemical synthesis, recombinant expression, culture media, etc.).

[0074] As used herein, the amino acid residue abbreviations are as follows: alanine (Ala; A), asparagine (Asn; N), aspartic acid (Asp; D), arginine (Arg; R), cysteine (Cys; C), glutamic acid (Glu; E), glutamine (Gln; Q), glycine (Gly; G), histidine (His; H), isoleucine (lie; I), leucine (Leu; L), lysine (Lys; K), methionine (Met; M), phenylalanine (Phe; F), proline (Pro; P), serine (Ser; S), threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y), and valine (Val; V).

[0075] In all embodiments of the polypeptides disclosed herein, any N-terminal methionine residue is optional (i.e., the N-terminal methionine residue can be present or absent). In all embodiments of the polypeptides disclosed herein, any C-terminal glycine residue is optional (i.e., the C-terminal glycine residue can be present or absent).

[0076] The term "conservative substitution" when used in reference to proteins is intended to reflect an amino acid substitution that does not significantly alter the activity (specificity or binding affinity) of the molecule. Generally, a conservative amino acid substitution involves the replacement of one amino acid by another that has similar chemical properties (e.g., charge or hydrophobicity). The following six groups each contain amino acids that are typical conservative substitutions for one another: 1) Alanine (A), Serine (S), Threonine (T); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W). Polypeptides encompassed by the present disclosure include those having one or more conservative substitutions relative to the amino acid sequences provided herein.

[0077] The percent identity between two sequences can be calculated by the following method: the number of matches in the sequence pair is multiplied by 100 and divided by the length of the aligned region (including gaps). Identity scores count only perfect matches, not degrees of similarity between amino acids. Only internal gaps are included in the length, not gaps at the ends of the sequences. Percent identity = (matches x 100) / length of aligned region (with gaps). "Alkyl" means a monoradical, branched or straight chain, non-cyclic, saturated hydrocarbon radical. Exemplary alkyl groups include methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, t-butyl, octyl, decyl, cyclopentyl, and cyclohexyl. In some cases, an alkyl group has 1 to 24 carbon atoms, for example, 1 to 12, 1 to 6, or 1 to 3 carbon atoms.

[0078] "Alkenyl" refers to a monoradical, branched or straight-chain, acyclic hydrocarbon group containing a carbon-carbon double bond. Exemplary alkenyl groups include ethenyl, n-propenyl, isopropenyl, n-butenyl, isobutenyl, octenyl, decenyl, tetradecenyl, hexadecenyl, eicosanenyl, and tetracosanenyl.

[0079] "Alkynyl" refers to a monoradical, branched or straight-chain, acyclic hydrocarbon group containing a carbon-carbon triple bond. Exemplary alkynyl groups include ethynyl and n-propynyl.

[0080] "Cycloalkyl" refers to a monoradical, cyclic, saturated hydrocarbon group. Similarly, "cycloalkenyl" refers to a monoradical and cyclic group having a carbon-carbon double bond, while "cycloalkynyl" refers to a monoradical and cyclic group having a carbon-carbon triple bond.

[0081] "Heterocyclyl" refers to a monoradical, cyclic group containing a heteroatom (e.g., O, S, N) as a ring atom and which is not aromatic (i.e., distinguishes heterocyclyl from heteroaryl). Exemplary heterocyclyl groups include piperidinyl, tetrahydrofuranyl, dihydrofuranyl, and thioxanyl.

[0082] "Aryl" refers to an aromatic group containing at least one aromatic ring, wherein each atom in the ring is a carbon atom, i.e., none of the ring atoms are heteroatoms (e.g., O, S, N). In some cases, the aryl group has a second aromatic ring, e.g., an aromatic ring fused to the first aromatic ring. Exemplary aryl groups are phenyl, naphthyl, biphenyl, diphenyl ether, diphenylamine, and benzophenone.

[0083] "Heteroaryl" refers to an aromatic group containing at least one aromatic ring, wherein at least one atom in the aromatic ring is a heteroatom (e.g., O, S, N). Exemplary heteroaryl groups include those obtained from pyridine, pyrimidine, furan, thiophene, or benzothiophene by removal of a hydrogen atom.

[0084] The term "substituted" means the replacement of one or more hydrogens from an atom (e.g., from a C or N atom) with a different group. For example, a hydrogen atom on a phenyl group (-C6H5) can be replaced with a methyl group to form a -C6H4CH3 group. Thus, a -C6H4CH3 group can be considered a substituted aryl group. As another example, two hydrogen atoms from the second carbon of a propyl group (-CH2CH2CH3) can be replaced with an oxygen atom to form a -CH2C(O)CH3 group, which can be considered a substituted alkyl group. However, replacement of a hydrogen atom on a propyl group (-CH2CH2CH3) with a methyl group (e.g., to give -CH2CH(CH3)CH3) is not considered "substitution" as used herein because both the starting and end groups are alkyl groups. However, if the propyl group is replaced with a methoxy group, to give a -CH2CH(OCH3)CH3 group, the entire group can no longer be considered an "alkyl" group, and thus is a "substituted alkyl" group. Thus, to be considered a substituent, the replacing group is of a different type than the original group. Furthermore, unless described as substituted, groups are presumed to be unsubstituted. For example, the terms "alkyl" and "unsubstituted alkyl" are used interchangeably herein.

[0085] Exemplary substituents include alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, acyl, alkoxy, amino, azido, carbonyl, carboxyl, cyano, ether, halo, hydroxyl, nitro, sulfonate, and substituted forms thereof.

[0086] In some cases, a substitution itself can be further substituted with one or more groups. For example, -C6H4CH2CH3 can be considered a substituted aryl group, i.e., an aryl group substituted with an ethyl group, which is an alkyl group. Furthermore, the ethyl group itself can be substituted with a pyridyl group to form -C6H4CH2CH2C5H5N, which can also be considered a substituted aryl group when the term is used herein. In some cases, a substituent is not substituted with any other groups.

[0087] Also described herein are diradical groups, i.e., in contrast to the monoradical groups described above such as alkyl and aryl. The term “alkylene” refers to the diradical form of an alkyl group, i.e., an alkylene is a diradical, branched or straight chain, cyclic or acyclic, saturated hydrocarbon group. Exemplary alkylene groups include dimethylene (-CH2-, which is also known as methylene), 1,2- diethylene (-CH2CH2-), and 1,1-diethylene (i.e., a CHCH3 moiety in which the first atom has two single bonds to two different groups). The term “arylene” refers to the diradical form of an aryl group, e.g., 1,4-diylbenzene refers to a C6H4 moiety in which the two hydrogens located at the para positions of each other are removed and replaced with single bonds to other groups. The terms “alkenylene,” “alkynylene,” “heteroarylene,” and “heterocycloalkylene” are also used herein.

[0088] “Alkoxy” refers to a group of the formula -O(alkyl). Similar groups can be derived from alkenyl, alkynyl, aryl, heteroaryl, and other groups.

[0089] “Amino” refers to a group of the formula -NR X R Y wherein R X and R Y are each independently H or a non-hydrogen substituent. Exemplary non-hydrogen substituents include alkyl groups (e.g., methyl, ethyl, and isopropyl).

[0090] “Hydroxyl” refers to a group of the formula -OH.

[0091] “Halo” and “halogen” refer to a chloro, bromo, fluoro, and iodo group.

[0092] “Haloalkyl” refers to an alkyl group in which a hydrogen atom has been replaced with a halogen.

[0093] “Nitro” refers to a group of the formula -NO2.

[0094] Unless otherwise indicated, a reference to an atom is intended to include all isotopes of that atom. For example, a reference to H includes 1 H, 2 H (i.e., D or deuterium), and 3 H (i.e., tritium), and a reference to C includes 12 C and all other isotopes of carbon (e.g., 13 C), both. Unless otherwise indicated, a group includes all possible stereoisomers.

[0095] Numerical ranges include the numbers defining the range. Polypeptide

[0096] The polypeptides disclosed herein are based on a polypeptide known as LuxSit-i (SEQ ID NO: 1, Figure 1). LuxSit-i is an optimized version of LuxSit (Latin: Let there be light). LuxSit is a de novo designed synthetic luciferase with no significant sequence similarity to naturally occurring luciferases. LuxSit is based on the topology of the NTF2 (nuclear transport factor 2)-like protein superfamily, which does not have luciferase activity but contains multiple pockets that are compatible in size and structure for binding to luciferase substrates such as Diphenylterazine (DTZ). LuxSit was generated by (i) optimizing the core region of the binding pocket while allowing changes, such as substitutions and / or deletions, in more flexible regions of the protein to identify the optimal scaffold compatible with the binding pocket; followed by (ii) screening the active site for DTZ while maintaining scaffold stability. See Nature 614, 774-780 (2023). LuxSit has the following secondary structure defining the protein scaffold:

[0097] H1-L1-H2-L2-B1-L3-B2-L4-H3-L5-B3-L6-B4-L7-B5-L8-B6, where “H” is a helix domain, “L” is a loop domain, and “B” is a beta strand domain.

[0098] LuxSit comprises the following catalytic dyads: (i) a D residue at position 18 in the H1 domain and an R residue at position 2 in the B3 domain (also referred to as Asp18-Arg65), which form dyad 1; and (ii) a Y residue at position 14 in the H1 domain and an H residue at position 9 in the B5 domain (also referred to as Tyr14-His98), which form dyad 2.

[0099] The amino acid sequence of LuxSit is set forth in SEQ ID NO: 92: (M)SEEQIRQFL RRF Y EALD SG D ADTAASLF H PGV TIHL WDG VTF TSR EEFR EWFERLFST R KDA QREIKSL EVR GDT VEVH VQLHATH NGQ KHTVDAT H HW HFR GNRVTEV RVHINPT(G) (SEQ ID NO: 1)

[0100] LuxSit-i offers many advantages over naturally occurring luciferases, such as small size, stability, robust folding, and high activity. An optimized version of LuxSit was created with the following substitutions R60S / A96L / M110V relative to SEQ ID NO: 92 and is referred to as LuxSit-i. LuxSit and LuxSit-i are described in Nature 614, 774-780 (2023).

[0101] The amino acid sequence of LuxSit-i is shown as SEQ ID NO: 1: (M)SEEQIRQFL RRF Y EALD SG D ADTAASLF H PGV TIHL WDG VTF TSR EEFR EWFERLFST S KDA QREIKSL EVR GDT VEVH VQLHATH NGQ KHTVDLT H HW HFR GNR VTEV RVHINPT(G) (SEQ ID NO: 1)

[0102] In each of the annotated sequences shown as SEQ ID NO: 1 and 92: (a) the bold and underlined residues are Bin 1 (catalytic residues) Y14 (residue 14 of the H1 domain) + H98 (residue 9 of the B5 domain); (b) the bold residues are Bin 2 (catalytic residues) D18 (residue 9 of the H1 domain) + R65 (residue 2 of the B3 domain); (c) the italicized residues are core packing (recognition residues) F13 (residue 13 of the H1 domain), I35 (residue 2 of the B1 domain), W38 (residue 1 of the L3 domain), F49 (residue 4 of the H3 domain), V81 (residue 6 of the B4 domain), L83 (residue 8 of the B4 domain), V94 (residue 5 of the B5 domain), A / L97 (residue 8 of the B5 domain), W100 (residue 11 of the B5 domain), M / V110 (residue 5 of the B6 domain), V112 (residue 7 of the B6 domain); and (d) The underlined, but not bolded, positions are the loop domains or residues immediately adjacent that facilitate cleavage of the enzyme or insertion of other functional domains.

[0103] The amino acids in parentheses can or can not be present. FIG. 1 shows the secondary structure arrangement H1-L1-H2-L2-B1-L3-B2-L4-H3-L5-B3-L6-B4-L7-B5-L8-B6 mapped onto the amino acid sequence of LuxSit-i. Polypeptides having luciferase activity LuxSit-i variants

[0104] The polypeptides described herein include one or more changes in the amino acid sequence of LuxSit-i that result in an improvement in one or more properties of the protein compared to LuxSit-i.

[0105] In certain aspects, the polypeptides have improved activity compared to LuxSit-i. For example, the luciferase activity of the polypeptides is at least 10% higher than the luciferase activity of LuxSit-i, e.g., at least 20% higher, at least 30% higher, at least 40% higher, at least 50% higher, at least 60% higher, at least 70% higher, at least 80% higher, at least 90% higher, at least 100% higher, at least 150% higher, or up to 150% higher, or up to 180% higher, or up to 200% higher than the luciferase activity of LuxSit-i. Luciferase activity can be measured using any suitable assay, including the assays provided herein. Luciferase activity can be measured using a luciferin substrate, e.g., DTZ, coelenterazine, furimazine, coelenterazine-n, coelenterazine-f, coelenterazine-h, coelenterazine-hcp, coelenterazine-cp, coelenterazine-c, coelenterazine-e, coelenterazine-fcp, dideoxycoelenterazine, coelenterazine-i, coelenterazine-icp, coelenterazine-v, and 2-methylcoelenterazine, or another luciferin substrate or analog thereof. Luciferase activity can be measured using a compound disclosed herein.

[0106] In certain aspects, the polypeptides have improved stability at high temperatures compared to LuxSit-i. For example, the polypeptides are stable at higher temperatures compared to LuxSit-i. Stability can be measured by enzyme activity and / or protein misfolding measured over a period of time. In certain embodiments, static light scattering (SLS) can be used to measure stability. In certain embodiments, the polypeptides disclosed herein are more stable than LuxSit-i at temperatures above 37ºC. In certain embodiments, the polypeptides disclosed herein are more stable than LuxSit-i at temperatures above 37ºC as measured by SLS.

[0107] In certain aspects, the polypeptides have improved specificity compared to LuxSit-i. For example, the specificity for luciferin substrate can be 2-fold, 3-fold, 5-fold, 10-fold compared to LuxSit-i.

[0108] In certain aspects, the polypeptides have improved yield compared to LuxSit-i. For example, the polypeptides are expressed at higher levels and / or have lower levels of aggregated or misfolded protein when expressed in standard expression systems (e.g., E. Coli, yeast, mammalian cell lines, etc.) compared to LuxSit-i. Figure 40 shows the improvement in yield of LuxSit-i variants compared to LuxSit-i expressed in E. Coli (BL21) cultures. 1 L cultures were used for expression of LuxSit-i variants and LuxSit-i.

[0109] In certain aspects, the polypeptides provided herein have the same secondary structure as LuxSit and LuxSit-i: H1-L1-H2-L2-B1-L3-B2-L4-H3-L5-B3-L6-B4-L7-B5-L8-B6. "H" is a helix domain, "L" is a loop domain, and "B" is a beta strand domain. In the polypeptides provided herein, the H1 domain is at least 18 or 19 amino acids in length; residue 14 of the H1 domain is Y, D, or E, and residue 9 of the H1 domain is D or E; the B3 domain is at least 6, 7, 8, 9, or 10 amino acids in length, and residue 2 of the B3 domain is R; and the B5 domain is at least 10, 11, 12, 13, or 14 amino acids in length, and residue 9 of the B5 domain is H or N. Thus, catalytic dyad 1 and dyad 2 are unchanged.

[0110] In the polypeptides provided herein, in some aspects, one or more core packing can not be changed. In some aspects, residue 13 of domain H1 is F; residue 1 of domain L3 is W; residue 5 of domain B5 is V or another hydrophobic residue; residue 8 of domain B5 is A or L or another hydrophobic residue; and / or residue 11 of domain B5 is W. In further aspects, residue 2 of domain B1 is I or another hydrophobic residue; residue 4 of domain H3 is F; residue 6 of domain B4 is V or another hydrophobic residue; residue 8 of domain B4 is L or another hydrophobic residue; residue 5 of domain B6 is M or V or another hydrophobic residue; and / or residue 7 of domain B6 is V or another hydrophobic residue.

[0111] In certain aspects, the H1 domain is 19 amino acids in length; the H2 domain is 7 amino acids in length; the B1 domain is 4 amino acids in length; the B2 domain is 4 amino acids in length; the H3 domain is 14 amino acids in length; the B3 domain is 10 amino acids in length; the B4 domain is 12 amino acids in length; the B5 domain is 14 amino acids in length; and the B6 domain is 12 or 13 amino acids in length. The loop domains can be any length and can include any insertions of residues or functional domains deemed appropriate relative to the sequences exemplified herein, including but not limited to metal binding domains, drug binding domains, GPCR receptors, protein switches, and small molecule binding domains.

[0112] In certain aspects, the H1 domain present in the polypeptides disclosed herein has an amino acid sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence: MSEEQIRQFLRRFYEALDS (SEQ ID NO: 2738) or SISEEQIRQFLRRFYEALDS (SEQ ID NO: 2739) or IPEEQIRQFLRRFYEALDS (SEQ ID NO: 2740) or EISEEQIRQFLRRFYEALDS (SEQ ID NO: 2741).

[0113] In certain aspects, the H2 domain present in the polypeptides disclosed herein has an amino acid sequence that is at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence: ADTAASL (SEQ ID NO: 2742).

[0114] In certain aspects, the B1 domain present in the polypeptides disclosed herein has an amino acid sequence that is at least 50%, 75%, or 100% identical to the amino acid sequence: TIHL (SEQ ID NO: 2743).

[0115] In certain aspects, the B2 domain present in the polypeptides disclosed herein has an amino acid sequence that is at least 50%, 75%, or 100% identical to the amino acid sequence: GVTF (SEQ ID NO: 2744).

[0116] In certain aspects, the B3 domain present in the polypeptides disclosed herein has an amino acid sequence that is at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence: WREIKSLEVR (SEQ ID NO: 2746).

[0117] In certain aspects, the B3 domain present in the polypeptides disclosed herein has an amino acid sequence that is at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence: WREIKSLEVR (SEQ ID NO: 2746).

[0118] In certain aspects, the B4 domain present in the polypeptides disclosed herein has an amino acid sequence that is at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence: TVEVHVQLHFTL (SEQ ID NO: 2747) or TVVVVVRLDFTL (SEQ ID NO: 2748).

[0119] In certain aspects, the B5 domain present in the polypeptides disclosed herein has an amino acid sequence that is at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence: QKHTVDLTHHFHFR (SEQ ID NO: 2749) or QKHTVILTHVFRFR (SEQ ID NO: 2750).

[0120] In certain aspects, the B6 domain present in the polypeptides disclosed herein has an amino acid sequence that is at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence: RVTEVRVHINPTG (SEQ ID NO: 2751) or RVTEVRVEIVPV (SEQ ID NO: 2752).

[0121] In certain aspects, the LI, L2, L3, L4, L5, L6, L7, and L8 domains are at least 1, 2, 3, 4, or 5 amino acids in length and comprise any amino acid and optionally are at most 5 amino acids in length. In certain aspects, the LI, L2, L3, L4, L5, L6, L7, and L8 domains comprise an insertion that does not alter the overall protein conformation.

[0122] In certain aspects, some of the LuxSit-i variants provided herein having the same secondary structure arrangement as LuxSit-i have an amino acid sequence that is at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence: MSEEQIRQFLRRFYEALDSGDADTAASLFHPGVTIHLWDGVTFTSREEFREWFERLFSTSKDAQREIKSLEVRGDTVEVHVQLHATHNGQKHTVDLTHHWHFRGNRVTEVRVHINPTG (SEQ ID NO: 1). LuxSit-i variants having substitutions in the B4 domain

[0123] In certain aspects, the protein having luciferase activity can comprise the secondary structure arrangement H1-L1-H2-L2-B1-L3-B2-L4-H3-L5-B3-L6-B4-L7-B5-L8-B6 as described herein, wherein the B4 domain is at least 12 amino acids in length. In certain aspects, residue 10 of the B4 domain is F, L, Y, I, K, or M. In certain aspects, residue 10 of the B4 domain is F. In contrast, residue 10 of the B4 domain of LuxSit-i is A. Residue 10 of B4 can also be referred to by the position of the corresponding amino acid in the B4 domain in SEQ ID NO: 1, wherein residue 10 in the B4 domain is position 85 in SEQ ID NO: 1.

[0124] The protein having luciferase activity can have an amino acid sequence that is at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 1, wherein residue 85, when numbered relative to SEQ ID NO: 1, is F, Y, L, I, K, or M.

[0125] In all aspects, the relative position relative to SEQ ID NO: 1 can be determined by aligning the amino acid sequence with the amino acid sequence of SEQ ID NO: 1.

[0126] In another aspect, residue 12 of the B4 domain is F, L, R, D, M, Q, or V. In certain aspects, residue 12 of the B4 domain is F. In contrast, residue 12 of the B4 domain of LuxSit-i is H. Residue 12 of B4 can also be referred to by the position of the corresponding amino acid in the B4 domain of SEQ ID NO: 1, where residue 12 in the B4 domain is position 87 in SEQ ID NO: 1.

[0127] In certain aspects, the protein having luciferase activity can have an amino acid sequence that is at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 1, wherein residue 87 is F, L, R, D, M, Q, or V when numbered relative to SEQ ID NO: 1.

[0128] In another aspect, residue 12 of the B4 domain is F, L, R, D, M, Q, or V, and residue 10 of the B4 domain is F, L, Y, I, K, or M. In certain aspects, residue 10 of the B4 domain is F and residue 12 of the B4 domain is F.

[0129] In certain aspects, the protein having luciferase activity can have an amino acid sequence that is at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% identical to the amino acid sequence of SEQ ID NO: 1, wherein residue 87 is F, L, R, D, M, Q, or V and residue 85 is F, Y, L, I, K, or M when numbered relative to SEQ ID NO: 1.

[0130] In certain aspects, the protein having luciferase activity comprises an amino acid sequence that is at least 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 1, and (i) one or more of the following substitutions relative to SEQ ID NO: 1 : E3D, I6T / K, Y14W, E15G, S19R, L28S / F, G32R / D / A / E / H, T42I, F43L / G, S45A, L56R / K / Q, F57V, T59K, Q64W / H, K61P / E, V77Y, E78W, Q82T / K, A85F / Y / L / I / M, T86A, H87L / V, H99L, W100F / Y / L, R106L, V107I, T108N / D, and H113F; and / or E3, I6, Y14, E15, S19, L28, G32, T42, F43, S45, L56, F57, T59, K61, Q64, V77, E78, Q82, A85, T86, H92, L96, H99, W100, R106, T108, and H113; and / or (ii) lacks one or more lysine residues, or lacks lysine residues, relative to SEQ ID NO: 1. In certain embodiments, the protein lacks a lysine residue present in SEQ ID NO: 1, wherein the lysine residue is replaced with another amino acid (e.g., R, Q, T, S, L, Y, etc.).

[0131] In certain embodiments, the protein having luciferase activity comprises an amino acid sequence that is at least 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 1, and comprises: (i) one or more of the following substitutions relative to SEQ ID NO: 1 : E3D, I6T / K, Y14W, E15G, S19R, L28S / F, G32R / D / A / E / H, T42I, F43L / G, S45A, L56R / K / Q, F57V, T59K, Q64W / H, K61P / E, V77Y, E78W, Q82T / K, A85F / Y / L / I / M, T86A, H87L / V, H99L, W100F / Y / L, R106L, V107I, T108N / D, and H113F; and / or (ii) lacks lysine residues relative to SEQ ID NO: 1 and comprises one or more of the following substitutions: F9, D23, H30, H36, V41, R46, R55, L56, Q64, K68, H80, Q82, H84, A85, H87, H92, T97, H98, H99, W100, H101, R103, T108, E109, H113, and I114.

[0132] In certain embodiments, the protein lacks a lysine residue, which is replaced with another amino acid (e.g., R, Q, T, S, L, Y, etc.).

[0133] In certain embodiments, the amino acid sequence of the protein comprises all of the following substitutions: F9V / S / N, D23I, H30D, H36T, V41T, R46V, R55S, L56Q, Q64L, K68S, H80T, Q82R, H84S, A85F, H87R, H92S, T97L, H98Q, H99L, W100F, H101K / R, R103V, T108D / V, E109A, H113Y, and I114V. LuxSit-i variants having substitutions in the B4 and B5 domains

[0134] In certain aspects, the protein having luciferase activity can comprise the secondary structure arrangement H1-L1-H2-L2-B1-L3-B2-L4-H3-L5-B3-L6-B4-L7-B5-L8-B6 as described herein, wherein residue 12 of the B4 domain is F, L, R, D, M, Q, or V and / or residue 10 of the B4 domain is F, L, Y, I, K, or M, and the length of the B5 domain is at least 11, 12, 13, or 14 amino acids, and residue 11 of the B5 domain is F, Y, or L. In contrast, residue 11 of the B5 domain in LuxSit-i is W. Residue 11 of B5 can also be referred to by the position of the corresponding amino acid in the B5 domain in SEQ ID NO: 1, where residue 11 in the B5 domain is position 100 in SEQ ID NO: 1.

[0135] In certain aspects, residue 11 of the B5 domain is F, Y, or L; residue 10 of the B4 domain is F, L, Y, I, K, or M; and residue 12 of the B4 domain is F, L, R, D, M, Q, or V. In certain aspects, residue 11 of the B5 domain is F or Y; residue 10 of the B4 domain is F or L, and residue 12 of the B4 domain is D, F, or L; and / or residue 1 of the B3 domain is W / L / H.

[0136] In certain aspects: (i) residue 11 of the B5 domain is F, Y, or L; (ii) residue 10 of the B4 domain is F, L, Y, I, K, or M; (iii) residue 12 of the B4 domain is F, L, R, D, M, Q, or V; (vi) residue 9 of the H1 domain is D, K, L, N, R, S, T, Q, V, or Y; (v) residue 2 of the L2 domain is not H, and further optionally wherein residue 2 of the L2 domain is D, F, K, L, N, R, S, T, Q, V, or Y; (vi) residue 3 of the B1 domain is not H, and further optionally wherein residue 3 of the B1 domain is D, F, K, L, N, R, S, T, Q, V, or Y; (vii) residue 2 of the B2 domain is D, F, K, L, N, R, S, T, Q, or Y; (viii) residue 1 of the H3 domain is D, F, K, L, N, S, T, Q, V, or Y; (ix) residue 5 of the B4 domain is not H, and further optionally wherein residue 5 of the B4 domain is D, F, K, L, N, R, S, T, Q, V, or Y; (x) residue 9 of the B4 domain is not H, and further optionally wherein residue 9 of the B4 domain is D, F, K, L, N, R, S, T, Q, V, or Y; (xi) residue 8 of the B5 domain is D, F, K, L, N, R, S, Q, V, or Y; (xii) residue 9 of the B5 domain is not H, and further optionally wherein residue 9 of the B5 domain is D, F, K, L, N, R, S, T, Q, V, or Y; (xiii) residue 10 of the B5 domain is not H, and further optionally wherein residue 10 of the B5 domain is D, F, K, L, N, R, S, T, Q, V, or Y; (xiv) residue 12 of the B5 domain is not H, and further optionally wherein residue 12 of the B5 domain is D, F, K, L, N, R, S, T, Q, V, or Y; (xv) residue 14 of the B5 domain is D, F, K, L, N, S, T, Q, V, or Y; (xvi) residue 3 of the B6 domain is D, F, K, L, N, R, S, Q, V, or Y; (xvii) residue 4 of the B6 domain is D, F, K, L, N, R, S, T, Q, V, or Y; (xviii) residue 8 of the B6 domain is not H, and further optionally wherein residue 8 of the B6 domain is D, F, K, L, N, R, S, T, Q, V, or Y; and / or (xix) residue 9 of the B6 domain is D, F, K, L, N, R, S, T, Q, V, or Y.

[0137] In certain aspects: (i) residue 11 of the B5 domain is F; (ii) residue 10 of the B4 domain is F, L, Y, I, K, or M; (iii) residue 12 of the B4 domain is R; (vi) residue 9 of the H1 domain is N; (v) residue 2 of the L2 domain is not H, and further optionally wherein residue 2 of the L2 domain is D; (vi) residue 3 of the B1 domain is not H, and further optionally wherein residue 3 of the B1 domain is T; (vii) residue 2 of the B2 domain is T; (viii) residue 1 of the H3 domain is V; (ix) residue 5 of the B4 domain is not H, and further optionally wherein residue 5 of the B4 domain is T; (x) residue 9 of the B4 domain is not H, and further optionally wherein residue 9 of the B4 domain is S; (xi) residue 8 of the B5 domain is L; (xii) residue 9 of the B5 domain is not H, and further optionally wherein residue 9 of the B5 domain is Q; (xiii) residue 10 of the B5 domain is not H, and further optionally wherein residue 10 of the B5 domain is L; (xiv) residue 12 of the B5 domain is not H, and further optionally wherein residue 12 of the B5 domain is K; (xv) residue 14 of the B5 domain is V; (xvi) residue 3 of the B6 domain is V; (xvii) residue 4 of the B6 domain is D; (xviii) residue 8 of the B6 domain is not H, and further optionally wherein residue 8 of the B6 domain is Y; and / or (xix) residue 9 of the B6 domain is T.

[0138] In certain aspects: (i) the H1 domain is at least 18 or 19 amino acids in length; residue 9 of the H1 domain is N, T, S, H, R, C, L, D, V, A, Q, G, E, K, I, N, or M, residue 9 of the H1 domain corresponding to position 9 of SEQ ID NO: 1, which in contrast is F; (ii) residue 1 of the B3 domain is L, W, or H, residue 1 of the B3 domain corresponding to position 64 of SEQ ID NO: 1, which in contrast is Q; (iii) residue 10 of the B4 domain is F, Y, L, I, K or M, which corresponds to position 85 of SEQ ID NO: 1, in comparison to the amino acid at position 85 of SEQ ID NO: 1 which is A; (iv) residue 12 of the B4 domain is F, L, R, D, M, Q or V; (v) residue 10 of the B5 domain is L; (vi) residue 3 of the B6 domain is D or N; (vii) residue 8 of the B6 domain is Y, F or L; and / or (viii) residue 11 of the B5 domain is F or Y.

[0139] In certain aspects, in addition to the amino acids specified in (i)-(viii) above: (i) residue 9 of the H1 domain is N, T, S, H, R, C, L, D, V, A, Q, G, E, K, I, N or M; (ii) residue 1 of the B3 domain is L, W or H; (iii) residue 10 of the B4 domain is F, Y, L, I, K or M; (iv) residue 12 of the B4 domain is F, D, Y, L, I, K or M; (v) residue 10 of the B5 domain is L; (vi) residue 3 of the B6 domain is D or N; (vii) residue 8 of the B6 domain is Y, F or L; and (viii) residue 11 of the B5 domain is F or Y.

[0140] In certain aspects, (i) residue 9 of the H1 domain is N, T, S, H, R, C, L, D, V, A, Q, G, E, K, I, N or M; (ii) residue 1 of the B3 domain is L, W or H; (iii) residue 10 of the B4 domain is F, Y, L, I, K or M; (iv) residue 12 of the B4 domain is F, L, R, D, M, Q or V; (v) residue 10 of the B5 domain is L; (vi) residue 3 of the B6 domain is D or N; (vii) residue 8 of the B6 domain is Y, F or L; (viii) residue 11 of the B5 domain is F or Y; and / or (ix) residue 2 of the L2 domain is not H, and further optionally wherein residue 2 of the L2 domain is D, F, L, Q, R, S, T, W, or Y. Further, in certain embodiments, the protein comprising the amino acids specified in (i) through (ix) above is further mutated to replace one or more histidines with other amino acids, for example: (x) residue 3 of the B1 domain is not H, and further optionally wherein residue 3 of the B1 domain is D, F, L, Q, R, S, T, W, or Y; (xi) residue 5 of the B4 domain is not H, and further optionally wherein residue 5 of the B4 domain is D, F, L, Q, R, S, T, W, or Y; (xii) residue 9 of the B4 domain is not H, and further optionally wherein residue 9 of the B4 domain is D, F, L, Q, R, S, T, W, or Y; (xiii) residue 3 of the B5 domain is not H, and further optionally wherein residue 3 of the B5 domain is D, F, L, Q, R, S, T, W, or Y; (xiv) residue 9 of the B5 domain is not H, and further optionally wherein residue 9 of the B5 domain is D, F, L, Q, R, S, T, W, or Y; (xiv) residue 10 of the B5 domain is not H, and further optionally wherein residue 10 of the B5 domain is D, F, L, Q, R, S, T, W, or Y; (xvi) residue 12 of the B5 domain is not H, and further optionally wherein residue 12 of the B5 domain is D, F, L, Q, R, S, T, W, or Y; and / or (xvii) residue 8 of the B6 domain is not H, and further optionally wherein residue 8 of the B6 domain is D, F, L, Q, R, S, T, W, or Y.

[0141] In certain aspects, (i) residue 9 of the H1 domain is N; (ii) residue 1 of the B3 domain is L; (iii) residue 10 of the B4 domain is F; (iv) residue 12 of the B4 domain is R; (v) residue 10 of the B5 domain is L; (vi) residue 3 of the B6 domain is D; (vii) residue 8 of the B6 domain is Y; (viii) residue 11 of the B5 domain is F; (ix) residue 2 of the L2 domain is not H, and further optionally wherein residue 2 of the L2 domain is D; (x) residue 3 of the B1 domain is not H, and further optionally wherein residue 3 of the B1 domain is T; (xi) residue 5 of the B4 domain is not H, and further optionally wherein residue 5 of the B4 domain is T; (xii) residue 9 of the B4 domain is not H, and further optionally wherein residue 9 of the B4 domain is S; (xiii) residue 3 of the B5 domain is not H, and further optionally wherein residue 3 of the B5 domain is S; (xiv) residue 9 of the B5 domain is not H, and further optionally wherein residue 9 of the B5 domain is Q; (xiv) residue 10 of the B5 domain is not H, and further optionally wherein residue 10 of the B5 domain is L; (xvi) residue 12 of the B5 domain is not H, and further optionally wherein residue 12 of the B5 domain is R; and / or (xvii) residue 8 of the B6 domain is not H, and further optionally wherein residue 8 of the B6 domain is Y.

[0142] In certain aspects, (i) residue 9 of the H1 domain is N, T, S, H, R, C, L, D, V, A, Q, G, E, K, I, N, or M; (ii) residue 1 of the B3 domain is L, W, or H; (iii) residue 10 of the B4 domain is F, Y, L, I, K, or M; (iv) residue 12 of the B4 domain is F, L, R, D, M, Q, or V; (v) residue 10 of the B5 domain is L; (vi) residue 8 of the B6 domain is Y, F, or L; and / or (vii) residue 11 of the B5 domain is F or Y; (viii) residue 2 of the H2 domain is A, F, I, K, L, N, R, S, T, Q, V, or Y; (ix) residue 2 of the L2 domain is not H, and further optionally wherein residue 2 of the L2 domain is A, D, F, I, K, L, N, R, S, T, Q, V, or Y; (x) residue 3 of the B1 domain is not H, and further optionally wherein residue 3 of the B1 domain is A, D, F, I, K, L, N, R, S, T, Q, V, or Y; (xi) residue 2 of the B2 domain is A, D, F, I, K, L, N, R, S, T, Q, or Y; (xii) residue 1 of the H3 domain is A, D, F, I, K, L, N, S, T, Q, V, or Y; (xiii) residue 10 of the H3 domain is A, D, F, I, K, L, N, S, T, Q, V, or Y; (xiv) residue 11 of the H3 domain is A, D, F, I, K, N, R, S, T, Q, V, or Y; (xv) residue 5 of the B3 domain is A, D, F, I, L, N, R, S, T, Q, V, or Y; (xvi) residue 5 of the B4 domain is not H, and further optionally wherein residue 5 of B4 is A, D, F, I, K, L, N, R, S, T, Q, V, or Y; (xvii) residue 7 of the B4 domain is A, D, F, I, L, N, R, S, T, V, or Y; (xviii) residue 9 of the B4 domain is not H, and further optionally wherein residue 9 of B4 is A, D, F, I, L, K, N, R, S, T, Q, V, or Y; (xix) residue 8 of the B5 domain is A, D, F, I, L, K, N, R, S, Q, or Y; (xx) residue 9 of the B5 domain is not H, and further optionally wherein residue 9 of B5 is A, D, F, I, L, K, N, R, S, Q, V, or Y; (xxi) residue 12 of the B5 domain is not H, and further optionally wherein residue 12 of B5 is A, D, F, I, L, K, N, R, S, Q, V, or Y; (xxii) residue 14 of the B5 domain is A, D, F, I, L, K, N, S, Q, V, or Y; (xxiii) residue 3 of the B6 domain is A, D, F, I, L, K, N, R, S, Q, V, or Y; (xxiv) residue 4 of the B6 domain is A, D, F, I, L, K, N, R, S, Q, V, or Y; and / or (xxv) residue 9 of the B6 domain is A, D, F, L, K, N, R, S, Q, V, or Y.

[0143] In certain aspects, (i) residue 9 of the H1 domain is N; (ii) residue 1 of the B3 domain is L; (iii) residue 10 of the B4 domain is F; (iv) residue 12 of the B4 domain is R; (v) residue 10 of the B5 domain is L; (vi) residue 8 of the B6 domain is Y; (vii) residue 11 of the B5 domain is F; (viii) residue 2 of the H2 domain is I; (ix) residue 2 of the L2 domain is not H, and further optionally wherein residue 2 of the L2 domain is D; (x) residue 3 of the B1 domain is not H, and further optionally wherein residue 3 of the B1 domain is T; (xi) residue 2 of the B2 domain is T; (xii) residue 1 of the H3 domain is V; (xiii) residue 10 of the H3 domain is S; (xiv) residue 11 of the H3 domain is Q; (xv) residue 5 of the B3 domain is S; (xvi) residue 5 of the B4 domain is not H, and further optionally wherein residue 5 of the B4 is T; (xvii) residue 7 of the B4 domain is R; (xviii) residue 9 of the B4 domain is not H, and further optionally wherein residue 9 of the B4 is S; (xix) residue 8 of the B5 domain is L; (xx) residue 9 of the B5 domain is not H, and further optionally wherein residue 9 of the B5 is Q; (xxi) residue 12 of the B5 domain is not H, and further optionally wherein residue 12 of the B5 is K; (xxii) residue 14 of the B5 domain is V; (xxiii) residue 3 of the B6 domain is V; (xxiv) residue 4 of the B6 domain is A; and / or (xxv) residue 9 of the B6 domain is V.

[0144] In other aspects, the polypeptide can have an amino acid sequence wherein: (i) residue 7 of the H2 domain is S; (ii) residue 4 of the L2 domain is H; (iii) residue 10 of the B3 domain is R; (iv) residue 1 of the B3 domain is L, W, or H; (v) residue 7 of the B4 domain is K; (vi) residue 10 of the B4 domain is F, Y, L, I, K, or M; (vii) residue 12 of the B4 domain is F, D, Y, L, I, K, or M; (viii) residue 3 of the B6 domain is D or N; (ix) residue 8 of the B6 domain is Y, F, or L; and (x) residue 11 of the B5 domain is W, Y, or F.

[0145] One or more amino acids that can be present at a particular position in a domain of a polypeptide of the present disclosure and have luciferase activity; its position relative to SEQ ID NO: 1; and the amino acid at that position in SEQ ID NO: 1 are as follows:

[0146] In certain aspects, a protein having luciferase activity can have an amino acid sequence that is at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, or 95% identical to the amino acid sequence of SEQ ID NO: 1, wherein when numbered relative to SEQ ID NO: 1, residue 87 is F, L, R, D, M, Q, or V; residue 85 is F, Y, L, I, K, or M, and residue 100 is F, Y, or L. LuxSit-i variants having substitutions in the H1 domain

[0147] In certain aspects, the protein having luciferase activity can have the secondary structure arrangement H1-L1-H2-L2-B1-L3-B2-L4-H3-L5-B3-L6-B4-L7-B5-L8-B6 as described herein, wherein the length of the H1 domain is at least 18 or 19 amino acids; residue 9 of the H1 domain is T, S, H, R, C, L, V, A, Q, G, E, K, I, N, or M. In contrast, residue 9 of the H1 domain in LuxSit-i is F. Residue 9 of H1 can also be referred to by the position of the corresponding amino acid in the H1 domain in SEQ ID NO: 1, wherein residue 9 in the H1 domain is position 9 in SEQ ID NO: 1. In certain aspects, the protein having luciferase activity can have an amino acid sequence that is at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, or 95% identical to the amino acid sequence of SEQ ID NO: 1, wherein residue 9 is T, S, H, R, C, L, V, A, Q, G, E, K, I, N, or M when numbered relative to SEQ ID NO: 1.

[0148] In certain aspects, the protein having luciferase activity comprises an amino acid sequence that is at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 1, wherein the amino acid at position 9 is any amino acid other than F, wherein position 9 is numbered based on SEQ ID NO: 1. In some embodiments, the amino acid at position 9 is D, E, Q, R, S, T, H, I, L, V, A, G, C, N, K, or M.

[0149] In certain aspects, the protein can further comprise a B4 domain that is at least 12 amino acids in length, and residue 10 of the B4 domain is F, Y, L, I, K, or M; and / or residue 12 of the B4 domain is L, R, D, M, Q, or V.

[0150] In certain aspects, residue 9 of the H1 domain is V, residue 10 of the B4 domain is F, Y, L, I, K, or M, and residue 12 of the B4 domain is L, R, D, M, Q, or V. In certain aspects, residue 9 of the H1 domain is V, residue 10 of the B4 domain is F, and residue 12 of the B4 domain is L. The B3 domain is at least 6, 7, 8, 9, or 10 amino acids in length, and residue 1 of the B3 domain is W or H. Residue 1 of the B3 domain is W.

[0151] In certain aspects, the polypeptide can comprise one or more amino acids in the following listed domains. Also listed is its position relative to SEQ ID NO: 1 and the amino acid at that position in SEQ ID NO: 1.

[0152] In certain embodiments, the protein can further comprise a substitution at position H30, H36, H80, H84, H87, H92, H98, H99, or H101 with another amino acid (e.g., R, Q, T, S, L, Y, etc.). In some embodiments, the protein further comprises one or more of the following substitutions: H30D, H36T, H80T, H84S, H87R, H92S, H98Q, H99L, and H101R. In other embodiments, the protein further comprises a substitution at one or more of positions V41, R46, T97, R103, E109, and I114. In other embodiments, the protein further comprises one or more of the following substitutions: V41T, R46V, T97L, R103V, E109D, and I114T. LuxSit-i variants having substitutions in the B3 domain

[0153] In certain aspects, the protein having luciferase activity can have the secondary structure arrangement H1-L1-H2-L2-B1-L3-B2-L4-H3-L5-B3-L6-B4-L7-B5-L8-B6 as described herein, the B3 domain is at least 6, 7, 8, 9, or 10 amino acids in length, and residue 1 of the B3 domain is W or H. In contrast, residue 1 of the B3 domain in LuxSit-i is Q. Residue 1 of the B3 domain can also be referred to by the position of the corresponding amino acid in the B3 domain of SEQ ID NO: 1, where residue 1 of the B3 domain is position 64 in SEQ ID NO: 1. Proteins having amino acid sequence identity to LuxSit-i variants

[0154] In certain aspects, a protein having luciferase activity can have an amino acid sequence that is at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 1, wherein residue 64 is W or H when numbered relative to SEQ ID NO: 1.

[0155] In certain embodiments, a protein of the present disclosure having luciferase activity can have an amino acid sequence that is at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of a LuxSit-i variant disclosed herein (e.g., a LuxSit-i variant listed in Table 5).

[0156] In certain embodiments, a protein of the present disclosure having luciferase activity can have an amino acid sequence that is at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 2-91, 93-143, 2601-2604, 2663, 2665, 2682-2732, and 2753-2769.

[0157] In certain embodiments, a protein of the present disclosure can have an amino acid sequence that is at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 2668-2671, wherein the protein does not have significant luciferase activity. The protein can have luciferase activity when associated with a complementary polypeptide (e.g., a fragment having an amino acid sequence that is at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of any one of SEQ ID NOs: 2605, 2607, 2615-2617, 2619, 2621, 2623, 2625, 2627, 2629, 2631, 2633, 2635, 2637, 2638, 2640-2655, 2672-2673, 2676, 2678, and 2734).

[0158] In certain embodiments, a protein of the present disclosure can have an amino acid sequence that is at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of any LuxSit-i variant or fragment thereof disclosed herein. In certain embodiments, a protein of the present disclosure can have an amino acid sequence that is at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of any LuxSit-i variant or fragment thereof disclosed herein, and the protein can comprise one or more substitutions relative to the amino acid sequence set forth in SEQ ID NO: 1, wherein the one or more substitutions are conservative amino acid substitutions.

[0159] In certain embodiments, a protein of the present disclosure can have an amino acid sequence that is at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of any of the LuxSit-i variants disclosed herein or fragments thereof, and the protein lacks a lysine residue, wherein the lysine residue present in SEQ ID NO: 1 is replaced with another amino acid (such as R, Q, T, S, L, Y, etc.). In certain embodiments, a protein of the present disclosure can have an amino acid sequence that is at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 1 or fragments thereof, the protein lacks a lysine residue relative to SEQ ID NO: 1 and contains an arginine or histidine in place of the lysine.

[0160] In certain embodiments, a protein of the present disclosure comprises the substitution F9V / S / N, and optionally one or more of the following substitutions: Q64W, A85F, and H87L.

[0161] In certain embodiments, a protein of the present disclosure comprises the substitution F9N, and optionally one or more of the following substitutions: Q64L, A85F, H87R, H99L, W100F, T108D, and H113Y.

[0162] In certain embodiments, a protein of the present disclosure comprises the substitution F9N, and optionally one or more of the following substitutions: H30D, H36T, Q64L, H80T, H84S, A85F, H87R, H92S, H98Q, H99L, W100F, H101R, T108D, and H113Y.

[0163] In certain embodiments, a protein of the present disclosure comprises the substitution F9N, and optionally one or more of the following substitutions: H30D, H36T, L56Q, Q64L, H80T, H84S, A85F, H87R, H92S, H98Q, H99L, W100F, H101K / R, T108D, and H113Y.

[0164] In certain embodiments, the protein of the present disclosure comprises the substitution F9N, and optionally one or more of the following substitutions: D23I, H30D, H36T, V41T, R46V, R55S, L56Q, Q64L, K68S, H80T, Q82R, H84S, A85F, H87R, H92S, T97L, H98Q, H99L, W100F, H101K / R, R103V, T108D / V, E109A, H113Y, and I114V, further optionally wherein the amino acid sequence does not comprise lysine.

[0165] The sequences of the LuxSit-i variants are set forth in SEQ ID NOs: 2-91, 93-143, 2601-2604, 2663, or 2665 in Table 5. Conjugated proteins

[0166] The LuxSit-i variants disclosed herein can be conjugated to another moiety. The moiety can be a small molecule, a peptide, a polypeptide, a nucleic acid, or a lipid. The LuxSit-i variants can also be tagged with sequences for localization to a cellular compartment, a cell membrane, or for secretion. The LuxSit-i variants disclosed herein can be used as biosensors by conjugating a moiety to the N-terminus, the C-terminus, or conjugated between the N-terminus and the C-terminus. The moiety can be conjugated directly to the LuxSit-i variant, for example, via a peptide bond to the N-terminus and / or the C-terminus and / or an amino acid side chain, or can be conjugated to the LuxSit-i body via a linker. The linker can be a polymer, for example, an amino acid linker or a sugar linker.

[0167] A variety of linkers can be used, and the linker can include an alkyl, a methylene carbon chain, an ether, a polyether, an alkyl amide linker, a peptide linker, a modified peptide linker, a poly(ethylene glycol) (PEG) linker, a streptavidin-biotin or avidin-biotin linker, a polyamino acid (e.g., polylysine), a functionalized PEG, a polysaccharide, a glycosaminoglycan, an oligonucleotide linker, a phospholipid derivative, an alkenyl chain, an alkynyl chain, a disulfide, or combinations thereof. In some embodiments, the linker is cleavable (e.g., enzymatic (e.g., TEV protease site), chemical, light-induced cleavage, etc.).

[0168] In certain aspects, the moiety can be a heterologous amino acid sequence. In certain aspects, the moiety is conjugated to the LuxSit-i variant post-translation. In certain aspects, the moiety is conjugated to the LuxSit-i variant during translation, e.g., the nucleic acid can encode a fusion protein comprising the Lux-Sit-i variant and the moiety.

[0169] In certain aspects, the heterologous amino acid sequence comprises a protein binding domain, such as a protein binding domain that binds IL-17RA (e.g., IL-17A) or an IL-17A binding domain of IL-17RA, a Jun binding domain of Erg or an EG binding domain of Jun; a potassium channel voltage sensing domain (e.g., a domain useful for detecting protein conformational changes, a GTPase binding domain of Cdc42 or rac target or other GTPase binding domain), a domain associated with kinase or phosphatase activity (e.g., regulatory myosin light chain, PKC delta, pleckstrin-harboring PH and DEP domains, other phosphorylation recognition domains and substrates); a glucose binding protein domain, a glutamate / aspartate binding protein domain, a PKA or cAMP-dependent binding substrate, an InsP3 receptor, GKI, PDE, an estrogen receptor ligand binding domain, apoK1-er or a calmodulin binding domain.

[0170] In certain aspects, a fusion protein comprising a LuxSit-i variant fused to a heterologous amino acid sequence can be a biosensor. The biosensor can be used to detect binding of a GTPase, such as Cdc42 or Rac, to EBFP, EGFP PAK fragment, Raichu-Rac, Raichu-Cdc42, integrin alpha v beta 3, IBB of importin-a, DMCA of CRaf1, or NBD-Ras (for Ras activation), binding domain of Ras / Rap Ral RBD with Ras isoprenylation sequence. In one embodiment, the biosensor detects PI(4,5)P2 (e.g., using Ph-PCLdelta1, PH-GRP1), PI(4,5)P2 or PI(4)P (e.g., PH-OSBP), PI(3,4,5)P3 (e.g., using PH-ARNO, or PH-BTK, or PH-Cell adhesion protein 1), PI(3,4,5)P3 or PI(3,4)P2 (e.g., using PH Akt), PI(3)P (e.g., using FYVE-EEA1), or Ca2+(cytosolic) (e.g., using calmodulin or C2 domain of PKC).

[0171] In one aspect, the fusion protein comprising a LuxSit-i variant is fused to a protein domain. In one embodiment, the domain is one that has a phosphorylated tyrosine (e.g., in Src, AbI, and EGFR), detects phosphorylation of ErbB2, phosphorylation of tyrosine in Src, AbI, and EGFR, activation of MKA2 (e.g., using MK2), cAMP-induced phosphorylation, activation of PKA (e.g., using KID of CREG), phosphorylation of CrkII (e.g., using SH2 domain pTyr peptide), binding of bZIP transcription factors and REL proteins (e.g., bFos and bJun, ATF2 and Jun), or p65 NFκB, or microtubule binding (e.g., using kinesin).

[0172] The LuxSit-i variants disclosed herein, as well as the circularly permuted versions of LuxSit-i and LuxSit-i variants, and the self-complementary components of LuxSit-I, LuxSit-i variants, and the circularly permuted versions of LuxSit-i and LuxSit-i variants, can be conjugated to an antibody or antigen-binding fragment thereof.

[0173] The LuxSit-i variants disclosed herein, as well as the circularly permuted versions of LuxSit-i and LuxSit-i variants, can include deletion of residues at the N- or C-terminus or both, e.g., 1 to 3 or more residues at the N-terminus and 1 to 6 or more residues at the C-terminus, prior to the original (e.g., prior to circular permutation), as well as including sequences that interact directly or indirectly with a molecule of interest, such as a molecule described herein. Self-complementary multipartite proteins having luciferase activity

[0174] A self-complementary multipartite protein having luciferase activity is provided. In certain aspects, the multipartite protein can have two self-complementary components or three self-complementary components.

[0175] Self-complementarity refers to the ability of two or more polypeptides to form a complex with one another to restore an enzymatic activity that is not present or is substantially not present when the two or more polypeptides are not associated. The complementary polypeptides can require assistance to form a stable complex (e.g., from an interaction element), e.g., to place the polypeptides in the proper conformation for complementation, to co-localize the complementary polypeptides, to lower the interaction energy of the polypeptides, etc.

[0176] A multi-part protein refers to a protein complex in which the polypeptide components of the multi-part protein are in direct and / or indirect contact with one another. In one aspect, direct contact means that two or more molecules are in close enough proximity such that attractive noncovalent interactions between the molecules (e.g., van der Waals forces, hydrogen bonding, ionic, and hydrophobic interactions, etc.) influence the interaction of the molecules. An example of direct contact can include a multi-part protein comprising, from N-terminus to C-terminus, a first polypeptide component, a linker, and a second polypeptide component, wherein the first polypeptide component and the second polypeptide component are associated and have luciferase activity, and are separated and lack or have significantly reduced cleavage activity after cleavage of the linker. In one aspect, indirect contact means that two or more molecules interact when a bridging moiety conjugated to the two or more molecules brings the two or more molecules in close enough proximity in a stable complex such that attractive noncovalent interactions between the molecules (e.g., van der Waals forces, hydrogen bonding, ionic, and hydrophobic interactions, etc.) influence the interaction of the molecules. Self-complementary multi-part proteins having two or more components

[0177] In certain aspects, a self-complementary multi-part protein comprises at least a first polypeptide component and a second polypeptide component, wherein the at least first polypeptide component and the second polypeptide component are not covalently linked or are covalently linked via a linker (e.g., a cleavable linker), wherein the first polypeptide component and the second polypeptide component collectively comprise a secondary structure arrangement H1-L1-H2-L2-B1-L3-B2-L4-H3-L5-B3-L6-B4-L7-B5-L8-B6, wherein each domain is as described herein, and (a) each H and B domain is entirely present within one polypeptide component of the first polypeptide component or the second polypeptide component, (b) the first polypeptide component and the second polypeptide component do not comprise all of the H and B domains, (c) the relative order of the H, L, and B domains is unchanged from the order shown in the secondary structure arrangement H1-L1-H2-L2-B1-L3-B2-L4-H3-L5-B3-L6-B4-L7-B5-L8-B6 when present in either the first polypeptide component or the second polypeptide component, and (d) the first component and the second component do not have detectable luciferase activity or have luciferase activity that is lower than the luciferase activity of the self-complementary multi-part protein when not present in the self-complementary multi-part protein.

[0178] In certain aspects, the first polypeptide component and the second polypeptide component comprise a secondary structure arrangement as shown in Table 1: Table 1:

[0179] The L domain in parentheses (i) is present in one but not both of the first and second components, (ii) is split between the first and second components, or (iii) is absent.

[0180] In certain aspects, one or both of the first and second components comprises an additional domain. The additional domain can be covalently linked to one or both of the first and second components. The domain can be a small molecule, a peptide, a polypeptide, a nucleic acid, a lipid, an aptamer, and the like.

[0181] In certain aspects, the first component is a fusion protein comprising a first domain, and the second component is a fusion protein comprising a second domain. The H and B domains present in the first and second components can be as described herein, such as those having substitutions relative to the H and B domains of LuxSit-i.

[0182] In some embodiments, the first and second components have high affinity for each other and form a high affinity two-component protein with luciferase activity through direct interaction. In other words, the two components form a stable complex with luciferase activity when in close proximity (e.g., in a polypeptide, in a cell, in a cell lysate, in a cell-free solution, etc.).

[0183] In some embodiments, the first and second components have low affinity for each other and form a two-component protein with luciferase activity through indirect interaction mediated by a binding pair. In other words, the two components form a stable complex with luciferase activity when each component is conjugated to a member of a binding pair, and the interaction between the binding pair members allows the two-component protein with luciferase activity to form.

[0184] The binding pair can be a ligand and a receptor; an antigen and an antibody; self-complementary enzyme fragments, such as beta-galactosidase; biotin-avidin; two complementary nucleic acids; two polypeptides capable of dimerization (e.g., homodimers, heterodimers, etc.); and the like.

[0185] In some embodiments, the self-complementary multi-part protein comprises, from N- to C-terminus: a first polypeptide component, a linker, and a second polypeptide component or a second polypeptide component, a linker, and a first polypeptide component, and has luciferase activity. The linker can be a cleavable linker. For example, the linker can comprise a cleavage site for a protease. In the presence of the protease, the linker is cleaved, resulting in separation of the first polypeptide component and the second polypeptide component and a loss or substantial reduction in luciferase activity compared to the luciferase activity of the self-complementary multi-part protein. In certain embodiments, the protease can be a neurotoxin and the self-complementary multi-part protein can be used to detect the presence of the protease. In certain embodiments, the self-complementary multi-part protein can comprise a spacer between the linker and the first component and / or the second component. In certain embodiments, the neurotoxin cleavage site comprises a Clostridium botulinum neurotoxin (BoNT) or a tetanus neurotoxin cleavage site. Self-complementary multi-part proteins having three or more components

[0186] In certain aspects, a self-complementary multi-part protein having luciferase activity as provided herein comprises at least a first polypeptide component, a second polypeptide component, and a third polypeptide component, wherein the at least first polypeptide component, the second polypeptide component, and the third polypeptide component are not covalently linked or are covalently linked via one or more linkers (e.g., one or more cleavable linkers), wherein the first polypeptide component, the second polypeptide component, and the third polypeptide component collectively comprise a secondary structure arrangement H1-L1-H2-L2-B1-L3-B2-L4-H3-L5-B3-L6-B4-L7-B5-L8-B6, wherein each domain is as described herein, and (a) each H and B domain is entirely present within one polypeptide component of the first polypeptide component, the second polypeptide component, or the third polypeptide component, (b) the first polypeptide component, the second polypeptide component, and the third polypeptide component do not comprise all of the H and B domains, (c) the relative order of the H, L, and B domains when present in the first polypeptide component, the second polypeptide component, or the third polypeptide component is unchanged relative to the order in the secondary structure arrangement H1-L1-H2-L2-B1-L3-B2-L4-H3-L5-B3-L6-B4-L7-B5-L8-B6, and (d) the first polypeptide component, the second polypeptide component, and the third polypeptide component do not have detectable luciferase activity or have a luciferase activity that is lower than the luciferase activity of the self-complementary multi-part protein when not present in the self-complementary multi-part protein.

[0187] In certain aspects, the H and B domains of the protein are separated at the L domain into a first polypeptide component, a second polypeptide component, and a third polypeptide component, where the separation point is the N-terminus of the L domain, the C-terminus of the L domain, or within the L domain. Thus, the first component can comprise the H1 domain, the second component can comprise the H2 domain, and the third component can comprise the remainder of the domains, where the L1 domain can be split between the first component, the second component, between the two components, or absent from both components, and the L2 domain can be split between the second component, the third component, between the two components, or absent from both components.

[0188] A non-limiting list of three component systems is provided below:

[0189] In certain aspects, at least one of the first component, the second component, and the third component comprises an additional domain. In certain aspects, the additional domain is covalently linked to at least one of the first component, the second component, and the third component.

[0190] In certain aspects, the first component is a fusion protein comprising a first domain, the second component is a fusion protein comprising a second domain, and the third component is a fusion protein comprising a third domain. The H and B domains present in the first component, the second component, and the third component can be as described herein, such as those with substitutions relative to the H and B domains of LuxSit-i. Circularly permuted polypeptides having luciferase activity

[0191] In certain embodiments, a circularly permuted polypeptide having luciferase activity is disclosed. The N-terminus and the C-terminus of the circularly permuted polypeptide differ from the N-terminus and the C-terminus, respectively, of a protein having luciferase activity and comprising the secondary structure arrangement H1-L1-H2-L2-B1-L3-B2-L4-H3-L5-B3-L6-B4-L7-B5-L8-B6, where the H, B, and L domains are as shown for LuxSit-i or a variant of LuxSit-i as described herein. The N-terminus and the C-terminus of the protein having luciferase activity are connected by a linker sequence, and the circularly permuted polypeptide comprises the following secondary structure arrangement: H2-L2-B1-L3-B2-L4-H3-L5-B3-L6-B4-L7-B5-L8-B6-linker-H1-(L1) (I), B1-L3-B2-L4-H3-L5-B3-L6-B4-L7-B5-L8-B6-linker-H1-L1-H2-(L2) (II), B2-L4-H3-L5-B3-L6-B4-L7-B5-L8-B6-linker-H1-L1-H2-L2-B1-(L3) (III), H3-L5-B3-L6-B4-L7-B5-L8-B6-linker-H1-L1-H2-L2-B1-L3-B2-(L4) (IV), B3-L6-B4-L7-B5-L8-B6-linker-H1-L1-H2-L2-B1-L3-B2-L4-H3-(L5) (V), B4-L7-B5-L8-B6-linker-H1-L1-H2-L2-B1-L3-B2-L4-H3-L5-B3-(L6) (VI), B5-L8-B6-linker-H1-L1-H2-L2-B1-L3-B2-L4-H3-L5-B3-L6-B4-(L7) (VII) or B6-linker-H1-L1-H2-L2-B1-L3-B2-L4-H3-L5-B3-L6-B4-L7-B5-(L8) (VIII), wherein the L domain in parentheses is present at the C-terminus or the N- terminus, or split between the C-terminus and the N-terminus, or is not present.

[0192] In some embodiments, the B5 domain comprises an amino acid sequence at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence: QKHTVDLTHHWHFR or QKHTVILTHVFRFR. In some embodiments, the B4 domain comprises an amino acid sequence at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence: TVEVHVQLHATH or TVVVVVRLDFTL.

[0193] In some aspects, the length of the linker is 10-100 amino acids, for example, the length is 10-90, 10-80, 10-70, 10-60, or 10-50 amino acids. In some aspects, the linker comprises secondary structure H4-L9. In some aspects, the linker comprises secondary structure H4-L9-H5-L10. In some aspects, the linker comprises secondary structure H4-L9-H5-L10-H6-L11. H4, H5, and H6 can be a helical domain of any amino acid sequence that provides a helical structure. L9, L10, and L11 can be linker sequences, and the length range can be 1, 2, 3, 4, 5, or more amino acids.

[0194] In some aspects, the linker comprises an amino acid sequence that is at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acids: VDDVEEVLARVLEEGERLVERLRAERPEA; TGEEPEKPEFKETFGPS; VESEEELPAALARAEELGRELLERTLAEEGAGGPP; APSLDEESIEARVAEARRLAEERLAELGDPPP; TGEEPEPPEFRERFGPSA; DLSPEAIEAAIAKALARADALLAELGAPPP; TGEEPERPEFVERFGPSS; SLDEAAIEAAIARARARADELLAELGAPPA; CPSLDEASIAAAIAEAEALAAERLAELGAPPP; TGEEPEPPEFRERFGPSS; or DPDEETRLAAAREALERAGVPEEMRRAALELLERGERELFRPSA.

[0195] The present disclosure also encompasses split component multi-part proteins comprising at least two components or at least three components produced by splitting a cyclic permutation polypeptide described herein. The B domain, the H domain, and the L domain can be as specified herein.

[0196] In some aspects, the cyclic permutation protein is derived from a LuxSit-i variant having the amino acid sequence set forth in SEQ ID NO: 2600, and has the amino acid sequence set forth in SEQ ID NO: 2227 or 2236, and has luciferase activity similar to SEQ ID NO: 2600.

[0197] The circularly permuted proteins provided herein can be used in methods analogous to those described herein for the LuxSit-i variants and in methods known in the art for using luciferase. Split forms of the circularly permuted proteins can be used in methods known in the art and described herein for self-complementary multi-part proteins.

[0198] In certain aspects, the polypeptides encompassed by the present disclosure include polypeptides having an amino acid sequence that is at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid of any of the polypeptides provided herein.

[0199] In certain aspects, the circularly permuted proteins comprise an amino acid sequence that is at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 144-2599.

[0200] In certain aspects, the first component encompassed by the present disclosure includes a first component having an amino acid sequence that is at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid of any of the first components provided herein.

[0201] In certain aspects, the second component encompassed by the present disclosure includes a second component having an amino acid sequence that is at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid of any of the second components provided herein.

[0202] In certain aspects, the third component encompassed by the present disclosure includes a third component having an amino acid sequence that is at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid of any of the third components provided herein. Nucleic acids

[0203] In some aspects, where the polypeptides are relatively short, e.g., comprising one or a few H domains or B domains, such polypeptides can be synthesized using synthetic chemistry. In other aspects, the present disclosure provides nucleic acids comprising nucleotide sequences encoding the polypeptides described herein. These nucleic acids can be used for cell-free transcription and translation. The nucleotide sequences encoding the subject polypeptides can be operably linked to one or more regulatory elements, such as promoters and enhancers, that allow the nucleotide sequences to be expressed in recombinant cells that are genetically modified to produce the polypeptides.

[0204] Suitable promoter and enhancer elements are known in the art. For expression in bacterial cells, suitable promoters include, but are not limited to, lacI, lacZ, T3, T7, gpt, lambda P, and trc. For expression in eukaryotic cells, suitable promoters include, but are not limited to, the cytomegalovirus immediate early promoter; the herpes simplex thymidine kinase promoter; the early and late SV40 promoters; promoters present in the long terminal repeat from retroviruses; the mouse metallothionein-I promoter; and the like.

[0205] The nucleotide sequences encoding the subject polypeptides can be present in expression vectors and / or cloning vectors. Expression vectors can include selectable markers, origins of replication, and other features that provide for replication and / or maintenance of the vector. A large number of suitable vectors and promoters are known to those of skill in the art; many are commercially available for the production of subject recombinant constructs. Vectors are provided by way of example. Bacterial: pBs, phagescript, PsiX174, pBluescript SK, pBs KS, pNH8a, pNH16a, pNH18a, pNH46a (Stratagene, La Jolla, Calif., USA); pTrc99A, pKK223-3, pKK233-3, pDR540, and pRIT5 (Pharmacia, Uppsala, Sweden). Eukaryotic: pWLneo, pSV2cat, pOG44, PXR1, pSG (Stratagene), pSVK3, pBPV, pMSG, and pSVL (Pharmacia). Expression vectors typically have convenient restriction sites located near the promoter sequence to provide for the easy insertion of nucleic acid sequences encoding polypeptides. Selectable markers that are functional in the expression host cells can be present.

[0206] In some cases, a nucleic acid (e.g., as described herein) can be introduced into a cell, e.g., by contacting the cell with the nucleic acid. A cell into which a nucleic acid has been introduced will be generally referred to herein as a genetically modified cell. Various nucleic acid delivery methods can be employed, including but not limited to, e.g., naked nucleic acid delivery, viral delivery, chemical transfection, biolistics, etc.

[0207] Nucleic acids of the disclosure can be provided in a kit. The kit can comprise additional components, such as a reconstitution buffer for resuspending a nucleic acid provided in the kit in lyophilized form. Host cells

[0208] The disclosure provides isolated genetically modified cells (e.g., in vitro cells, ex vivo cells, cultured cells, etc.) that are genetically modified with a subject nucleic acid. In some aspects, a subject isolated genetically modified cell can produce a subject polypeptide. In some cases, a genetically modified cell can be used to screen and / or discover protein-protein interactions; protein-drug interactions; protein-nucleic acid interactions; etc.

[0209] Suitable cells include eukaryotic cells, such as mammalian cells, insect cells, yeast cells; and prokaryotic cells, such as bacterial cells. Introduction of a subject nucleic acid into the host cell can be achieved, e.g., by calcium phosphate precipitation, DEAE dextran-mediated transfection, liposome-mediated transfection, electroporation, or other known methods. Kits

[0210] Aspects of the disclosure include kits for measuring luciferase activity of a luciferase. The kits can comprise components for measuring luciferase activity. The components can be present in separate compartments, e.g., in separate vials. Aspects of the disclosure include kits for measuring luciferase activity of a polypeptide having luciferase activity and / or a self-complementary multi-part protein having luciferase activity. In certain aspects, the kits can comprise one or more of a polypeptide, a first component, a second component, and / or a third component as disclosed herein.

[0211] Aspects of the disclosure include kits comprising one or more nucleic acids encoding a polypeptide, a first component, a second component, and / or a third component.

[0212] In certain aspects, a kit can comprise an assay buffer suitable for measuring luciferase activity. A kit can comprise one or more container means, such as vials, test tubes, etc., each containing a different polypeptide, substrate, assay buffer, etc., used in a method for measuring luciferase activity. For example, one of the containers can comprise a polypeptide having luciferase activity or a polynucleotide encoding the polypeptide (e.g., in the form of a vector). A second container can contain a substrate for the polypeptide. The assay buffer can be any suitable buffer, such as the solutions described in the present disclosure.

[0213] A kit can comprise a luciferin substrate, such as DTZ, coelenterazine, furimazine, coelenterazine-n, coelenterazine-f, coelenterazine-h, coelenterazine-hcp, coelenterazine-cp, coelenterazine-c, coelenterazine-e, coelenterazine-fcp, dideoxy-coelenterazine, coelenterazine-i, coelenterazine-icp, coelenterazine-v, and 2-methyl-coelenterazine, or another luciferin substrate or an analog thereof.

[0214] In certain aspects, a compound of Formula (I) disclosed herein can be provided as part of a kit. In some embodiments, a kit can comprise one or more luciferases (in the form of a polypeptide, a polynucleotide, or both as disclosed herein) and a bioluminescent luciferin substrate of Formula (I).

[0215] A kit can also comprise one or more buffers, such as the solutions or assay buffers disclosed herein. A kit can comprise instructions to enable a user to perform an assay, such as those disclosed herein. In certain aspects, a kit comprises instructions for a method for detecting luminescence in a cell, the method comprising contacting the cell with a luciferin analog, wherein the luciferin analog is a compound of Formula (I) or a stereoisomer, tautomer, or salt thereof; and detecting luminescence. In certain aspects, the cell comprises a living cell. In certain aspects, the cell is in vivo, ex vivo, or in vitro.

[0216] In certain aspects, a kit comprises a luciferin analog, wherein the luciferin analog is a compound of Formula (I), or a stereoisomer, tautomer, or salt thereof. In certain aspects, the kit further comprises a polypeptide having luciferase activity as disclosed herein. In certain aspects, the kit further comprises a buffer reagent.

[0217] In certain aspects, a kit can comprise a luciferin substrate. In certain aspects, a kit can comprise a luciferin substrate of Formula (I): (I) wherein R 1 , R 2 , and R 3 are independently selected from the group consisting of: C3-6 cycloalkyl; aryl; C 1-3 Alkyl, halogen, C 1-3 An aryl group substituted with at least one of a haloalkyl, hydroxyl, alkoxy, nitro, or amino alcohol; a 5- to 10-membered heteroaryl group having 1 to 3 independently selected cyclic heteroatoms chosen from O, S, Se, and N; and a heterocycle; in: If R 1 If it is aryl, then R 2 and R 3 Selected independently from: C 3-6 cycloalkyl; aryl; C 1-3 Alkyl, halogen, C 1-3 An aryl group substituted with at least one of haloalkyl, hydroxyl, alkoxy or nitro groups; and a 5- to 10-membered heteroaryl group having 1 to 3 cyclic heteroatoms independently selected from O, S, Se and N; If R 3 If it is aryl, then R 1 and R 2 Selected independently from: C 3-6 cycloalkyl; aryl; C 1-3 Alkyl, halogen, C 1-3 An aryl group substituted with at least one of a haloalkyl, hydroxyl, alkoxy, nitro, or amino alcohol; a 5-membered heteroaryl group having 1-3 independently selected cyclic heteroatoms chosen from O, S, Se, and N; a 10-membered heteroaryl group having 1-3 independently selected cyclic heteroatoms chosen from O, S, and Se; a 6-membered heteroaryl group having 1-3 independently selected cyclic heteroatoms chosen from O, S, Se, and N; a heterocycle, and If R 2 If it is aryl, then R 1 and R 3 Selected independently from: C 3-6 cycloalkyl; aryl; C 1-3 Alkyl, halogen, C 1-3 An aryl group substituted with at least one of a haloalkyl, hydroxyl, alkoxy, nitro or amino alcohol; a 5-membered heteroaryl group having 1 to 3 cyclic heteroatoms independently selected from O, S, Se and N; a 10-membered heteroaryl group having 1 to 3 cyclic heteroatoms independently selected from O, S, Se and N; and a heterocycle.

[0218] In some respects, R 1 and R 2 It is aryl. In some respects, R 2 and R 3 It is aryl. In some respects, R 1 and R 3 It is an aryl group.

[0219] In some respects, R 1 R2 and any of R 3 is selected from C 3-6 cycloalkyl. C 3-6 Cycloalkyl includes cycloalkyl groups having 3 to 6 carbon atoms, such as cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl. In certain aspects, any of R 1 , R 2 , and R 3 is selected from cyclopropyl.

[0220] In certain aspects, any of R 1 , R 2 , and R 3 is selected from aryl substituted with at least one of C 1-3 alkyl, halogen, C 1-3 haloalkyl, hydroxyl, alkoxy, nitro, or amino alcohol.

[0221] In certain aspects, any of R 1 , R 2 , and R 3 is selected from aryl substituted with C 1-3 alkyl. C 1-3 Alkyl includes straight-chain or branched alkyl groups having 1 to 3 carbon atoms, such as methyl, ethyl, n-propyl, and isopropyl. In certain aspects, any of R 1 , R 2 , and R 3 is selected from aryl substituted with methyl.

[0222] In certain aspects, R 1 , R 2 , and R 3 is selected from aryl substituted with halogen. Halogen groups include halogen atoms, such as fluorine (F), chlorine (Cl), bromine (Br), and iodine (I). In certain aspects, any of R 1 , R 2 , and R 3 is selected from aryl substituted with fluorine.

[0223] In certain aspects, any of R 1 , R 2 , and R 3 is selected from aryl substituted with C 1-3 haloalkyl. C 1-3 Haloalkyl includes straight-chain or branched haloalkyl groups having 1 to 3 carbon atoms obtained by substituting one or more hydrogen atoms with a halogen atom, such as fluoromethyl, difluoromethyl, trifluoromethyl, chloromethyl, and dichloromethyl. In certain aspects, any of R 1 , R 2 , and R 3 is selected from aryl substituted with trifluoromethyl.

[0224] In certain aspects, any of R 1 , R 2 , and R 3 is selected from aryl substituted with hydroxyl. In certain aspects, any of R 1 , R 2 , and R 3 is selected from aryl substituted with alkoxy. Alkoxy includes straight or branched chain alkyl groups having 1 to 3 carbon atoms, such as methoxy, ethoxy, n-propoxy, and isopropoxy. In certain aspects, any of R 1 , R 2 , and R 3 is selected from aryl substituted with methoxy.

[0225] In certain aspects, any of R 1 , R 2 , and R 3 is selected from aryl substituted with nitro.

[0226] In certain aspects, any of R 1 , R 2 , and R 3 is selected from aryl substituted with halogen and hydroxyl. In certain aspects, any of R 1 , R 2 , and R 3 is selected from aryl substituted with fluorine and hydroxyl.

[0227] In certain aspects, any of R 1 , R 2 , and R 3 is selected from 5-10 membered heteroaryl having 1-3 ring heteroatoms independently selected from O, S, Se, and N. 5-10 membered heteroaryl includes pyrrole, furan, thiophene, selenophene, pyridine, imidazole, thiazole, isothiazole, oxazole, isoxazole, quinoline, and isoquinoline. In certain aspects, R 3 is selected from 6-membered heteroaryl having N heteroatoms, such as pyridine. In certain aspects, R 1 and R 2 are not 6-membered heteroaryl having N heteroatoms, such as pyridine.

[0228] In certain aspects, R 1 is aryl, R 2 is aryl, and R 3 is cyclopropyl. In certain aspects, R 1 is aryl, R 2 is aryl, and R 3 is aryl substituted with methyl. In certain aspects, R 1 is aryl, R 2 is aryl, and R 3 is aryl substituted with fluorine. In certain aspects, R1 is aryl, R 2 is aryl and R 3 is aryl substituted with trifluoromethyl. In certain aspects, R 1 is aryl, R 2 is aryl and R 3 is aryl substituted with hydroxy. In certain aspects, R 1 is aryl, R 2 is aryl and R 3 is aryl substituted with methoxy. In certain aspects, R 1 is aryl, R 2 is aryl and R 3 is aryl substituted with nitro. In certain aspects, R 1 is aryl, R 2 is aryl and R 3 is selected from a 5-membered heteroaryl having O, S, Se, or N heteroatoms.

[0229] In certain aspects, R 1 is aryl, R 2 is aryl and R 3 is a 6-membered heteroaryl having N heteroatoms. In certain aspects, R 2 is aryl, R 3 is aryl and R 1 is cyclopropyl. In certain aspects, R 2 is aryl, R 3 is aryl and R 1 is aryl substituted with methyl. In certain aspects, R 2 is aryl, R 3 is aryl and R 1 is aryl substituted with fluoro. In certain aspects, R 2 is aryl, R 3 is aryl and R 1 is aryl substituted with trifluoromethyl. In certain aspects, R 2 is aryl, R 3 is aryl and R 1 is aryl substituted with hydroxy. In certain aspects, R 2 is aryl, R 3 is aryl and R 1 is aryl substituted with methoxy. In certain aspects, R 2 is aryl, R 3 is aryl and R 1 is aryl substituted with nitro. In certain aspects, R 2 is aryl, R 3 is aryl and R 1 is selected from a 5-membered heteroaryl having O, S, Se, or N heteroatoms.

[0230] In certain aspects, R is aryl, R is aryl, and R is cyclopropyl. In certain aspects, R is aryl, R is aryl, and R is methyl. In certain aspects, R is aryl, R is aryl, and R is fluorine. In certain aspects, R is aryl, R is aryl, and R is trifluoromethyl. In certain aspects, R is aryl, R is aryl, and R is hydroxyl. In certain aspects, R is aryl, R is aryl, and R is methoxy. In certain aspects, R is aryl, R is aryl, and R is nitro. In certain aspects, R is aryl, R is aryl, and R is selected from 5-membered heteroaryl having O, S, Se, or N heteroatom. 1 is aryl, R 3 is aryl and R 2 is cyclopropyl. In certain aspects, R 1 is aryl, R 3 is aryl and R 2 is aryl substituted with methyl. In certain aspects, R 1 is aryl, R 3 is aryl and R 2 is aryl substituted with fluorine. In certain aspects, R 1 is aryl, R 3 is aryl and R 2 is aryl substituted with trifluoromethyl. In certain aspects, R 1 is aryl, R 3 is aryl and R 2 is aryl substituted with hydroxyl. In certain aspects, R 1 is aryl, R 3 is aryl and R 2 is aryl substituted with methoxy. In certain aspects, R 1 is aryl, R 3 is aryl and R 2 is aryl substituted with nitro. In certain aspects, R 1 is aryl, R 3 is aryl and R 2 is selected from 5-membered heteroaryl having O, S, Se, or N heteroatom.

[0231] In certain aspects, R 1 is aryl substituted with hydroxyl, R 2 is aryl substituted with methoxy, and R 3 is aryl. In certain aspects, R 1 is aryl substituted with hydroxyl, R 2 is aryl substituted with hydroxyl, and R 3 is aryl. In certain aspects, R 1 is aryl substituted with hydroxyl, R 2 is aryl substituted with fluorine, and R 3 is aryl. In certain aspects, R 1 is aryl substituted with hydroxyl, R 2 is selected from 5-membered heteroaryl having O, S, Se, or N heteroatom, and R 3 is aryl. In certain aspects, R 1 is aryl substituted with hydroxyl, R 2 is imidazole, and R 3 is aryl. In certain aspects, R 1 is aryl substituted with hydroxyl, R 2is selected from 10-membered heteroaryl having O, S, Se, or N heteroatoms, and R 3 is aryl. In certain aspects, R 1 is aryl substituted with hydroxyl, R 2 is quinoline, and R 3 is aryl. In certain aspects, R 1 is aryl substituted with hydroxyl, R 2 is 6-membered heteroaryl having N heteroatoms, and R 3 is aryl. In certain aspects, R 1 is aryl substituted with hydroxyl, R 2 is pyridine, and R 3 is aryl.

[0232] Representative compounds of Formula (I) include, but are not limited to:

[0233] Compounds can exist as stereoisomers where asymmetric or chiral centers are present. Stereoisomers are "R" or "S" depending on the configuration of substituents around the chiral carbon atom. The terms "R" and "S" as used herein are as defined by IUPAC 1974 Recommendations for Section E, Fundamental Stereochemistry, Pure Appl. Chem., 1976, 45: 13-30. The present disclosure contemplates various stereoisomers and mixtures thereof, and these are specifically included within the scope of the invention. Stereoisomers include enantiomers and diastereomers and mixtures of enantiomers or diastereomers. Single individual stereoisomers of a compound can be synthesized from commercially available starting materials containing asymmetric or chiral centers, or prepared from racemic mixtures by resolution methods well known to those of ordinary skill in the art. These resolution methods are exemplified by: (1) attachment of the mixture of enantiomers to a chiral auxiliary, separation of the resulting mixture of diastereomers by recrystallization or chromatography, and optional release of the optically pure product from the auxiliary, as described in Furniss, Hannaford, Smith, and Tatchell, "Vogels Text book of Practical Organic Chemistry", 5thEdition (1989), Longman Scientific & Technical, Essex CM20 2JE, England, or (2) direct separation of the mixture of optical enantiomers on a chiral chromatographic column, or (3) fractional recrystallization methods.

[0234] It will be appreciated that compounds can have tautomeric forms as well as geometric isomers, and these also form an aspect of the present invention. Properties of compounds of formula (I)

[0235] Compounds of formula (I) are bioluminescent luciferin substrates that can be used by luciferases or photoproteins to produce luminescence. Bioluminescent luciferin substrates as described herein can have improved properties, such as better luminescence than diphenyltriazine (DTZ) and better serum stability. Bioluminescent luciferin substrates as described herein can also exhibit better solubility than DTZ.

[0236] As used herein, "luminescence" refers to detectable electromagnetic radiation, typically UV, IR, or visible light radiation, produced when the excited product of an exergonic chemical process reverts to its ground state with the emission of light. Chemiluminescence is luminescence produced by a chemical reaction. Bioluminescence is chemiluminescence produced by a chemical reaction that uses a biomolecule or synthetic form or analog thereof as a substrate and / or enzyme.

[0237] As used herein, "bioluminescence", as a type of chemiluminescence, refers to the emission of light by biomolecules, particularly proteins. A necessary condition for bioluminescence is the presence of molecular oxygen, either bound or free, in the presence of an oxygenase enzyme, luciferase, which acts on a substrate, luciferin. Bioluminescence is produced by an enzyme, luciferase, which acts on a substrate, luciferin (a bioluminescent luciferin substrate), in the presence of molecular oxygen and converts the substrate to an excited state, which releases energy in the form of detectable electromagnetic radiation when returning to a lower energy level.

[0238] Luminescence is the light output of luciferase under suitable conditions, e.g., in the presence of a suitable substrate such as a diphenyltriazine analog. The light output can be measured as a momentary or near-momentary measurement of light output at the start of the luminescence reaction, which can be initiated upon addition of luciferin substrate. In various embodiments, the luminescence reaction is carried out in solution. In other embodiments, the luminescence reaction is carried out on a solid support. The solution can contain lysate, e.g., from cells in a prokaryotic or eukaryotic expression system. In other embodiments, expression occurs in a cell-free system, or the luciferase protein is secreted into the extracellular medium, such that in the latter case, lysate need not be produced. In some embodiments, the reaction is initiated by injection of suitable materials (e.g., diphenyltriazine analog, buffer, etc.) into a reaction chamber (e.g., a well of a multi-well plate such as a 96-well plate) containing the luminescent protein. In other embodiments, the luciferase and / or diphenyltriazine analog (e.g., a compound of Formula (I)) is introduced into a host, and the luminescence measurement is carried out on the host or a portion thereof, which can include the entire organism or its cells, tissues, explants, or extracts. The reaction chamber can be located in a reading device, which can measure the light output, e.g., using a luminometer or photomultiplier tube. The light output or luminescence can also be measured over time, e.g., for a period of seconds, minutes, hours, etc., in the same reaction chamber. The light output or luminescence can be reported as an average over time, a half-life of signal decay, a sum of the signal over a period of time, or a peak output. Luminescence can be measured in relative light units (RLU).

[0239] The terms "luminescence" and "bioluminescence" are used interchangeably herein. Synthesis of compounds of Formula (I)

[0240] A method of preparing a bioluminescent luciferin substrate is disclosed, wherein the luciferin substrate comprises an imidazopyrazine scaffold. Typically, the method comprises modifying position C2, C6 or C8 of the imidazopyrazine scaffold.

[0241] In certain embodiments, the method is performed according to Scheme I below:

[0242] Scheme I

[0243] In Scheme I, R 1 , R 2 and R 3 are the same as defined above; NBS is N-bromosuccinimide; DCM is dichloromethane; Br2 is bromine; Pyr is pyridine; EtOH is ethyl alcohol or ethanol; and R 1 B(OH)2and R 2 B(OH)2is boronic acid.

[0244] The method of preparing a compound of Formula (I) uses a Suzuki coupling reaction as the key reaction. Utility

[0245] The compounds of the present disclosure can be used in any manner in which luciferin substrates have been used. For example, they can be used in a bioluminescent method that employs a luciferin substrate to detect one or more molecules (e.g., an enzyme, a co-factor of an enzyme reaction, an enzyme substrate, an enzyme inhibitor, an enzyme activator, or an OH radical) or one or more conditions (e.g., redox conditions) in a sample. The sample can include an animal (e.g., a vertebrate), a plant, a fungus, a physiological fluid (e.g., blood, plasma, urine, mucus secretion), a cell, a cell lysate, a cell supernatant, or a purified fraction of a cell (e.g., a subcellular fraction). The presence, amount, spectral profile, emission kinetics, or specific activity of such a molecule can be detected or quantified. The molecule can be detected or quantified in solution (including a multiphasic solution (e.g., an emulsion or a suspension)) or on a solid support (e.g., a particle, a capillary, or an assay vessel).

[0246] In certain aspects, the compound of Formula (I) can be used to detect luminescence in living cells. In some aspects, a luciferase can be expressed in the cells (as a reporter or otherwise), and the cells treated with a bioluminescent luciferin substrate (e.g., a compound of Formula (I)) that penetrates the cells in culture reacts with the luciferase and produces luminescence. In some embodiments, a compound of Formula (I) containing a chemical modification known to increase the stability of native diphenyl tetrazines in culture media can be synthesized and used in a more robust live cell luciferase-based reporter assay. In other aspects, samples containing luciferase and a compound of Formula (I) (including cells, tissues, animals, etc.) can be assayed using various microscopy and imaging techniques. Luciferin substrates

[0247] The present disclosure provides luciferin substrates. The luciferin substrate can be a compound of Formula (la): (Ia) or a stereoisomer, tautomer, or salt thereof, wherein: X 1 -X 2 is independently selected from: halogen, hydroxyl, haloalkyl, alkyl, or nitro; provided that: when X 2 is hydrogen, then X 1 is selected from: haloalkyl, alkyl, or nitro; when X 1 is hydroxyl, then X 2 is selected from halogen.

[0248] In certain aspects, X 2 is hydrogen and X 1 is haloalkyl. Haloalkyl includes straight chain or branched haloalkyl groups obtained by replacing one or more hydrogen atoms with a halogen atom, such as fluoromethyl, difluoromethyl, trifluoromethyl, chloromethyl, and dichloromethyl. In certain aspects, X 1 is trifluoromethyl.

[0249] In certain aspects, X 2 is hydrogen and X 1 is alkyl. Alkyl includes straight chain or branched alkyl groups, such as methyl, ethyl, n-propyl, and isopropyl. In certain aspects, X 1 is methyl.

[0250] In certain aspects, X 2 is hydrogen and X 1 is nitro. In certain aspects, X 2 is hydrogen and X 1is halogen. In certain aspects, X 2 is selected from fluorine, chlorine, bromine, or iodine. In certain aspects, X 1 is hydroxyl and X 2 is fluorine.

[0251] In certain aspects, the luciferin substrate can be a compound of Formula (Ib): (Ib) or a stereoisomer, tautomer, or salt thereof, wherein: R 1 is selected from the group consisting of: cycloalkyl, 5-membered heteroaryl having 1-3 ring heteroatoms independently selected from O, S, Se, and N; and 10-membered heteroaryl having 1-3 ring heteroatoms independently selected from O, S, Se, and N; or R 1 is selected from the group consisting of: .

[0252] In certain aspects, R 1 is cycloalkyl. Cycloalkyl includes cycloalkyl groups such as cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl. In certain aspects, R 1 is cyclopropyl.

[0253] In certain aspects, R 1 is 5-membered heteroaryl having 1-3 ring heteroatoms independently selected from O, S, Se, and N. In certain aspects, R 1 is selected from the group consisting of pyrrole, furan, thiophene, selenophene, imidazole, thiazole, and oxazole. In certain aspects, R 1 is 10-membered heteroaryl having 1-3 ring heteroatoms independently selected from O, S, Se, and N. In certain aspects, R 1 is quinoline.

[0254] In certain aspects, R 1 is In certain aspects, R 1 is In certain aspects, R 1 is In certain aspects, R 1 is .

[0255] In certain aspects, the luciferin substrate can be a compound of Formula (Ic): (Ic) or a stereoisomer, tautomer, or salt thereof, wherein: R 3Selected from: cycloalkyl groups, 5-membered heteroaryl groups having 1-3 cyclic heteroatoms independently selected from O, S, Se and N; and 10-membered heteroaryl groups having 1-3 cyclic heteroatoms independently selected from O, S, Se and N.

[0256] In some respects, R 3 It is a cycloalkyl group. Cycloalkyl groups include cycloalkyl groups such as cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl. In some respects, R 3 It is cyclopropyl.

[0257] In some respects, R 3 It is a 5-membered heteroaryl group having 1-3 independently selected cyclic heteroatoms chosen from O, S, Se, and N. In some respects, R 3 Selected from pyrrole, furan, thiophene, selenophene, imidazole, thiazole, and oxazole. In some respects, R 3 It is a 10-membered heteroaryl group having 1-3 independently selected cyclic heteroatoms chosen from O, S, Se, and N. In some respects, R 3 It is quinoline.

[0258] In some respects, luciferin substrates can be compounds of formula (Id): (Id) Or its stereoisomers, tautomers, or salts, wherein: X 2 -X 3 Independently selected from: hydrogen, halogen, or hydroxyl. X 4 It is an alkoxy group; The condition is X 2 -X 3 One of them is hydrogen.

[0259] In some respects, X 2 It is hydrogen and X 3 It's a halogen. In some respects, X 2 It is hydrogen and X 3 Selected from fluorine, chlorine, bromine, or iodine. In some respects, X 2 It is hydrogen and X 3 It's fluorine. In some respects, X 2 It is hydrogen and X 3 It is a hydroxyl group. In some respects, X 3 It is hydrogen and X 2 It's a halogen. In some respects, X 3 It is hydrogen and X 2 Selected from fluorine, chlorine, bromine, or iodine. In some respects, X 3 It is hydrogen and X 2 It's fluorine. In some respects, X 3 It is hydrogen and X2 is hydroxy.

[0260] In certain aspects, the luciferin substrate can be a compound of Formula (Ie): (Ie) or a stereoisomer, tautomer, or salt thereof, wherein: R 1 is selected from: or R 2 is selected from: .

[0261] In certain aspects, R 1 is In certain aspects, R 1 is In certain aspects, R 1 is In certain aspects, R 1 is In certain aspects, R 1 is .

[0262] In certain aspects, the luciferin substrate can be a compound selected from: . Methods

[0263] The methods disclosed herein include imaging cells expressing a polypeptide using a polypeptide having luciferase activity as disclosed herein. In certain aspects, the polypeptide having luciferase activity can be expressed as a fusion protein for imaging cells expressing a protein of interest fused to the polypeptide. The polypeptides having luciferase activity as disclosed herein can be used to image live mammalian cells (e.g., mammals).

[0264] The methods disclosed herein include assays using a self-complementary multi-part protein having luciferase activity to detect a molecular interaction (e.g., transient association, stable association, complex formation, etc.) between a first moiety and a second moiety. The first moiety and the second moiety can be a peptide, a protein, a nucleic acid, a small molecule, etc. The first moiety can be conjugated to a first polypeptide component of the self-complementary multi-part, and the second moiety conjugates a second polypeptide component to another moiety, where the two components do not stably associate and do not produce a signal (e.g., substantially no signal) in the absence of a molecular interaction between the first moiety and the second moiety, but stably associate to form the self-complementary multi-part protein and produce a detectable (e.g., bioluminescent) signal when the first moiety and the second moiety interact. In such embodiments, assembly of the self-complementary multi-part protein is operated by the molecular interaction of the first moiety and the second moiety. If the first moiety and the second moiety engage in a sufficiently stable interaction, the self-complementary multi-part protein having luciferase activity is formed and a bioluminescent signal is produced. If the first moiety and the second moiety fail to engage in a sufficiently stable interaction, the self-complementary multi-part protein having luciferase activity is not formed or is formed only weakly, and no or substantially reduced bioluminescent signal is produced (e.g., substantially undetectable, substantially not detectable, differentially detectable, etc. as compared to a stable control signal). In some embodiments, the magnitude of the detectable bioluminescent signal is proportional (e.g., directly proportional) to the amount, strength, favorability, and / or stability of the molecular interaction between the first moiety and the second moiety. In certain aspects, the first moiety can be a protein and the second moiety can be a small molecule, or vice versa. In certain aspects, the first moiety is a protein and is conjugated to a first component, where the first component is larger than a second component, and the second component is conjugated to a second moiety that is a small molecule, or vice versa.

[0265] The methods disclosed herein include assays using a self-complementary multi-part protein having luciferase activity to detect a molecular interaction (e.g., transient association, stable association, complex formation, etc.) between a first moiety and a second moiety. The methods can involve the use of a first polypeptide component and a second polypeptide component that can associate to form a self-complementary multi-part protein having luciferase activity when the first component or the second component, or both, is conjugated to a moiety, and do not associate when one or more moieties are bound to another moiety. For example, the first polypeptide component can be fused to a first moiety, and can associate with a second polypeptide component to form a self-complementary multi-part protein having luciferase activity. However, when a moiety (e.g., a ligand) interacts with the first moiety, the first component and the second component can no longer associate to form a self-complementary multi-part protein having luciferase activity.

[0266] In some aspects, the interaction is detected in a living cell, in vivo, or in vitro by detecting a bioluminescent signal emitted by the cell. In some embodiments, the interaction is detected outside of a living cell, wherein the first component and the second component are secreted by the cell. In some embodiments, the interaction is detected in a living organism, within a cell, or within a tissue of the living organism.

[0267] In some aspects, a change in the interaction resulting from a change in the environment of the cell is detected by detecting a difference in the emitted bioluminescent signal relative to a control cell in the absence of the changed environment. In some embodiments, the changed environment is a result of adding or removing a molecule (e.g., a drug) from the culture medium.

[0268] The polypeptides having luciferase activity as described herein (e.g., LuxSit-i variants, LuxSit-i variant-derived self-complementary polypeptide proteins, circularly permuted LuxSit-i, and LuxSit-i variants) can be used for a number of purposes, including but not limited to detecting the amount or presence of a particular molecule (a biosensor), isolating a particular molecule, detecting a conformational change in a particular molecule, e.g., due to binding, phosphorylation, or ionization, facilitating high or low throughput screening, detecting inter-protein, protein-DNA, or other protein-based interactions, or selecting or evolving a biosensor. For example, a polypeptide having luciferase activity or a fusion thereof can be used to detect the amount, presence, or activity of a particular kinase (e.g., by inserting a kinase site into the protein), RNAi (e.g., by inserting a sequence suspected of being recognized by RNAi into the coding sequence of the protein, then monitoring reporter gene activity after addition of the RNAi), or a protease (such as for detecting the presence of a particular viral protease, which in turn is an indicator of the presence of a virus or an antibody) in, e.g., an in vitro or cell-based assay; screening for inhibitors (e.g., protease inhibitors); identifying recognition sites or detecting substrate specificity, e.g., using a luciferase having a selected recognition sequence or a library of polypeptides having luciferase activity and having a plurality of different sequences with a single molecule of interest or a plurality of molecules (e.g., a library); selecting or evolving a biosensor or a molecule of interest, e.g., a protease; or detecting inter-protein interactions via complementation or binding, e.g., in an in vitro or cell-based method. In one aspect, a polypeptide having luciferase activity comprising an inserted amino acid sequence is contacted with a random library or a mutant library of molecules, and the molecules that interact with the inserted amino acid sequence are identified. In another aspect, a library of polypeptides having luciferase activity with a plurality of insertions is contacted with a molecule, and the polypeptides having luciferase activity that interact with the molecule are identified. In one embodiment, a polypeptide having luciferase activity or a fusion thereof can be used to detect the amount or presence of cAMP or cGMP (e.g., by inserting a cAMP or cGMP binding site into a polypeptide having luciferase activity), screen for inhibitors or activators of, e.g., cAMP or cGMP, inhibitors or activators of cAMP that bind to the cAMP binding site, or inhibitors or activators of G protein-coupled receptors (GPCRs), identify recognition sites or detect substrate specificity (e.g., using a polypeptide having a selected recognition sequence and having luciferase activity or a library of polypeptides having a plurality of different sequences and having luciferase activity with a single molecule of interest or a plurality of molecules (e.g., a library)), select or evolve a cAMP or cGMP binding site, or be used for whole animal imaging, e.g., in an in vitro or cell-based assay.

[0269] Also encompassed herein are methods of monitoring the expression, localization, and / or trafficking of a molecule in a cell and monitoring changes in the microenvironment within a cell using a polypeptide having luciferase activity or a fusion protein thereof. In one aspect, the polypeptide having luciferase activity comprises a recognition site for a molecule, and when the molecule interacts with the recognition site results in an increase in activity, and thus can be used to detect or determine the presence or amount of the molecule. For example, in one aspect, the polypeptide having luciferase activity comprises an internal insertion containing two domains that interact with each other under certain conditions. In one embodiment, one domain in the insertion contains an amino acid that can be phosphorylated, and the other domain is a phosphoamino acid binding domain. In the presence of the appropriate kinase or phosphatase, the two domains in the insertion interact and change the conformation of the polypeptide having luciferase activity, resulting in a detectable change in the activity of the modified luciferase. In another embodiment, the modified luciferase comprises a recognition site for a molecule, and when the molecule interacts with the recognition site results in an increase in activity, and thus can be used to detect or determine the amount or presence of another molecule.

[0270] In certain aspects, the method for detecting luminescence in a cell further comprises contacting the cell with a polypeptide having luciferase activity. In certain aspects, the polypeptide is fused to a targeting moiety that specifically binds to the cell. In certain aspects, the targeting moiety is a peptide, a lipid, a protein, or a small molecule. In certain aspects, the targeting moiety is an antibody or antigen binding fragment thereof, a receptor, a ligand, or a substrate.

[0271] In certain aspects, after contacting the cell with the polypeptide having luciferase activity, the cell is contacted with a luciferin analog, wherein the luciferin analog is a compound described herein or a stereoisomer, tautomer, or salt thereof. In certain aspects, the cell is in a tissue sample. In certain aspects, the cell is in a subject.

[0272] In certain aspects, the method comprises contacting a tissue with a polypeptide having luciferase activity and fused to a targeting moiety and contacting the tissue with a luciferin analog, wherein the luciferin analog is a compound of Formula (I) or a stereoisomer, tautomer, or salt thereof, or any of the compounds described herein; and detecting the localization of the polypeptide in the tissue.

[0273] In certain aspects, the method comprises administering to a subject a polypeptide having luciferase activity and fused to a targeting moiety for localizing the polypeptide to a cell, administering to the subject a luciferin analog, wherein the luciferin analog is a compound of Formula (I) or a stereoisomer, tautomer, or salt thereof, or any of the compounds described herein; and detecting luminescence to determine the localization of the polypeptide in the cell. In certain aspects, the subject is a mammal, a primate, or a human.

[0274] In certain aspects, a method for detecting luminescence in a transgenic animal comprises administering a luciferin analog to the transgenic animal, wherein the luciferin analog is a compound of Formula (I) or a stereoisomer, tautomer, or salt thereof, or any of the compounds described herein; and detecting luminescence. In certain aspects, the transgenic animal expresses a polypeptide having luciferase activity. Assay solutions

[0275] Assay buffers that increase and stabilize signal output of luciferase assays are described. In certain aspects, the assay buffer can comprise imidazole, e.g., about 10 mM-1000 mM imidazole, about 50 mM-1000 mM imidazole, about 10 mM-500 mM imidazole, about 50 mM-500 mM imidazole, about 75 mM-250 mM imidazole, about 75 mM-150 mM imidazole, or about 100 mM imidazole. In certain aspects, the assay buffer can have a pH of about 8, e.g., about pH 6-pH 9, about pH 7-pH 9, or about pH 7.5-8.5, such as pH 7.6, 7.8, 8.0, 8.2, or 8.4.

[0276] In certain aspects, the assay buffer results in a signal from luciferase activity that is at least 10% higher, e.g., at least 20% higher, at least 30% higher, at least 40% higher, at least 50% higher, at least 60% higher, at least 70% higher, at least 80% higher, at least 90% higher, at least 100% higher, at least 150% higher, or up to 150% higher, or up to 180% higher, or up to 200% higher, than a signal obtained using an assay buffer that does not comprise imidazole.

[0277] In certain aspects, the assay buffer can comprise a buffer, e.g., phosphate buffered saline, Tris, histidine buffer, N-(2-hydroxyethyl)piperazine-N-(2-ethanesulfonic acid) (HEPES), 2-(N-morpholino)ethanesulfonic acid (MES), 2-(N-morpholino)ethanesulfonic acid sodium salt (MES), 3-(N-morpholino)propanesulfonic acid (MOPS), N-tris[hydroxymethyl]methyl-3-aminopropanesulfonic acid (TAPS), and the like. In certain aspects, the assay buffer can comprise one or more of: a stabilizer; an antifoam agent; an antioxidant; and a reducing agent.

[0278] In certain aspects, the stabilizer can be an alcohol, e.g., propylene glycol; an antifoam agent, such as an alcohol (cetostearyl alcohol), an insoluble oil (castor oil), a stearate, a polydimethylsiloxane and other siloxane derivatives, an ether, and a glycol; an antioxidant, e.g., ascorbic acid, glutathione, cysteine, methionine, or citric acid; a reducing agent, e.g., thiourea.

[0279] In certain aspects, the kits can comprise an assay buffer and a luciferin substrate of the present disclosure. The luciferin substrate can be any luciferin substrate, such as a luciferin substrate of the present disclosure.

[0280] In certain aspects, the kits provided herein can comprise one or more of the polypeptides, substrates, and assay buffers provided herein. Examples

[0281] The following examples are provided to illustrate, but not limit, any embodiments provided by the present disclosure. Example 1: LuxSit-i variants having improved stability

[0282] The LuxSit-i (SEQ ID NO: 1) sequence is provided in FIG. 1. The secondary structure H1-L1-H2-L2-B1-L3-B2-L4-H3-L5-B3-L6-B4-L7-B5-L8-B6 is mapped onto the primary structure. The LuxSit-i (SEQ ID NO: 1) sequence was derived from LuxSit as described in Yeh, A. HW. et al. De novo design of luciferases using deep learning. Nature 614, 774-780 (2023). The amino acid sequence of LuxSit is set forth in SEQ ID NO: 92.

[0283] Single saturation mutagenesis (SSM) was performed to evaluate the effect of single mutations on the stability of LuxSit-i (SEQ ID NO: 1) by yeast display. The SSM library was transformed into electrocompetent cells of strain EBY100. 20 million cells were labeled with anti-myc FITC antibody to detect protein expression on the surface of the yeast by measuring fluorescence emission. Cells were sorted using a Sony SH800 cell sorter, and cells showing protein expression (FITC signal) were collected and cultured. DNA was extracted from the sorted cells using a conventional DNA extraction routine. The DNA library was sequenced using nanopore technology. Single amino acid substitutions that increased protein stability were enriched after sorting.

[0284] Single saturation mutagenesis (SSM) was performed to evaluate the effect of single mutations on LuxSit-i-i stability (via yeast display). The SSM library was transformed into electrocompetent cells of strain EBY100. 20 million cells were labeled with anti-myc FITC antibody to detect protein expression on the yeast surface by measuring the fluorescent emission of FITC. The yeast were incubated with a mixture of 3.5 uM trypsin and 1.4 uM chymotrypsin (1 / 729 dilution of stock solution) for 15 min at 25ºC. Cells were sorted using a Sony SH800 cell sorter, and cells showing protein expression (FITC signal) were collected and grown after protease incubation. DNA was extracted from the sorted cells using a regular DNA extraction protocol. The DNA library was sequenced using Nanopore technology. Single amino acid substitutions that increase protein stability were enriched after sorting.

[0285] Single saturation mutagenesis (SSM) was performed to evaluate the effect of single mutations on LuxSit-i stability (via yeast display). The SSM library was transformed into electrocompetent cells of strain EBY100. 20 million cells were labeled with anti-myc FITC antibody to detect protein expression on the yeast surface by measuring the fluorescent emission of FITC. The yeast were incubated with a mixture of 10 uM trypsin and 4 uM chymotrypsin (1 / 243 dilution of stock solution) for 15 min at 25ºC. Cells were sorted using a Sony SH800 cell sorter, and cells showing protein expression (FITC signal) were collected and grown after protease incubation. DNA was extracted from the sorted cells using a regular DNA extraction protocol. The DNA library was sequenced using Nanopore technology. Single amino acid substitutions that increase protein stability were enriched after sorting.

[0286] Single saturation mutagenesis (SSM) was performed to evaluate the effect of single mutations on LuxSit-i stability (via yeast display). The SSM library was transformed into electrocompetent cells of strain EBY100. 20 million cells were labeled with anti-myc FITC antibody to detect protein expression on the yeast surface by measuring the fluorescent emission of FITC. The yeast were incubated with a mixture of 15 uM trypsin and 6 uM chymotrypsin (1 / 162 dilution of stock solution) for 15 min at 25ºC. Cells were sorted using a Sony SH800 cell sorter, and cells showing protein expression (FITC signal) were collected and grown after protease incubation. DNA was extracted from the sorted cells using a regular DNA extraction protocol. The DNA library was sequenced using Nanopore technology. Single amino acid substitutions that increase protein stability were enriched after sorting.

[0287] Single saturation mutagenesis (SSM) was performed to evaluate the effect of single mutations on LuxSit-i stability (by yeast display). The SSM library was transformed into electrocompetent cells of strain EBY100. 20 million cells were labeled with anti-myc FITC antibody to detect protein expression on the surface of the yeast by measuring the fluorescent emission of FITC. The yeast was incubated with a mixture of 30 uM trypsin and 12 uM chymotrypsin (1 / 81 dilution of stock solution) for 15 min at 25ºC. Cells were sorted using a Sony SH800 cell sorter, and cells showing protein expression (FITC signal) were collected and grown after the protease incubation. DNA was extracted from the sorted cells using a regular DNA extraction protocol. The DNA library was sequenced using Nanopore technology. Single amino acid substitutions that increased protein stability were enriched after sorting. Example 2: LuxSit-i variants having improved activity

[0288] SSM was performed to evaluate the effect of single mutations on luciferase activity of LuxSit-i. Figure 3 graphically presents the mutation frequency at positions found to be beneficial for increasing luciferase activity in SSM.

[0289] Table 2 summarizes the properties of exemplary LuxSit-i variants. Amino acid substitutions are relative to SEQ ID NO: 1 (LuxSit-i). Stability and brightness are relative to LuxSit-i. Table 2:

[0290] MBIO-158 comprises a single amino acid substitution W100F relative to SEQ ID NO: 1.

[0291] Single amino acid substitutions that improve luciferase activity and / or stability were combined and luciferase activity and stability were determined. Exemplary variants are listed in Table 3. Table 3: Stability and brightness are relative to LuxSit-i:

[0292] Figure 4A provides data for luciferase activity of MBIO-148, MBIO-158, and LuxSit-i. MBIO-148, MBIO-158, and Luxsit-i were serially diluted starting from a final well concentration of 10,000 pM. Each dilution was combined with diphenyltriazine (DTZ) to achieve a final well concentration of 10 µM. The points shown are from the initial reading after the start plate read.

[0293] Figure 4B provides data for luciferase activity of MBIO-148, MBIO-301, MBIO-302, and LuxSit-i. MBIO-148, MBIO-301, MBIO-302, and LuxSit-i were serially diluted starting from a final well concentration of 500 pM. Each dilution was combined with DTZ to achieve a final well concentration of 10 µM. The points shown are from the initial reading after the start plate read.

[0294] Table 4 provides the substitutions present in these and additional variants relative to SEQ ID NO: 1.

[0295] Table 4:

[0296] Table 6 provides the substitutions present in LuxSit-i variants relative to the sequence of LuxSit-i shown in SEQ ID NO: 1. The LuxSit-i variants exhibit increased luciferase activity compared to LuxSit-i.

[0297] Table 6:

[0298] Table 7 lists LuxSit-i variants that show increased luciferase activity and / or increased stability relative to LuxSit-I. The substitutions relative to the sequence of LuxSit-i shown in SEQ ID NO: 1 are shown. Table 7 lists single mutants. The mutants were obtained from error prone PCR.

[0299] Table 7:

[0300] Table 8 lists LuxSit-i variants that show increased luciferase activity and / or increased stability relative to LuxSit-i. The substitutions relative to the sequence of LuxSit-i shown in SEQ ID NO: 1 are shown. Table 8 lists double mutants or triple mutants. The mutants were obtained from error prone PCR.

[0301] Table 8.

[0302] Table 9 lists the mutant sequences obtained from artificial combinations of some of the most common mutations listed in Table 7 and Table 8. Brightness and / or stability are improved relative to LuxSit-i and the single mutants or double mutants or triple mutants.

[0303] Table 9

[0304] Table 10 lists mutants obtained from a combinatorial library containing the most common mutations listed in Table 7 and Table 8. MBIO-301 (having the sequence set forth in SEQ ID NO: 104) has the highest luciferase activity.

[0305] Table 10

[0306] Table 11 lists variants derived from MBIO-301. The listed histidine residues in MBIO-301 were substituted. MBIO-2466, which contains the mutation H98Q in the MBIO-301 sequence, shows more than 5-fold higher luciferase activity than MBIO-301. Table 11 lists the substitutions relative to SEQ ID NO: 1.

[0307] Table 11.

[0308] Table 12 lists variants derived from MBIO-2466. MBIO-3073 has the highest luciferase activity. Table 12 lists the substitutions relative to SEQ ID NO: 1.

[0309] Table 12.

[0310] Table 13 lists variants derived from MBIO-2859. MBIO-4039 has the highest luciferase activity and is the most stable variant. Table 13 lists the substitutions relative to SEQ ID NO: 1.

[0311] Table 13. The sequences of the mutants of LuxSit-i are set forth in SEQ ID NOs: 2-91, 93-143, 2601-2604, 2663, 2665, and 2682-2732. FIGS. 37A-37H show the enzyme activity of the listed variants of LuxSit-i. Example 2: Circularly permuted proteins having luciferase activity

[0312] Initial designs were generated using RosettaFold repair. Using MBIO-148 / SEQ ID NO: 2600 as input, repair sequences ranging from 10-50 amino acids in length were generated.

[0313] SEQ ID NO: 2600 (MBIO-148) - the secondary structure arrangement is H1-L1-H2-L2-B1-L3-B2-L4-H3-L5-B3-L6-B4-L7-B5-L8-B6, where "H" is a helical domain, "L" is a loop domain, and "B" is a beta strand domain. Three types of domains are delineated in the protein sequence: MSEEQIRQFLRRFYEALDS GD ADTAASL FHPGV TIHL WD GVTF TS REEFREWFERLFST SKDA WREIKSLEVR GD TVEVHVQLHFTL NG QKHTVDLTHHFHFR GN RVTEVRVHINPTG

[0314] The helical domains are indicated by smaller font size. The loop domains are italicized and underlined. The beta strand domains are in bold.

[0315] The contig designated for repair, i.e., the order of the SEQ ID NO: 2600 domain junctions is residues 89-117, linker, 1-88; where the linker is a variable repair sequence of length 10-50. The secondary structure of the contig is as follows: B5-L8-B6-linker-H1-L1-H2-L2-B1-L3-B2-L4-H3-L5-B3-L6-B4-(L7) (VII). The L7 domain in parentheses (i) exists at the C-terminus, (ii) exists at the N-terminus, (iii) is split between the C-terminus and the N-terminus, or (iii) is absent.

[0316] In one round of design, all beta sheet residues that face outward from the protein active site were mutated to valine prior to repair to facilitate structure packing of the repair against the rest of the protein.

[0317] After repair, sequence redesign was performed on each repaired design using ProteinMPNN. MPNN was only allowed to change the residues of the above repair and any mutated valine residues; the rest of the enzyme, including the active site residues, were left intact. 20 sequences were output for generation for each repair.

[0318] All MPNN redesigned sequences were alpha folded using single sequence prediction with 3 cycles. The top design of the original SEQ ID: 2600 structure was selected for further testing by pLDDT and low RMSD.

[0319] Provided below are selected circular dismutase proteins each having the arrangement B5-L8-B6-Linker-H1-L1-H2-L2-B1-L3-B2-L4-H3-L5-B3-L6-B4-(L7) (VII). In these examples, the L7 domain in parentheses is split between the C-terminus and the N-terminus. The sequence in bold and underlined indicates the linker sequence. The linker sequence is also referred to as the repair sequence. The helical domains are indicated by smaller font size. The loop domains are italicized and underlined. The beta strand domains are in bold. 2209- Repair sequence length 31: G QKHTVDLTHHFHFR GN RVTEVRVHITP VDDVEEVLARVLEEGERLVERLRAERPEA SIPEEQIRQFLRRFYEALDS GD ADTAASL FHPGV TIHL WD GVTF TS REEFREWFERLFST SKDA WREIKSLEVR GD TVEVHVQLHFTL N 263- Repair sequence length 17 G QKHTVDLTHHFHFR GN RVTEVRVHITP TGEEPEKPEFKETFGPS SIPEEQIRQFLRRFYEALDS GD ADTAASL FHPGV TIHL WD GVTF TS REEFREWFERLFST SKDA WREIKSLEVR GD TVEVHVQLHFTL N 2284- Repair sequence length 35 G QKHTVDLTHHFHFR GN RVTEVRVHITP VESEEELPAALARAEELGRELLERTLAEEGAGGPP SIPEEQIRQFLRRFYEALDS GD ADTAASL FHPGV TIHL WD GVTF TS REEFREWFERLFST SKDA WREIKSLEVR GD TVEVHVQLHFTL N 2236 - repair sequence length 32 G QKHVVVLVHTFRFR GN RVTEVRVEIIP APSLDEESIEARVAEARRLAEERLAELGDPPP SISEEQIRQFLRRFYEALDS GD ADTAASL FHPGV TIHL WD GVTF TS REEFREWFERLFST SKDA WREIVELRVR GD TVVVVVVLHFTL N 259 - repair sequence length 17 G QKHTVDLTHHFHFR GN RVTEVRVHIRP TGEEPEPPEFRERFGPSA IPEEQIRQFLRRFYEALDS GD ADTAASL FHPGV TIHL WD GVTF TS REEFREWFERLFST SKDA WREIKSLEVR GD TVEVHVQLHFTL N 2221 - repair sequence length 32 G QKHVVVLVHTFVFR GN RVTEVRVEIFPAP DLSPEAIEAAIAKALARADALLAELGAPPP SISEEQIRQFLRRFYEALDS GD ADTAASL FHPGV TIHL WD GVTF TS REEFREWFERLFST SKDA WREIVSLRVV GD TVVVVVVLHFTL N 256 - repair sequence length 17 G QKHTVDLTHHFHFR GN RVTEVRVHIEP TGEEPERPEFVERFGPSS IPEEQIRQFLRRFYEALDS GD ADTAASL FHPGV TIHL WD GVTF TS REEFREWFERLFST SKDAWREIKSLEVR GD TVEVHVQLHFTL N 2237 - repair sequence length 32 G QKHVVVLVHTFVFR GN RVTEVRVEIFPVP SLDEAAIEAAIARARARADELLAELGAPPA SISEEQIRQFLRRFYEALDS GD ADTAASL FHPGV TIHL WD GVTF TS REEFREWFERLFST SKDA WREIVSLRVV GD TVVVVVVLHFTL N 2227 - repair sequence length 32 G QKHVVVLVHTFRFR GN RVTEVEVEIIP CPSLDEASIAAAIAEAEALAAERLAELGAPPP SISEEQIRQFLRRFYEALDS GD ADTAASL FHPGV TIHL WD GVTF TS REEFREWFERLFST SKDA WREIVSLRVV GD TVVVVVVLHFTL N 264 - repair sequence length 17 G QKHTVDLTHHFHFR GN RVTEVRVHIEP TGEEPEPPEFRERFGPSS IPEEQIRQFLRRFYEALDS GD ADTAASL FHPGV TIHL WD GVTF TS REEFREWFERLFST SKDA WREIKSLEVR GD TVEVHVQLHFTL N 2493 - repair sequence length 45 G QKHTVILTHVFRFR GN RVTEVRVEIVPVP DPDEETRLAAAREALERAGVPEEMRRAALELLERGEREL FRPSA IPEEQIRQFLRRFYEALDS GD ADTAASLFHPGV TIHL WD GVTF TS REEFREWFERLFST SKDA WREILALVVD GD TVVVVVRLDFTL N

[0320] Figure 6A provides kinetic curves for the circularly permuted LuxSit-i variants over one hour. The variants were diluted in PBS and combined 1 : 1 with the DTZ substrate, resulting in a final well concentration of 5 nM protein / variant and 10 µM DTZ.

[0321] Figure 6B shows the initial RLU values for the circularly permuted LuxSit variants. The variants were diluted in PBS and combined 1 : 1 with the DTZ substrate, resulting in a final well concentration of 5 nM protein and 10 µM DTZ.

[0322] Circularly permuted proteins having SEQ ID NOs: 2227 and 2236 have luciferase activity similar to SEQ ID NO: 2600.

[0323] Additional examples of circularly permuted proteins are provided in Appendix B, which is incorporated by reference herein in its entirety. Example 3: Split LuxSit-i variants

[0324] LuxSit-i variants, circularly permuted LuxSit-i, and circularly permuted LuxSit-i variants are split into two fragments. In some embodiments, the split point is placed such that two fragments of unequal length are produced, a small fragment and a large fragment.

[0325] In certain embodiments, a circularly permuted LuxSit-i or circularly permuted LuxSit-i variant having the following secondary structure arrangement: B1-L3-B2-L4-H3-L5-B3-L6-B4-L7-B5-L8-B6- linker-H1-L1-H2-(L2) (II) or B2-L4-H3-L5-B3-L6-B4-L7-B5-L8-B6-linker-H1-L1-H2-L2-B1- (L3) (III) is split into two components. The L domain in parentheses is present at the C-terminus or N-terminus, or split between the C-terminus and N-terminus, or is not present.

[0326] Luminescent activity of high affinity two-component luciferase variants fused to the rapamycin-inducible FRB:FKBP system is shown in Figure 7. Luminescent activity of low affinity two-component luciferase variants fused to the rapamycin-inducible FRB:FKBP system is shown in Figure 8.

[0327] The sequences of the two-component luciferase variants are set forth in Table 13. These components are also referred to as fragments. In certain embodiments, the two-component luciferase variants differ in the size of their sequences. In certain embodiments, the smallest small component is about 5 to 6 amino acids in size, and the largest small component is about 30 to 40 amino acids in size. In certain embodiments, the smallest large component is about 70 to 80 amino acids in size, and the largest large component is about 110 amino acids in size.

[0328] Table 13A. Lists the small and large components of the two-component luciferase variants. “smLux” refers to the smaller small component relative to the component that complements it to increase luciferase activity. “lgLux” refers to the large component that complements the activity of the smLx.

[0329] Table 13A:

[0330] Table 13A also lists polypeptides that have the same length as the LuxSit-i variants disclosed herein and have one or more mutations that inactivate them. These polypeptides are referred to as darkbits in Table 13A. These polypeptides restore activity when associated with any of the small fragments disclosed herein (e.g., any of the smluxes of Table 13A). In certain embodiments, the present disclosure provides a kit comprising an lgLux and an smLux or a nucleic acid encoding an lgLux and a nucleic acid encoding an smLux as disclosed herein, wherein the lgLux comprises an amino acid sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence of an lgLux disclosed herein (e.g., in Table 13A, Table 19, Table 20, Table 21, or Table 22), and the smLux comprises an amino acid sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence of an smLux disclosed herein (e.g., in Table 13A or Table 18).

[0331] Also provided herein are one or both of a first nucleic acid encoding a first protein and a second nucleic acid encoding a second protein, wherein: (i) the first protein comprises an amino acid sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence: MSEEQIRQFLRRFYEALDSGDADTAASLFHPGVTIHLWDGVTFT (SEQ ID NO: 2605) and the second protein comprises an amino acid sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence: SREEFREWFERLFSTSKDAWREIKSLEVRGDTVEVHVQLHFTLNGQKHTVDLTHHFHFRGNRVTEVRVHINPTGLE (SEQ ID NO: 2606) (ii) the first protein comprises an amino acid sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence: MSEEQIRQFLRRFYEALDSGDADTAASLFHPGVTIHLWDGVTFTSREEFREWFERLFSTSKDAWREIKSLEVRG (SEQ ID NO: 2608) and the second protein comprises an amino acid sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence: DTVEVHVQLHFTLNGQKHTVDLTHHFHFRGNRVTEVRVHINPTGLE (SEQ ID NO: 2607); (iii) the first protein comprises an amino acid sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence: MSEEQIRQFLRRFYEALDSGDADTAASLFHPGVTIHLWDGVTFTSREEFREWFERLFSTSKDAWREIKSLEVRGDTVEVHVQLHFTLNGQKHTVDLTHHFHFRG (SEQ ID NO: 2610), and the second protein comprises an amino acid sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence: NRVTEVRVHINPTGLE (SEQ ID NO: 2609); (iv) the first protein comprises an amino acid sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence: SEEQIRQNLRRFYEALDSGDADTAASLFHPGVTIHLWDGVTFTSREEFREWFERLFSTSKDALREIKSLEVRGDTVEVHVQLHFTRNGQKHTVDLTHLFHFR (SEQ ID NO: 2614), and the second protein comprises an amino acid sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence: NRVDEVRVYIN (SEQ ID NO: 2615) or NRVDEVRVYINPT (SEQ ID NO: 2616); (v) the first protein comprises at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence: GQKHVVVLVHTFRFRG (SEQ ID NO: 2617), and the second protein comprises at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence: NRVTEVRVEIIPAPSLDEESIEARVAEARRLAEERLAELGDPPPSISEEQIRQFLRRFYEALDSGDADTAASLFHPGVTIHLWDGVTFTSREEFREWFERLFSTSKDAWREIVELRVRGDTVVVVVVLHFTLN (SEQ ID NO: 2618); (vi) the first protein comprises at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence: NRVTEVRVEIIPAP (SEQ ID NO: 2619), and the second protein comprises at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence: SLDEESIEARVAEARRLAEERLAELGDPPPSISEEQIRQFLRRFYEALDSGDADTAASLFHPGVTIHLWDGVTFTSREEFREWFERLFSTSKDAWREIVELRVRGDTVVVVVVLHFTLN GQKHVVVLVHTFRFRG (SEQ ID NO: 2620); (vii) the first protein comprises at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence: SLDEESIEARVAEARRLAEERLAELGDPP (SEQ ID NO: 2621), and the second protein comprises at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence: PSISEEQIRQFLRRFYEALDSGDADTAASLFHPGVTIHLWDGVTFTSREEFREWFERLFSTSKDAWREIVELRVRGDTVVVVVVLHFTLNGQKHVVVLVHTFRFRGNRVTEVRVEIIPAP (SEQ ID NO: 2622); (viii) the first protein comprises at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence: PSISEEQIRQFLRRFYEALDSG (SEQ ID NO: 2623), and the second protein comprises at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence: DADTAASLFHPGVTIHLWDGVTFTSREEFREWFERLFSTSKDAWREIVELRVRGDTVVVVVVLHFTLNGQKHVVVLVHTFRFRGNRVTEVRVEIIPAPSLDEESIEARVAEARRLAEERLAELGDPP (SEQ ID NO: 2624); (ix) the first protein comprises at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence: DADTAASLFHP (SEQ ID NO: 2625), and the second protein comprises at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence: GVTIHLWDGVTFTSREEFREWFERLFSTSKDAWREIVELRVRGDTVVVVVVLHFTLNGQKHVVVLVHTFRFRGNRVTEVRVEIIPAPSLDEESIEARVAEARRLAEERLAELGDPPPSISEEQIRQFLRRFYEALDSG (SEQ ID NO: 2626); (x) the first protein comprises at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence: GVTIHLW (SEQ ID NO: 2627), and the second protein comprises at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence: DGVTFTSREEFREWFERLFSTSKDAWREIVELRVRGDTVVVVVVLHFTLNGQKHVVVLVHTFRFRGNRVTEVRVEIIPAPSLDEESIEARVAEARRLAEERLAELGDPPPSISEEQIRQFLRRFYEALDSGDADTAASLFHP (SEQ ID NO: 2628); (xi) the first protein comprises at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence: DGVTFT (SEQ ID NO: 2629), and the second protein comprises at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence: SREEFREWFERLFSTSKDAWREIVELRVRGDTVVVVVVLHFTLNGQKHVVVLVHTFRFRGNRVTEVRVEIIPAPSLDEESIEARVAEARRLAEERLAELGDPPPSISEEQIRQFLRRFYEALDSGDADTAASLFHPGVTIHLW (SEQ ID NO: 2630); (xi) the first protein comprises at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence: DGVTFT (SEQ ID NO: 2629), and the second protein comprises at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence: SREEFREWFERLFSTSKDAWREIVELRVRGDTVVVVVVLHFTLNGQKHVVVLVHTFRFRGNRVTEVRVEIIPAPSLDEESIEARVAEARRLAEERLAELGDPPPSISEEQIRQFLRRFYEALDSGDADTAASLFHPGVTIHLW (SEQ ID NO: 2630); (xiii) the first protein comprises at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence: DAWREIVELRVRG (SEQ ID NO: 2633), and the second protein comprises at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence: DTVVVVVVLHFTLNGQKHVVVLVHTFRFRGNRVTEVRVEIIPAPSLDEESIEARVAEARRLAEERLAELGDPPPSISEEQIRQFLRRFYEALDSGDADTAASLFHPGVTIHLWDGVTFTSREEFREWFERLFSTSK (SEQ ID NO: 2634); (xiv) the first protein comprises at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence: DTVVVVVVLHFTLN (SEQ ID NO: 2635), and the second protein comprises at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence: GQKHVVVLVHTFRFRGNRVTEVRVEIIPAPSLDEESIEARVAEARRLAEERLAELGDPPPSISEEQIRQFLRRFYEALDSGDADTAASLFHPGVTIHLWDGVTFTSREEFREWFERLFSTSK DAWREIVELRVRG (SEQ ID NO: 2636); or (xv) the first protein comprises at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence: MSGMSEEQIRQNLRRFYEALDSGDAITAASLFDPGVTITLWDGTTFTSVEEFREWFESQFSTSKDALREISSLEVRGDTVEVTVRLSFTRNGQKHTVDLLQLFKFV (SEQ ID NO: xx), and the second protein comprises at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence: MSGGNRVVAVRVYVNPT (SEQ ID NO: 2734).

[0332] Each fragment of the two-part luciferase variant was fused to the FRB FKBP protein and incubated with its complementary part in a 1 : 1 ratio. The full-length luciferase variant and the individual large split fragments were included as controls. All constructs were tested in E. coli cell lysate. Rapamycin was added to induce reconstitution of the two fragments, and diphenyltriazine (DTZ) was added as a luminescence substrate.

[0333] The small fragments listed in Table 13 can be complemented with the large fragments listed to form a complex that has higher enzymatic activity than the small fragment or large fragment on its own.

[0334] Figures 36A-36F provide results for split luxsit variant pairs screened for rapamycin-induced luminescence. All lglux constructs were in the form mcherry-28x-linker-FRB-33x-linker-lglux-His_tag, and all smlux constructs were in the form mcherry-FKBP-sm lux-His_tag. The 28x and 33x linkers are flexible GS sequences. Concentrations of each sample were calculated using mcherry fluorescence. Each sample was measured at 1 nM lglux + 1 nM smlux. Fold change was calculated by taking the difference in signal 15 min after adding 20 uM rapamycin or PBS to each sample. Substrate was added at a concentration of 50 uM in each sample. Maximum signal was reported 15 min after adding rapamycin for the (+) rapamycin condition, and baseline was reported 15 min after adding PBS for the (-) rapamycin condition. Data was collected in Corning 3600 opaque 96-well plates on a BioTek plate reader. Example 4: Optimized ("OB" or "OPT") buffers for measuring luciferase activity

[0335] Assay buffers that increase and stabilize signal output of luciferase assays were tested. The inclusion of imidazole increased the brightness of LuxSit at pH 8. The optimized buffer with the effect of increasing and stabilizing luciferase signal output (OPT1.0) had the following composition: IX PBS, 0.5% propylene glycol, 0.1% antifoam, 10 mM ascorbic acid, 35 mM thiourea, 100 mM imidazole, pH 8.0.

[0336] Figure 9A shows kinetic RLU values over the course of one hour comparing MBIO-302 diluted in PBS only or in optimized buffer. The in-well concentration was 185 pM enzyme and 10 μΜ DTZ. Figure 9B shows signal retention as a percentage of initial RLU values over the course of one hour for 185 pM MBIO-302 diluted in PBS only or in optimized buffer. The in-well concentration of DTZ was 10 μΜ.

[0337] Additional optimized buffers OB3.0 and OB2.0 were formulated in IX PBS: Table 14. OB3.0 Table 15. OB2.0 Example 5: Bioluminescent emission spectra of synthetic luciferin substrates employing the MBIO-301 enzyme

[0338] Bioluminescent emission spectra of synthetic luciferin substrates were measured. The luciferin substrates were incubated with MBIO-301 enzyme at 500 pM. Each molecule was incubated with IX phosphate buffered saline (PBS), 0.5% propylene glycol, 0.05% antifoam, 100 mM imidazole, 10 mM ascorbic acid buffer and emission spectra were obtained using a Synergy Hl plate reader.

[0339] Figure 10 shows bioluminescent emission spectra of synthetic luciferin substrates la, lb, lc, Id, lk, ln, and lp incubated with MBIO-301 enzyme at 500 pM. Figure 11 shows bioluminescent emission spectra of synthetic luciferin substrates 2a, 2b, 2c, 2d, 2f, 2h, and 2p incubated with MBIO-301 enzyme. Figure 12 shows bioluminescent emission spectra of synthetic luciferin substrates 3b and 3i incubated with MBIO-301 enzyme. Example 6: Bioluminescent emission spectra of synthetic luciferin substrates in the presence of 20% human serum

[0340] Bioluminescent emission of synthetic luciferin substrate was measured. Luciferin substrate was incubated with 500 pM MBIO-301 enzyme in the presence of 20% human serum. Each molecule was incubated with 20% human serum diluted in IX PBS, 0.5% propylene glycol, 0.05% antifoam, 100 mM imidazole, 10 mM ascorbic acid buffer. Bioluminescence was measured with a Synergy Hl plate reader. Figure 13 shows the bioluminescent emission of synthetic luciferin substrate incubated with 500 pM MBIO-301 enzyme in the presence of 20% human serum.

[0341] Figure 14 compares the bioluminescent emission of synthetic luciferin substrate lc and DTZ incubated with MBIO-301 under two assay conditions: 100% assay buffer (IX PBS, 0.5% propylene glycol, 0.05% antifoam, 100 mM imidazole, 10 mM ascorbic acid buffer) and 20% human serum + 80% assay buffer. Example 7: Bioluminescence emission spectra of synthetic luciferin substrates employing the split enzyme derived from MBIO-301

[0342] MBIO-301 enzyme was split into two components: MBIO-557 (small part) and MBIO-563 (large part). MBIO-557 has the following sequence: NRVDEVRVYINGSGS (SEQ ID NO: 2735) MBIO-563 has the following sequence: SEEQIRQNLRRFYEALDSGDADTAASLFHPGVTIHLWDGVTFTSREEFREWFERLFSTSKDALREIKSLEVRGDTVEVHVQLHFTRNGQKHTVDLTHLFHFRG (SEQ ID NO: 2736)

[0343] Bioluminescent emission of synthetic luciferin substrate was measured. Luciferin substrate was incubated with MBIO-301 derived split enzyme. The two-component MBIO-301 split luciferase was fused to the rapamycin-inducible FRB:FKBP system and incubated with its complementary part in a 1 : 1 ratio. Each molecule was incubated with IX PBS, 0.5% propylene glycol, 0.05% antifoam, 100 mM imidazole, 10 mM ascorbic acid buffer, 100 nM rapamycin and the emission signal was measured using a Synergy Hl plate reader. Figure 15 shows the bioluminescent emission of synthetic luciferin substrate incubated with MBIO-301 derived split enzyme.

[0344] In-hole concentration: 1 nM MBIO-557 (EGFP-FRB-small), 1 nM MBIO-563 (EGFP-FKBP-large), 10 µM substrate, 100 nM rapamycin. Table 16. Example 8: LuxSit-i variants lacking lysine residues

[0345] LuxSit-i variants were generated in which all lysine residues were substituted with another amino acid. Exemplary LuxSit-i variants were generated starting from the amino acid sequence of MBIO-4039 that lack lysine residues and retain activity, and which have the following sequence:

[0346] MBIO-4039_ no lysine residues

[0347] MSGMSEEQIRQNLRRFYEALDSGDAITAASLFDPGVTITLWDGTTFTSVEEFREWFESQFSTSRDALREISSLEVRGDTVEVTVRLSFTRNGQRHTVDLLQLFRFVGNRVVAVRVYVNPT (SEQ ID NO: 2737)

[0348] This no-lysine variant has only 76.9% sequence identity to LuxSit-i (SEQ ID NO: 1).

[0349] This MBIO-4039 was used to generate two fragments that can associate to form a functional enzyme:

[0350] MBIO-4039_LgLux_ no lysine residues

[0351] MSGMSEEQIRQNLRRFYEALDSGDAITAASLFDPGVTITLWDGTTFTSVEEFREWFESQFSTSKDALREISSLEVRGDTVEVTVRLSFTRNGQKHTVDLLQLFKFV (SEQ ID NO: 2733)

[0352] MBIO-4039_SmLux_ no lysine residues

[0353] MSGGNRVVAVRVYVNPT (SEQ ID NO: 2734)

[0354] These no-lysine variants can be used for applications such as ubiquitination analysis and assays for protein degradation. Example 9: LuxSit-i variants produced from MBIO-4039

[0355] LuxSit-i variant MBIO-4039 (SEQ ID NO: 2730) comprises the following mutations relative to LuxSit-i: F9N, D23I, H30D, H36T, V41T, R46V, R55S, L56Q, Q64L, K68S, H80T, Q82R, H84S, A85F, H87R, T97L, H98Q, H99L, W100F, H101K, R103V, T108V, E109A, H113Y, I114V.

[0356] Table 17 lists variants generated starting from the sequence of MBIO-4039 (SEQ ID NO: 2730).

[0357] Table 17:

[0358] MBIO-4517 exhibited the best properties among the variants generated from MBIO-4039.

[0359] MBIO-4517 and MBIO-4039 were split into large and small fragments and conjugated to proteins that bind to the rapamycin molecule (FKBP and FRB). Luciferase activity before and after addition of rapamycin is shown in Figure 46A. Plots comparing luciferase activity of split luciferase versions of MBIO-4517 and MBIO-4039 to the full-length version are shown in Figures 46B and 46C, respectively. Example 10: LuxSit split optimization

[0360] To optimize the function of the LuxSit split, a construct containing small Lux (SmLux), large Lux (LgLux), and the proteins that bind to the rapamycin molecule (FKBP and FRB) connected by a GS linker was expressed on the surface of yeast. When 50 uM of substrate 1c was added to yeast expressing this construct, a baseline signal was observed due to the reconstitution of SmLux and LgLux. When 20 uM of rapamycin was added, SmLux and LgLux were brought close to each other by FKBP and FRB, enhancing their reconstitution. As a result, an increase in signal was observed. See Figure 41.

[0361] Libraries of SmLux and LgLux were assembled and tested independently to identify variants with lower baseline and higher fold change.

[0362] SmLux_4039 (RVVAVRVYVNPTG; SEQ ID NO: 2770) was SmLux optimized. SmLux_4039 was mutated to generate MBIO-5343 (SEQ ID NO: 2771) and MBIO-5344 (SEQ ID NO: 2772). Table 18 shows that both mutants exhibit lower baseline activity and higher activity in the presence of rapamycin compared to SmLux_4039: LgLux_4039 (SEQ ID NO: 2773) was LgLux optimized. Sequences and properties of the generated variants (SEQ ID NO: 2797 to SEQ ID NO: 2805) are provided in Table 19 below. Table 19 Example 11: Luciferase activity of LuxSit-i variants

[0363] The luminescence of LuxSit variants MBIO-148 (SEQ ID NO: 2600), MBIO-301 (SEQ ID NO: 2603), MBIO-2466 (SEQ ID NO: 2703), MBIO-3073 (SEQ ID NO: 2711), MBIO-4039 (SEQ ID NO: 2730), MBIO-4517 (SEQ ID NO: 2753) obtained along the optimization process were tested with substrate DTZ or substrate 1c using buffer PBS, OPT1.0, OPT2.0 or OPT3.0 (see Example 4 - "OB" and "OPT" are used interchangeably). Figures 42A-42G show the enzyme activity of the LuxSit-i variants measured in different buffers: Phosphate Buffered Saline (PBS), OPT1.0, OPT2.0 and OPT3.0. MBIO-010 is LuxSit-i (SEQ ID NO: 1).

[0364] Figures 43A-43D show the enzyme activity of the listed LuxSit-i variants measured in different buffers using substrate 1c2t: Phosphate Buffered Saline (PBS), OPT1.0, OPT2.0 and OPT3.0.

[0365] While the subject proteins have been particularly shown and described with reference to certain embodiments of the present application, it will be understood by those skilled in the art that various changes in form and details can be made therein without departing from the scope of the present application encompassed by the appended claims. Example 12: LgLux stability optimization

[0366] As schematically depicted in Figure 44, LgLux mutants were generated by error-prone PCR (EP-PCR) and treated with proteases and their activities were measured.

[0367] Yeast expressing LgLux mutant libraries were incubated with proteases and screened to select for variants that are stable enough to resist proteolysis. Functional variants were selected by adding 50 uM of substrate 1c and 200 nM SmLux.

[0368] EP-PCR was performed on the nucleotide sequence encoding LgLux_4039 to identify protease stable mutants. The sequences (SEQ ID NO: 2806-2814) and activities of the mutants are summarized in Table 20 below.

[0369] Table 20: Example 13: LgLux stability optimization

[0370] As schematically depicted in Figure 45, LgLux sequences were designed computationally and their activities were measured.

[0371] Libraries of computationally generated LgLux variants were expressed in E. coli and tested in lysates. Screening was performed using 20 uM SmLux and 50 uM of substrate 1c.

[0372] The activities of the mutants are summarized in the table below:

[0373] The LgLux_4039 sequence is set forth in SEQ ID NO: 2773. MBIO-5103 through MBIO-5124, MBIO-5261 through MBIO-5295, MBIO-5101, and MBIO-5102 (SEQ ID NO: 2815-SEQ ID NO: 2873, respectively) are computationally generated LgLux. The sequences and activities of these LgLux are shown in Table 21 below.

[0374] Table 21:

[0375] MBIO-5358 and MBIO-5360 through MBIO-5381 (SEQ ID NO: 2774 - SEQ ID NO: 2796, respectively) are computationally generated LgLux. As shown in Table 22, most of the mutants exhibit lower baseline activity and similar or higher activity in the presence of rapamycin compared to LgLux_4039.

[0376] Table 22:

Claims

1. A protein having luciferase activity, said protein comprising a secondary structural arrangement H1-L1-H2-L2-B1-L3-B2-L4-H3-L5-B3-L6-B4-L7-B5-L8-B6, wherein "H" is a helical domain, "L" is a loop domain, and "B" is a β-chain domain, wherein: (a) The length of the H1 domain is at least 18 or 19 amino acids; the residue 9 of the H1 domain is T, S, H, R, C, L, V, A, Q, G, E, K, I, N, or M. or (a) The length of the B5 domain is at least 11, 12, 13, or 14 amino acids, and residue 11 of the B5 domain is F, Y, or L; and (b) The length of the B4 domain is at least 12 amino acids, and The residue 10 of the B4 domain is F, Y, L, I, K, or M; and / or Residue 12 of the B4 domain is F, L, R, D, M, Q, or V; and / or The protein in question lacks any lysine residues.

2. The protein according to claim 1, wherein residue 10 of the B4 domain is F, Y, L, I, K or M, and residue 12 of the B4 domain is L, R, D, M, Q or V.

3. The protein according to claim 1 or 2, wherein residue 11 of the B5 domain is F or Y.

4. The protein according to any one of claims 1-3, wherein residue 10 of the B4 domain is F or L, and residue 12 of the B4 domain is D, F, or L.

5. The protein according to any one of claims 1-4, wherein the length of the B3 domain is at least 6, 7, 8, 9, or 10 amino acids, and residue 1 of the B3 domain is W / L / H; optionally wherein: (i) The residue 9 of the H1 domain is D, K, L, N, R, S, T, Q, V or Y; (ii) The residue 2 of the L2 domain is not H, and further optionally the residue 2 of the L2 domain is D, F, K, L, N, R, S, T, Q, V or Y; (iii) The residue 3 of the B1 domain is not H, and further optionally the residue 3 of the B1 domain is D, F, K, L, N, R, S, T, Q, V or Y; (iv) The residue 2 of the B2 domain is D, F, K, L, N, R, S, T, Q or Y; (v) The residue 1 of the H3 domain is D, F, K, L, N, S, T, Q, V or Y; (vi) The residue 5 of the B4 domain is not H, and further optionally the residue 5 of the B4 domain is D, F, K, L, N, R, S, T, Q, V or Y; (vii) The residue 9 of the B4 domain is not H, and further optionally the residue 9 of the B4 domain is D, F, K, L, N, R, S, T, Q, V or Y; (viii) The residue 8 of the B5 domain is D, F, K, L, N, R, S, Q, V or Y; (ix) The residue 9 of the B5 domain is not H, and further optionally the residue 9 of the B5 domain is D, F, K, L, N, R, S, T, Q, V or Y; (x) The residue 10 of the B5 domain is not H, and further optionally the residue 10 of the B5 domain is D, F, K, L, N, R, S, T, Q, V or Y; (xi) The residue 12 of the B5 domain is not H, and further optionally the residue 12 of the B5 domain is D, F, K, L, N, R, S, T, Q, V or Y; (xii) The residue 14 of the B5 domain is D, F, K, L, N, S, T, Q, V or Y; (xiii) The residue 3 of the B6 domain is D, F, K, L, N, R, S, Q, V or Y; (xiv) The residue 4 of the B6 domain is D, F, K, L, N, R, S, T, Q, V or Y; (xv) Residue 8 of the B6 domain is not H, and further optionally, wherein residue 8 of the B6 domain is D, F, K, L, N, R, S, T, Q, V, or Y; and / or (xvi) The residue 9 of the B6 domain is D, F, K, L, N, R, S, T, Q, V or Y.

6. The protein according to claim 5, wherein residue 1 of the B3 domain is W / L.

7. The protein according to claim 1, wherein: (i) The length of the H1 domain is at least 18 or 19 amino acids; the residue 9 of the H1 domain is N, T, S, H, R, C, L, D, V, A, Q, G, E, K, I, N or M; (ii) The residue 1 of the B3 domain is L, W or H; (iii) The residue 10 of the B4 domain is F, Y, L, I, K or M; (iv) The residue 12 of the B4 domain is F, L, R, D, M, Q or V; (v) Residue 10 of the B5 domain is L; (vi) Residue 3 of the B6 domain is D or N; (vii) Residue 8 of the B6 domain is Y, F, or L; and / or (viii) Residue 11 of the B5 domain is F or Y; and Choose one of them: (ix) The second residue of the L2 domain is not H, and further optionally the second residue of the L2 domain is D, F, L, Q, R, S, T, W or Y; (x) The residue 3 of the B1 domain is not H, and further optionally the residue 3 of the B1 domain is D, F, L, Q, R, S, T, W or Y; (xi) The residue 5 of the B4 domain is not H, and further optionally the residue 5 of the B4 domain is D, F, L, Q, R, S, T, W or Y; (xii) The residue 9 of the B4 domain is not H, and further optionally the residue 9 of the B4 domain is D, F, L, Q, R, S, T, W or Y; (xiii) The residue 3 of the B5 domain is not H, and further optionally the residue 3 of the B5 domain is D, F, L, Q, R, S, T, W or Y; (xiv) The residue 9 of the B5 domain is not H, and further optionally the residue 9 of the B5 domain is D, F, L, Q, R, S, T, W or Y; (xv) The residue 10 of the B5 domain is not H, and further optionally the residue 10 of the B5 domain is D, F, L, Q, R, S, T, W or Y; (xvi) Residue 12 of the B5 domain is not H, and further optionally, wherein residue 12 of the B5 domain is D, F, L, Q, R, S, T, W, or Y; and / or (xvii) Residue 8 of the B6 domain is not H, and further optionally, residue 8 of the B6 domain is D, F, L, Q, R, S, T, W, or Y; and / or Choose one of them: (ix) The residue 2 of the H2 domain is A, F, I, K, L, N, R, S, T, Q, V or Y; (x) The residue 2 of the L2 domain is not H, and further optionally the residue 2 of the L2 domain is A, D, F, I, K, L, N, R, S, T, Q, V or Y; (xi) The residue 3 of the B1 domain is not H, and further optionally the residue 3 of the B1 domain is A, D, F, I, K, L, N, R, S, T, Q, V or Y; (xii) The residue 2 of the B2 domain is A, D, F, I, K, L, N, R, S, T, Q or Y; (xiii) The residue 1 of the H3 domain is A, D, F, I, K, L, N, S, T, Q, V or Y; (xiv) The residue 10 of the H3 domain is A, D, F, I, K, L, N, S, T, Q, V or Y; (xv) The residue 11 of the H3 domain is A, D, F, I, K, N, R, S, T, Q, V or Y; (xvi) The residue 5 of the B3 domain is A, D, F, I, L, N, R, S, T, Q, V or Y; (xvii) The 5th residue of the B4 domain is not H, and further optionally wherein the 5th residue of B4 is A, D, F, I, K, L, N, R, S, T, Q, V or Y; (xviii) The residue 7 of the B4 domain is A, D, F, I, L, N, R, S, T, V or Y; (xix) The residue 9 of the B4 domain is not H, and further optionally wherein the residue 9 of B4 is A, D, F, I, L, K, N, R, S, T, Q, V or Y; (xx) The residue 8 of the B5 domain is A, D, F, I, L, K, N, R, S, Q or Y; (xxi) The residue 9 of the B5 domain is not H, and further optionally wherein the residue 9 of B5 is A, D, F, I, L, K, N, R, S, Q, V or Y; (xxii) The residue 12 of the B5 domain is not H, and further optionally wherein the residue 12 of B5 is A, D, F, I, L, K, N, R, S, Q, V or Y; (xxiii) The residue 14 of the B5 domain is A, D, F, I, L, K, N, S, Q, V or Y; (xxiv) The residue 3 of the B6 domain is A, D, F, I, L, K, N, R, S, Q, V or Y; (xxv) The residue 4 of the B6 domain is A, D, F, I, L, K, N, R, S, Q, V, or Y; and / or (xxvi) The residue 9 of the B6 domain is A, D, F, L, K, N, R, S, Q, V or Y.

8. The protein according to claim 7, wherein: (i) The residue 9 of the H1 domain is N, T, S, H, R, C, L, D, V, A, Q, G, E, K, I, N or M; (ii) The residue 1 of the B3 domain is L, W or H; (iii) The residue 10 of the B4 domain is F, Y, L, I, K or M; (iv) The residue 12 of the B4 domain is F, D, Y, L, I, K or M; (v) Residue 10 of the B5 domain is L; (vi) Residue 3 of the B6 domain is D or N; (vii) Residue 8 of the B6 domain is Y, F, or L; and (viii) The residue 11 of the B5 domain is F or Y.

9. The protein according to claim 1, wherein: (i) The 7th residue of the H2 domain is S; (ii) Residue 4 of the L2 domain is H; (iii) Residue 10 of the B3 domain is R; (iv) The residue 1 of the B3 domain is L, W or H; (v) Residue 7 of the B4 domain is K; (vi) The residue 10 of the B4 domain is F, Y, L, I, K or M; (vii) The residue 12 of the B4 domain is F, D, Y, L, I, K or M; (viii) Residue 3 of the B6 domain is D or N; (ix) Residue 8 of the B6 domain is Y, F, or L; and (x) The residue 11 of the B5 domain is W, Y or F.

10. A protein having luciferase activity, said protein comprising a secondary structural arrangement H1-L1-H2-L2-B1-L3-B2-L4-H3-L5-B3-L6-B4-L7-B5-L8-B6, wherein "H" is a helical domain, "L" is a loop domain, and "B" is a β-chain domain, wherein: The length of the H1 domain is at least 18 or 19 amino acids; the residue 9 of the H1 domain is T, S, H, R, C, L, V, A, Q, G, E, K, I, N or M.

11. The protein of claim 10, wherein the length of the B4 domain is at least 12 amino acids, and The residue 10 of the B4 domain is F, Y, L, I, K, or M; and / or The residue 12 of the B4 domain is L, R, D, M, Q, or V.

12. The protein of claim 11, wherein residue 9 of the H1 domain is V, residue 10 of the B4 domain is F, Y, L, I, K or M, and residue 12 of the B4 domain is L, R, D, M, Q or V.

13. The protein of claim 11, wherein residue 9 of the H1 domain is V, residue 10 of the B4 domain is F, and residue 12 of the B4 domain is L.

14. The protein according to any one of claims 10-13, wherein the length of the B3 domain is at least 6, 7, 8, 9 or 10 amino acids, and the first residue of the B3 domain is W or H.

15. The protein of claim 14, wherein residue 1 of the B3 domain is W.

16. The protein according to claim 10, wherein: (i) Residue 19 of the H1 domain is R; (ii) The fourth residue of L2 is Q, P or H; (iii) Residue 11 of H3 is R or M; (iv) The residue 1 of the B3 domain is N, H, K, S, L, Q or Y; (v) The residue 10 of the B4 domain is F, Y, L, V, I, K or M; (vi) The residue 10 of the B5 domain is L; (vii) Residue 3 of the B6 domain is D or N; and / or (viii) The residue 8 of the B6 domain is Y, F or L.

17. The protein according to claim 16, wherein: (i) Residue 19 of the H1 domain is R; (ii) Residue 4 of L2 is Q; (iii) Residue 11 of H3 is R; (iv) The residue 1 of the B3 domain is N; (v) Residue 10 of the B4 domain is F; (vi) The residue 10 of the B5 domain is L; (vii) Residue 3 of the B6 domain is N; and / or (viii) Residue 8 of the B6 domain is Y.

18. The protein according to claim 16, wherein: (i) Residue 19 of the H1 domain is R; (ii) Residue 4 of L2 is Q; (iii) Residue 11 of H3 is R; (iv) The residue 1 of the B3 domain is N; (v) Residue 10 of the B4 domain is F; (vi) The residue 10 of the B5 domain is L; (vii) Residue 3 of the B6 domain is N; and (viii) Residue 8 of the B6 domain is Y.

19. The protein according to any one of claims 16-18, wherein residue 11 of the B5 domain is F or Y.

20. The protein of claim 19, wherein residue 11 of the B5 domain is F.

21. The protein according to any one of claims 16-20, wherein residue 9 of the H1 domain is E.

22. The protein according to claim 10, wherein: (i) Residue 9 of the H1 domain is A; (ii) Residue 19 of the H1 domain is R; (iii) The residue 1 of the B3 domain is N, H, K, S, L, Q or Y; (iv) The residue 10 of the B4 domain is F, Y, L, V, I, K or M; (v) The residue 12 of the B4 domain is L, R, D, M, Q or V; (vi) Residue 3 of the B6 domain is D or N; and / or (vii) The residue 8 of the B6 domain is Y, F or L.

23. The protein according to claim 10, wherein: (i) Residue 9 of the H1 domain is A; (ii) Residue 19 of the H1 domain is R; (iii) The residue 1 of the B3 domain is N; (iv) Residue 10 of the B4 domain is Y; (v) Residue 12 of the B4 domain is R; (vi) Residue 3 of the B6 domain is D or N; and / or (vii) Residue 8 of the B6 domain is Y.

24. The protein according to claim 22, wherein: (i) Residue 9 of the H1 domain is A; (ii) Residue 19 of the H1 domain is R; (iii) The residue 1 of the B3 domain is N; (iv) Residue 10 of the B4 domain is Y; (v) Residue 12 of the B4 domain is R; (vi) Residue 3 of the B6 domain is D or N; and (vii) Residue 8 of the B6 domain is Y.

25. The protein according to claim 10, wherein: (i) Residue 18 of the H1 domain is E; (ii) The residue 1 of the B3 domain is N, H, K, S, L, Q or Y; (iii) The residue 10 of the B4 domain is F, Y, L, V, I, K or M; (iv) The residue 12 of the B4 domain is L, R, D, M, Q or V; (v) Residue 3 of the B6 domain is D or N; and / or (vi) The residue 8 of the B6 domain is Y, F or L.

26. The protein according to claim 25, wherein: (i) Residue 18 of the H1 domain is E; (ii) The residue 1 of the B3 domain is H; (iii) Residue 10 of the B4 domain is Y; (iv) Residue 12 of the B4 domain is R; (v) Residue 3 of the B6 domain is D; and / or (vi) Residue 8 of the B6 domain is F.

27. The protein according to claim 25, wherein: (i) Residue 18 of the H1 domain is E; (ii) The residue 1 of the B3 domain is H; (iii) Residue 10 of the B4 domain is Y; (iv) Residue 12 of the B4 domain is R; (v) Residue 3 of the B6 domain is D; and (vi) Residue 8 of the B6 domain is F.

28. The protein according to any one of claims 25-27, wherein residue 9 of the H1 domain is K.

29. The protein according to claim 10, wherein: (i) The residue 1 of the B3 domain is N, H, K, S, L, Q or Y; (ii) The residue 10 of the B4 domain is F, Y, L, V, I, K, or M; and / or (iii) The residue 12 of the B4 domain is L, R, D, M, Q or V.

30. The protein according to claim 29, wherein: (i) The residue 1 of the B3 domain is K or Y; (ii) Residue 10 of the B4 domain is F; and / or (iii) The residue 12 of the B4 domain is V or R.

31. The protein according to claim 29, wherein: (i) The residue 1 of the B3 domain is K; (ii) Residue 10 of the B4 domain is F; and (iii) Residue 12 of the B4 domain is V; or (i) The first residue of the B3 domain is Y; (ii) Residue 10 of the B4 domain is F; and (iii) The residue 12 of the B4 domain is R.

32. The protein according to any one of claims 29-31, wherein residue 9 of the H1 domain is S or L.

33. The protein according to claim 10, wherein: (i) The residue 1 of the B3 domain is N, H, K, S, L, Q or Y; (ii) The residue 10 of the B4 domain is F, Y, L, V, I, K or M; (iii) Residue 12 of the B4 domain is L, R, D, M, Q, or V; and / or (iv) The residue 8 of the B6 domain is Y, F or L.

34. The protein of claim 33, wherein residue 3 of the B6 domain is D or N.

35. The protein according to claim 33 or 34, wherein (i) The first residue of the B3 domain is Y; (ii) Residue 10 of the B4 domain is V; (iii) Residue 12 of the B4 domain is V; (iv) Residue 8 of the B6 domain is Y, F, or L; and (v) Residue 3 of the B6 domain is D.

36. The protein according to claim 33 or 34, wherein... (i) The residue 1 of the B3 domain is L; (ii) The residue 10 of the B4 domain is F; (iii) Residue 12 of the B4 domain is F; (iv) Residue 8 of the B6 domain is Y, F, or L; (v) Residue 3 of the B6 domain is D; and (vi) The residue 10 of the B5 domain is L.

37. The protein according to claim 33, wherein: (i) Residue 11 of H3 is R or M; (ii) The residue 1 of the B3 domain is N, H, K, S, L, Q or Y; (iii) The residue 10 of the B4 domain is F, Y, L, V, I, K or M; (iv) The residue 12 of the B4 domain is L, R, D, M, Q, or V; and / or (v) Residue 8 of the B6 domain is Y, F, or L.

38. The protein according to claim 37, wherein: (i) Residue 11 of H3 is M; (ii) The residue 1 of the B3 domain is S; (iii) The residue 10 of the B4 domain is L; (iv) Residue 12 of the B4 domain is R; and (v) Residue 8 of the B6 domain is Y.

39. The protein according to any one of claims 33-38, wherein residue 9 of the H1 domain is G, N or I.

40. The protein according to claim 10, wherein: (i) Residue 19 of the H1 domain is R; (ii) The fourth residue of L2 is Q, P or H; (iii) Residue 11 of H3 is R or M; (iv) The residue 1 of the B3 domain is N, H, K, S, L, Q or Y; (v) The residue 10 of the B4 domain is F, Y, L, V, I, K, or M; and / or (vi) The residue 8 of the B6 domain is Y, F or L.

41. The protein according to claim 40, wherein: (vii) The residue 10 of the B5 domain is L; and (viii) The residue 3 of the B6 domain is D or N.

42. The protein according to claim 40 or 41, wherein residue 9 of the H1 domain is E or I.

43. The protein according to claim 10, wherein: (i) Residue 10 of the B3 domain is R; (ii) Residue 2 of L5 is Q; (iii) Residue 3 of the B6 domain is D or N; and / or (iv) The residue 8 of the B6 domain is Y, F or L.

44. The protein of claim 43, wherein residue 7 of the B4 domain is K.

45. The protein according to claim 43 or 44, wherein: i) Residue 10 of the B3 domain is R; (ii) Residue 2 of L5 is Q; (iii) Residue 3 of the B6 domain is D or N; (iv) Residue 8 of the B6 domain is Y, F, or L; and (v) Residue 7 of the B4 domain is K.

46. ​​The protein of claim 43, wherein the length of the B4 domain is at least 12 amino acids, and The residue 10 of the B4 domain is F, Y, L, I, K, or M; and / or The residue 12 of the B4 domain is F, L, R, D, M, Q, or V.

47. The protein according to claim 46, wherein: i) Residue 10 of the B3 domain is R; (ii) Residue 2 of L5 is Q; (iii) Residue 3 of the B6 domain is D or N; (iv) Residue 8 of the B6 domain is Y, F, or L; (v) The residue 10 of the B4 domain is F, Y, L, I, K, or M; and (vi) The residue 12 of the B4 domain is F, L, R, D, M, Q or V.

48. The protein according to any one of claims 43-47, wherein residue 9 of the H1 domain is T or Y.

49. A protein having luciferase activity, said protein comprising a secondary structural arrangement H1-L1-H2-L2-B1-L3-B2-L4-H3-L5-B3-L6-B4-L7-B5-L8-B6, wherein "H" is a helical domain, "L" is a loop domain, and "B" is a β-chain domain, wherein: The length of the B3 domain is at least 6, 7, 8, 9 or 10 amino acids, and the first residue of the B3 domain is W or H.

50. The protein of claim 49, wherein residue 1 of the B3 domain is W.

51. A protein having luciferase activity, said protein comprising a secondary structural arrangement H1-L1-H2-L2-B1-L3-B2-L4-H3-L5-B3-L6-B4-L7-B5-L8-B6, wherein "H" is a helical domain, "L" is a loop domain, and "B" is a β-chain domain, wherein: The length of the B4 domain is at least 12 amino acids, and residue 10 of the B4 domain is F, Y, L, I, K, or M.

52. The protein of claim 51, wherein residue 10 of the B4 domain is F.

53. A protein having luciferase activity, said protein comprising a secondary structural arrangement H1-L1-H2-L2-B1-L3-B2-L4-H3-L5-B3-L6-B4-L7-B5-L8-B6, wherein "H" is a helical domain, "L" is a loop domain, and "B" is a β-chain domain, wherein: The length of the B4 domain is at least 12 amino acids, and the residue 12 of the B4 domain is L, R, D, M, Q or V.

54. The protein of claim 53, wherein residue 12 of the B4 domain is L.

55. The protein according to any one of claims 1-54, wherein The length of the H1 domain is at least 18 or 19 amino acids; residue 14 of the H1 domain is Y, D or E, and residue 9 of the H1 domain is D or E; The length of the B3 domain is at least 6, 7, 8, 9, or 10 amino acids, and residue 2 of the B3 domain is R; and The length of the B5 domain is at least 11, 12, 13 or 14 amino acids, and residue 9 of the B5 domain is H or N.

56. The protein of claim 55, wherein residue 7 of the B5 domain is L.

57. The protein according to claim 55 or 56, wherein the length of the B6 domain is at least 9, 10, 11, 12 or 13 amino acids, and wherein residue 5 of the B6 domain is V.

58. The protein according to any one of claims 55-57, wherein residue 1 of the L5 domain is S.

59. The protein according to any one of claims 55-58, wherein residue 7 of the B5 domain is L, and residue 5 of the B6 domain is V.

60. The protein according to any one of claims 55-59, wherein residue 7 of the B5 domain is L, residue 5 of the B6 domain is V, and residue 1 of the L5 domain is S.

61. The protein according to any one of claims 53-60, wherein the length of the H2 domain is at least 5, 6, or 7 amino acids, the length of the H3 domain is at least 9, 10, 11, 12, 13, or 14 amino acids, the length of the B1 domain is at least 3 or 4 amino acids, the length of the B2 domain is at least 3 or 4 amino acids, and / or the length of the B4 domain is at least 12 amino acids.

62. The protein according to any one of claims 55-61, wherein: The length of the H1 domain is at least or at most 19 amino acids; The length of the H2 domain is at least or at most 7 amino acids; The length of the B1 domain is at least or at most 4 amino acids; The length of the B2 domain is at least or at most 4 amino acids; The length of the H3 domain is at least or at most 14 amino acids; The length of the B3 domain is at least or at most 10 amino acids; The length of the B4 domain is at least or at most 12 amino acids; The length of the B5 domain is at least or at most 14 amino acids; and The length of the B6 domain is at least or at most 12 or 13 amino acids.

63. The protein according to any one of claims 55-62, wherein: Residue 13 of domain H1 is F; Residue 1 of domain L3 is W; Residue 5 of domain B5 is V or another hydrophobic residue; and / or Residue 8 of domain B5 is A or L or another hydrophobic residue.

64. The protein according to any one of claims 62 or 63, wherein: Residue 2 of domain B1 is I or another hydrophobic residue; Residue 4 of domain H3 is F; Residue 6 of domain B4 is V or another hydrophobic residue; Residue 8 of domain B4 is L or another hydrophobic residue; Residue 5 of domain B6 is M or V or another hydrophobic residue; and / or Residue 7 of domain B6 is V or another hydrophobic residue.

65. The protein according to any one of claims 1-64, wherein: The H1 domain comprises the following amino acid sequence, which has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequences MSEEQIRQFLRRFYEALDS (SEQ ID NO: 2738), SISEEQIRQFLRRFYEALDS (SEQ ID NO: 2739), IPEEQIRQFLRRFYEALDS (SEQ ID NO: 2740), or EISEEQIRQFLRRFYEALDS (SEQ ID NO: 2741).

66. The protein according to any one of claims 1-65, wherein: The H2 domain contains the following amino acid sequence, which has at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence: ADTAASL.

67. The protein according to any one of claims 1-66, wherein: The B1 domain contains the following amino acid sequence, which has at least 50%, 75%, or 100% identity with the amino acid sequence TIHL.

68. The protein according to any one of claims 1-67, wherein: The B2 domain contains the following amino acid sequence, which has at least 50%, 75%, or 100% identity with the amino acid sequence GVTF.

69. The protein according to any one of claims 1-68, wherein: The H3 domain contains the following amino acid sequence, which has at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence: REEFREWFERLFST.

70. The protein according to any one of claims 1-69, wherein: The B3 domain comprises the following amino acid sequence, which has at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence WREIKSLEVR.

71. The protein according to any one of claims 1-70, wherein: The B4 domain contains the following amino acid sequence, which has at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence TVEVHVQLHFTL or TVVVVVRLDFTL.

72. The protein according to any one of claims 1-71, wherein: The B5 domain contains the following amino acid sequence, which has at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence QKHTVDLTHHFHFR or QKHTVILTHVFRFR.

73. The protein according to any one of claims 1-72, wherein: The B6 domain contains the following amino acid sequence, which has at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence RVTEVRVHINPTG or RVTEVRVEIVPV.

74. The protein according to any one of claims 1-73, wherein the length of the L1, L2, L3, L4, L5, L6, L7 and L8 domains is at least 1, 2, 3, 4 or 5 amino acids, and comprises any amino acid and optionally has a length of at most 5 amino acids.

75. The protein according to any one of claims 1-74, wherein the protein comprises the following amino acid sequence, wherein the amino acid sequence is: MSEEQIRQFLRRFYEALDSGDADTAASLFHPGVTIHLWDGVTFTSREEFREWFERLFSTSKDAQREIKSLEVRGDTVEVHVQLHATHNGQKHTVDLTHHWHFRGNRVTEVRVHINPTG (SEQ ID NO: 1) has at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity.

76. A protein having luciferase activity, said protein comprising an amino acid sequence that is at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:

1. in: (i)a) Residue 100 is F, Y, or L; and b) Residue 85 is F, Y, L, I, K, or M; and c) Residue 87 is L, R, D, M, Q, F, or V; or (ii)a) Residue 100 is F, Y, or L; and b) Residue 87 is L, R, D, M, Q, F, or V; or (iii)a) Residue 100 is F, Y, or L; and b) Residue 85 is F, Y, L, I, K, or M; or (iv) wherein the protein does not contain a lysine residue and contains another amino acid in place of the lysine residue present in SEQ ID NO: 1, and optionally the protein contains residues as shown in any of (i) - (iii).

77. The protein of claim 76, wherein the protein comprises a substitution W100F relative to SEQ ID NO:

1.

78. The protein according to claim 76 or 77, wherein the protein comprises a substituted A85F relative to SEQ ID NO:

1.

79. The protein according to any one of claims 76-78, wherein the protein comprises a substituted H87L relative to SEQ ID NO:

1.

80. The protein according to any one of claims 76-79, wherein the protein further comprises a substitution Q64W, Q64L or Q64H relative to SEQ ID NO:

1.

81. The protein according to any one of claims 1-80, wherein the protein further comprises an additional polypeptide domain fused to the protein.

82. The protein of claim 81, wherein the additional polypeptide domain is present at the N-terminus or C-terminus of the protein.

83. A self-complementary multipartic protein having luciferase activity, the self-complementary multipartic protein comprising at least a first polypeptide component and a second polypeptide component, wherein the at least first polypeptide component and the second polypeptide component are not covalently linked or covalently linked via a cleavable linker, wherein the first polypeptide component and the second polypeptide component collectively comprise a secondary structure arrangement H1-L1-H2-L2-B1-L3-B2-L4-H3-L5-B3-L6-B4-L7-B5-L8-B6, wherein each domain is defined as in any one of claims 1-74; Wherein (a) each H and B domain is entirely present in one polypeptide component of the first polypeptide component or the second polypeptide component, (b) neither the first polypeptide component nor the second polypeptide component contains all of the H and B domains, (c) when present in either the first polypeptide component or the second polypeptide component, the relative order of the H, L, and B domains does not change relative to the protein as defined in any one of claims 1-74, and (d) the first component and the second component, when not present in the self-complementary multipart protein, do not have detectable luciferase activity or have luciferase activity lower than that of the self-complementary multipart protein.

84. The self-complementary multiparticulate protein of claim 83, wherein the first polypeptide component and the second polypeptide component comprise a secondary structure arrangement as shown in Table 1: Table 1: Wherein the L-domain in parentheses (i) exists in one of the first component and the second component but not both, (ii) is split between the first component and the second component, or (iii) does not exist.

85. The self-complementary multipartic protein according to claim 83 or 84, wherein (i) one or both of the first component and the second component contain an additional domain, (ii) one or both of the first component and the second component contain an additional domain covalently linked to one or both of the first component and the second component, (iii) the first component is a fusion protein containing a first domain and the second component is a fusion protein containing a second domain, or (iv) the self-complementary multipartic protein comprises (a) the first component, a linker, and the second component from the N-terminus to the C-terminus, or (b) the second component, a linker, and the first component, wherein the self-complementary multipartic protein has luciferase activity, and wherein, upon cleavage of the linker, the self-complementary multipartic protein has significantly reduced cleavage activity or substantially undetectable cleavage activity.

86. A cyclic substitution polypeptide with luciferase activity, wherein: The N-terminus and C-terminus of the cyclic substitution polypeptide are different from those of the protein having luciferase activity and containing the secondary structural arrangement H1-L1-H2-L2-B1-L3-B2-L4-H3-L5-B3-L6-B4-L7-B5-L8-B6, respectively. "H" is a helical domain, "L" is a ring domain, and "B" is a β-chain domain. In the cyclic substitution polypeptide, the N-terminus and C-terminus of the protein with luciferase activity are linked by a linker sequence, and the cyclic substitution polypeptide comprises the following secondary structural arrangement: H2-L2-B1-L3-B2-L4-H3-L5-B3-L6-B4-L7-B5-L8-B6-connector-H1-(L1) (I), B1-L3-B2-L4-H3-L5-B3-L6-B4-L7-B5-L8-B6-connector-H1-L1-H2-(L2) (II), B2-L4-H3-L5-B3-L6-B4-L7-B5-L8-B6-connector-H1-L1-H2-L2-B1-(L3) (III), H3-L5-B3-L6-B4-L7-B5-L8-B6-connector-H1-L1-H2-L2-B1-L3-B2-(L4) (IV), B3-L6-B4-L7-B5-L8-B6-connector-H1-L1-H2-L2-B1-L3-B2-L4-H3-(L5) (V), B4-L7-B5-L8-B6-connector-H1-L1-H2-L2-B1-L3-B2-L4-H3-L5-B3-(L6) (VI), B5-L8-B6-Connector-H1-L1-H2-L2-B1-L3-B2-L4-H3-L5-B3-L6-B4-(L7) (VII) or B6-Connector-H1-L1-H2-L2-B1-L3-B2-L4-H3-L5-B3-L6-B4-L7-B5-(L8) (VIII), The L-domain in parentheses may exist at the C-terminus or N-terminus, or split between the C-terminus and N-terminus, or may not exist.

87. The cyclic substitution polypeptide of claim 86, wherein the linker comprises a secondary structure H4-L9.

88. The cyclic substitution polypeptide of claim 86, wherein the linker comprises a secondary structure H4-L9-H5-L10.

89. The cyclic substitution polypeptide of claim 86, wherein the linker comprises a secondary structure H4-L9-H5-L10-H6-L11.

90. The cyclic substitution polypeptide according to any one of claims 86-89, wherein H1, L1, H2, L2, B1, L3, B2, L4, H3, L5, B3, L6, B4, L7, B5, L8 and B6 are as described in any one of claims 1-74.

91. The cyclic substitution polypeptide according to any one of claims 86-87, wherein the B5 domain comprises an amino acid sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence QKHTVDLTHHWHFR or QKHTVILTHVFRFR.

92. The cyclic substitution polypeptide according to any one of claims 86-91, wherein the B4 domain comprises an amino acid sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence TVEVHVQLHATH or TVVVVVRLDFTL.

93. The cyclic substitution polypeptide according to any one of claims 86-92, wherein the cyclic substitution polypeptide comprises an amino acid sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence shown in any one of SEQ ID NO: 144-2599.

94. A fusion protein comprising a cyclic substitution polypeptide fused to another functional domain according to any one of claims 86-93.

95. A self-complementary multipartic protein having luciferase activity, the self-complementary multipartic protein comprising at least a first polypeptide component and a second polypeptide component, wherein the at least first polypeptide component and the second polypeptide component are not covalently linked or are covalently linked via a linker, optionally a cleavable linker, wherein the first polypeptide component and the second polypeptide component collectively comprise a secondary arrangement of cyclic substitution polypeptides as described in any one of claims 86-94. Wherein (a) each H and B domain is entirely present within one polypeptide component of the first polypeptide component or the second polypeptide component, (b) neither the first polypeptide component nor the second polypeptide component individually contains all of the H and B domains, (c) when present in either the first polypeptide component or the second polypeptide component, the relative order of the H, L, and B domains is unchanged relative to the cyclic substitution polypeptide as described in any one of claims 86-94, and (d) neither the first component nor the second component individually has detectable luciferase activity or individually has luciferase activity lower than that of the self-complementary multipart protein.

96. The self-complementary multipart protein of claim 95, wherein the H and B domains of the cyclic substitution polypeptide are separated into the first polypeptide component and the second polypeptide component at the L domain, wherein the separation point is the N-terminus of the L domain, the C-terminus of the L domain, or within the L domain.

97. The self-complementary multipart protein of claim 96, wherein the H and B domains of the cyclic substitution polypeptide (I) are separated into the first polypeptide component and the second polypeptide component at the L2 domain, the L3 domain, the L4 domain, the L5 domain, the L6 domain, the L7 domain, the L8 domain or the linker.

98. The self-complementary multipart protein of claim 96, wherein the H and B domains of the cyclic substitution polypeptide (II) are isolated into the first polypeptide component and the second polypeptide component at the L3 domain, the L4 domain, the L5 domain, the L6 domain, the L7 domain, the L8 domain, the linker, or the L1 domain.

99. The self-complementary multipart protein of claim 96, wherein the H and B domains of the cyclic substitution polypeptide (III) are isolated into the first polypeptide component and the second polypeptide component at the L4 domain, the L5 domain, the L6 domain, the L7 domain, the L8 domain, the linker, the L1 domain, or the L2 domain.

100. The self-complementary multipart protein of claim 96, wherein the H and B domains of the cyclic substitution polypeptide (IV) are isolated into the first polypeptide component and the second polypeptide component at the L5 domain, the L6 domain, the L7 domain, the L8 domain, the linker, the L1 domain, the L2 domain, or the L3 domain.

101. The self-complementary multipart protein of claim 96, wherein the H and B domains of the cyclic substitution polypeptide (V) are isolated into the first polypeptide component and the second polypeptide component at the L6 domain, the L7 domain, the L8 domain, the linker, the L1 domain, the L2 domain, the L3 domain, or the L4 domain.

102. The self-complementary multipart protein of claim 96, wherein the H and B domains of the cyclic substitution polypeptide (VI) are isolated into the first polypeptide component and the second polypeptide component at the L7 domain, the L8 domain, the linker, the L1 domain, the L2 domain, the L3 domain, the L4 domain, or the L5 domain.

103. The self-complementary multipart protein of claim 96, wherein the H and B domains of the cyclic substitution polypeptide (VII) are isolated into the first polypeptide component and the second polypeptide component at the L8 domain, the linker, the L1 domain, the L2 domain, the L3 domain, the L4 domain, the L5 domain, or the L6 domain.

104. The self-complementary multipart protein of claim 96, wherein the H and B domains of the cyclic substitution polypeptide (VIII) are isolated into the first polypeptide component and the second polypeptide component at the linker, the L1 domain, the L2 domain, the L3 domain, the L4 domain, the L5 domain, the L6 domain, or the L7 domain.

105. The self-complementary multipart protein according to any one of claims 95-104, wherein one or both of the first polypeptide component and the second polypeptide component are fused with an additional functional domain, optionally wherein the additional domain is covalently linked to one or both of the first component and the second component, optionally wherein the first component is a fusion protein comprising a first domain and the second component is a fusion protein comprising a second domain.

106. A self-complementary multipartic protein having luciferase activity, the self-complementary multipartic protein comprising at least a first polypeptide component, a second polypeptide component, and a third polypeptide component, wherein the at least first polypeptide component, the second polypeptide component, and the third polypeptide component are not covalently linked or are covalently linked via one or two linkers, optionally one or two cleavable linkers, wherein the first polypeptide component, the second polypeptide component, and the third polypeptide component collectively comprise a secondary structure arrangement H1-L1-H2-L2-B1-L3-B2-L4-H3-L5-B3-L6-B4-L7-B5-L8-B6, wherein each domain is defined as claimed in any one of claims 1-74; Wherein (a) each H and B domain is entirely present in one of the first, second, or third polypeptide components; (b) the first, second, and third polypeptide components do not contain all of the H and B domains; (c) when present in the first, second, or third polypeptide component, the relative order of the H, L, and B domains does not change relative to the protein as defined in any one of claims 1-74; and (d) the first, second, and third polypeptide components, when not present in the self-complementary multipart protein, do not have detectable luciferase activity or have luciferase activity lower than that of the self-complementary multipart protein.

107. The self-complementary multipart protein of claim 106, wherein the H and B domains of the protein as described in any one of claims 1-74 are separated at the L domain into the first polypeptide component, the second polypeptide component, and the third polypeptide component, wherein the separation point is the N-terminus of the L domain, the C-terminus of the L domain, or within the L domain, and optionally the L domain is not present in the first polypeptide component, the second polypeptide component, and the third polypeptide component.

108. The self-complementary multipart protein of claim 106 or 107, wherein at least one of the first component, the second component, and the third component comprises an additional domain, optionally wherein the additional domain is covalently linked to at least one of the first component, the second component, and the third component, optionally wherein the first component is a fusion protein comprising a first domain, the second component is a fusion protein comprising a second domain, and the third component is a fusion protein comprising a third domain.

109. A self-complementary multipartic protein having luciferase activity, the self-complementary multipartic protein comprising at least a first polypeptide component, a second polypeptide component, and a third component, wherein the at least first polypeptide component, the second polypeptide component, and the third component are not covalently linked or are covalently linked via one or two linkers, optionally one or two cleavable linkers, wherein the first polypeptide component, the second polypeptide component, and the third component collectively comprise a secondary arrangement of cyclic substitution polypeptides as described in any one of claims 86-94; Wherein (a) each H and B domain is completely present in one of the first polypeptide components, the second polypeptide component, or the third component; (b) the first polypeptide component, the second polypeptide component, and the third component individually do not contain all of the H and B domains; (c) when present in the first polypeptide component, the second polypeptide component, or the third component, the relative order of the H, L, and B domains does not change relative to the cyclic substitution polypeptide as described in any one of claims 86-94; and (d) the first component, the second component, and the third component individually do not have detectable luciferase activity or individually have luciferase activity lower than that of the self-complementary multipart protein.

110. The self-complementary multipart protein of claim 109, wherein the H and B domains of the cyclic substitution polypeptide are separated at the L domain into the first polypeptide component, the second polypeptide component, and the third component, wherein the separation point is the N-terminus of the L domain, the C-terminus of the L domain, or within the L domain.

111. The self-complementary multipart protein of claim 109 or 110, wherein one or more of the first polypeptide component, the second polypeptide component, and the third component are fused with an additional functional domain, optionally wherein the additional domain is covalently linked to one or more of the first component, the second polypeptide component, and the third component, optionally wherein the first component is a fusion protein containing a first domain, the second component is a fusion protein containing a second domain, and the third component is a fusion protein containing a third domain.

112. A protein having luciferase activity and comprising an amino acid sequence having at least 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO: 1, and (i) Amino acid substitutions relative to SEQ ID NO: 1 at one or more of the following positions: E3, I6, Y14, E15, S19, L28, G32, T42, F43, S45, L56, F57, T59, K61, Q64, V77, E78, Q82, A85, T86, H92, L96, H99, W100, R106, T108, and H113; and / or (ii) Contains another amino acid to replace the lysine residue present in SEQ ID NO:

1.

113. The protein of claim 112, wherein the protein comprises: (i) Replace one or more of the following: E3D, I6T / K, Y14W, E15G, S19R, L28S / F, G32R / D / A / E / H, T42I, F43L / G, S45A, L56R / K / Q, L56R / K / Q, F57V, T59K, Q64W / H, K61P / E, V77Y, E78W, Q82T / K, A85F / Y / L / I / M, T86A, H87L / V, H99L, W100F / Y / L, R106L, V107I, T108N / D, and H113F; or (ii) Amino acid substitutions relative to SEQ ID NO: 1 that lack lysine residues and are present at one or more of the following positions: F9, D23, H30, H36, V41, R46, R55, L56, Q64, K68, H80, Q82, H84, A85, H87, H92, T97, H98, H99, W100, H101, R103, T108, E109, H113, and I114, optionally wherein the lysine residue is replaced by arginine, and further optionally the amino acid sequence comprises all of the following: F9, D23, H30, H36, V41, R46, R55, L56, Q64, K68, H80, Q82, H84, A85, H87, H92, T97, H98, H99, W100, H101, R103, T108, E109, H113, and I114, ... Replacement for: F9V / S / N, D23I, H30D, H36T, V41T, R46V, R55S, L56Q, Q64L, K68S, H80T, Q82R, H84S, A85F, H87R, H92S, T97L, H98Q, H99L, W100F, H101K / R, R103V, T108D / V, E109A, H113Y and I114V.

114. A nucleic acid comprising a nucleotide sequence encoding a protein, polypeptide component, or fusion protein according to any of the preceding claims.

115. An expression vector comprising a nucleic acid according to claim 114 operatively connected to an expression control element.

116. A recombinant host cell comprising a protein, polypeptide component, fusion protein, nucleic acid, and / or expression vector according to any of the preceding claims.

117. A protein having luciferase activity and comprising an amino acid sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or 100% identity with the amino acid sequence of any one of SEQ ID NO: 2-91, 93-143, 2601-2604, 2663, 2665, or 2682-2732.

118. A protein having luciferase activity and comprising an amino acid sequence that is at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 1, wherein the amino acid at position 9 is any amino acid except F, and wherein position 9 is based on the numbering of SEQ ID NO:

1.

119. The protein of claim 118, wherein the amino acid at position 9 is D, E, Q, R, S, T, H, I, L, V, A, G, C, N, K or M.

120. The protein according to claim 118 or 119, the protein further comprising a substitution at position Q64, wherein position Q64 is based on SEQ ID NO: 1, and optionally wherein the substitution is Q64L / F / W / Q.

121. The protein according to any one of claims 118-120, the protein further comprising a substitution at position A85, wherein position A85 is based on SEQ ID NO: 1, and optionally wherein the substitution is A85F / Y / F / M / L / A.

122. The protein according to any one of claims 118-121, the protein further comprising a substitution at position H87, wherein position H87 is based on SEQ ID NO: 1, and optionally wherein the substitution is H87R / L / V / G / K.

123. The protein according to any one of claims 118-122, the protein further comprising a substitution at position H99, wherein position H99 is based on SEQ ID NO: 1, and optionally wherein the substitution is H99L.

124. The protein according to any one of claims 118-123, the protein further comprising a substitution at position W100, wherein position W100 is based on SEQ ID NO: 1, and optionally wherein the substitution is W100F / Y / L.

125. The protein according to any one of claims 118-124, the protein further comprising a substitution at position T108, wherein position T108 is based on SEQ ID NO: 1, and optionally wherein the substitution is T108N / D.

126. The protein according to any one of claims 118-125, the protein further comprising a substitution at position H113, wherein position H113 is based on SEQ ID NO: 1, and optionally wherein the substitution is H113F / Y.

127. The protein according to any one of claims 118-126, wherein the protein further comprises a substitution at position H30, H36, H80, H84, H87, H92, H98, H99 or H101.

128. The protein of claim 127, wherein the protein comprises one or more of the following substitutions: H30D, H36T, H80T, H84S, H87R, H92S, H98Q, H99L, and H101R.

129. The protein according to any one of claims 118-128, wherein the protein further comprises substitutions at one or more of positions V41, R46, T97, R103, E109, and I114.

130. The protein according to any one of claims 118-129, wherein the protein further comprises one or more of the following substitutions: V41T, R46V, T97L, R103V, E109D, and I114T.

131. The protein according to any one of claims 118-130, wherein the protein comprises a substitution F9V / S / N, and optionally comprises one or more of the following substitutions: Q64W, A85F, and H87L.

132. The protein according to any one of claims 118-130, wherein the protein comprises a substituted F9N, and optionally comprises one or more of the following substitutions: Q64L, A85F, H87R, H99L, W100F, T108D, and H113Y.

133. The protein of claim 118, wherein the protein comprises a substituted F9N, and optionally comprises one or more of the following substitutions: H30D, H36T, Q64L, H80T, H84S, A85F, H87R, H92S, H98Q, H99L, W100F, H101R, T108D, and H113Y.

134. The protein of claim 118, wherein the protein comprises a substituted F9N, and optionally comprises one or more of the following substitutions: H30D, H36T, L56Q, Q64L, H80T, H84S, A85F, H87R, H92S, H98Q, H99L, W100F, H101K / R, T108D, and H113Y.

135. The protein of claim 118, wherein the protein comprises a substituted F9N, and optionally comprises one or more of the following substitutions: D23I, H30D, H36T, V41T, R46V, R55S, L56Q, Q64L, K68S, H80T, Q82R, H84S, A85F, H87R, H92S, T97L, H98Q, H99L, W100F, H101K / R, R103V, T108D / V, E109A, H113Y, and I114V, further optionally wherein the amino acid sequence does not contain lysine.

136. A fusion protein comprising a protein fused to another protein according to any one of claims 1-85 and 94-135.

137. The protein according to any one of claims 1-85 and 94-136 or the polypeptide according to any one of claims 86-93, wherein the protein or the polypeptide comprises one or more non-naturally occurring amino acids.

138. The protein or polypeptide of claim 137, wherein the one or more non-naturally occurring amino acids are chemically modified forms of one or more naturally occurring amino acids.

139. The protein or polypeptide of claim 138, wherein one or more chemically modified amino acids comprise chemical modifications that introduce a chemical handle.

140. The protein according to any one of claims 1-85 and 94-136 or the polypeptide according to any one of claims 86-93, wherein the protein or the polypeptide comprises one or more cysteine ​​residues inserted at the N-terminus or C-terminus, or substitution of one or more amino acids by cysteine ​​residues.

141. A protein comprising an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with any one of the following amino acid sequences: ， ， ， ， Or a protein comprising an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with any one of the amino acid sequences shown in Table 13A, SEQ ID NO: 2733, SEQ ID NO: 2736, SEQ ID NO: 2734, SEQ ID NO: 2735, SEQ ID NO: 2770, SEQ ID NO: 2771, and SEQ ID NO: 2772, Tables 19, 20, 21, and 22.

142. A fusion protein comprising a protein according to claim 141 fused to another protein.

143. A nucleic acid comprising a nucleotide sequence encoding a protein according to claim 141 or 142.

144. An expression vector comprising a nucleic acid according to claim 143 operatively connected to an expression control element.

145. A recombinant host cell comprising one or more proteins according to claim 141 or 142, nucleic acids according to claim 143, and / or expression vectors according to claim 144.

146. One or both of the first nucleic acid encoding the first protein and the second nucleic acid encoding the second protein. The first protein comprises the following amino acid sequence, wherein the amino acid sequence has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with any of the following amino acid sequences: ， Or, the first protein may comprise an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with any one of the lgLux amino acid sequences shown in Table 13A, SEQ ID NO: 2733, SEQ ID NO: 2736, Table 19, Table 20, Table 21, and Table 22. and The second protein comprises an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with any of the amino acid sequences shown below: ， Alternatively, the second protein may comprise an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with any one of the smLux amino acid sequences shown in Table 13A, SEQ ID NO: 2734, SEQ ID NO: 2735, SEQ ID NO: 2770, SEQ ID NO: 2771, and SEQ ID NO: 2772.

147. One or both of the first nucleic acid encoding the first protein and the second nucleic acid encoding the second protein. in (i) The first protein comprises an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence: MSEEQIRQFLRRFYEALDSGDADTAASLFHPGVTIHLWDGVTFT (SEQ ID NO: 2605), and the second protein comprises an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence: SREEFREWFERLFSTSKDAWREIKSLEVRGDTVEVHVQLHFTLNGQKHTVDLTHHFHFRGNRVTEVRVHINPTGLE (SEQ ID NO: 2606). (ii) The first protein comprises an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence: MSEEQIRQFLRRFYEALDSGDADTAASLFHPGVTIHLWDGVTFTSREEFREWFERLFSTSKDAWREIKSLEVRG (SEQ ID NO: 2608), and the second protein comprises an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence: DTVEVHVQLHFTLNGQKHTVDLTHHFHFRGNRVTEVRVHINPTGLE (SEQ ID NO: 2607). (iii) The first protein comprises an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence: MSEEQIRQFLRRFYEALDSGDADTAASLFHPGVTIHLWDGVTFTSREEFREWFERLFSTSKDAWREIKSLEVRGDTVEVHVQLHFTLNGQKHTVDLTHHFHFRG (SEQ ID NO: 2610), and the second protein comprises an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence: NRVTEVRVHINPTGLE (SEQ ID NO: 2609); (iv) The first protein comprises an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence: SEEQIRQNLRRFYEALDSGDADTAASLFHPGVTIHLWDGVTFTSREEFREWFERLFSTSKDALREIKSLEVRGDTVEVHVQLHFTRNGQKHTVDLTHLFHFR (SEQ ID NO: 2614), and the second protein comprises an amino acid sequence: NRVDEVRVYIN (SEQ ID NO: 2615) or NRVDEVRVYINPT (SEQ ID NO: 2615). 2616) has an amino acid sequence with at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity; (v) The first protein contains at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence: GQKHVVVLVHTFRFRG (SEQ ID NO: 2617), and the second protein contains the amino acid sequence: NRVTEVRVEIIPAPSLDEESIEARVAEARRLAEERLAELGDPPPSISEEQIRQFLRRFYEALDSGDADTAASLFHPGVTIHLWDGVTFTSREEFREWFERLFSTSKDAWREIVELRVRGDTVVVVVVLHFTLN (SEQ ID NO: 2617). At least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with 2618); (vi) The first protein contains at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence: NRVTEVRVEIIPAP (SEQ ID NO: 2619), and the second protein contains the amino acid sequence: SLDEESIEARVAEARRLAEERLAELGDPPPSISEEQIRQFLRRFYEALDSGDADTAASLFHPGVTIHLWDGVTFTSREEFREWFERLFSTSKDAWREIVELRVRGDTVVVVVVLHFTLNGQKHVVVLVHTFRFRG (SEQ ID NO: 2619). At least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with 2620); (vii) The first protein contains at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence: SLDEESIEARVAEARRLAEERLAELGDPP (SEQ ID NO: 2621), and the second protein contains the amino acid sequence: PSISEEQIRQFLRRFYEALDSGDADTAASLFHPGVTIHLWDGVTFTSREEFREWFERLFSTSKDAWREIVELRVRGDTVVVVVVLHFTLNGQKHVVVLVHTFRFRGNRVTEVRVEIIPAP (SEQ ID NO: 2621). At least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with 2622); (viii) The first protein contains at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence: PSISEEQIRQFLRRFYEALDSG (SEQ ID NO: 2623), and the second protein contains at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence: DADTAASLFHPGVTIHLWDGVTFTSREEFREWFERLFSTSKDAWREIVELRVRGDTVVVVVLHFTLNGQKHVVVLVHTFRFRGNRVTEVRVEIIPAPSLDEESIEARVAEARRLAEERLAELGDPP (SEQ ID NO: 2624). (ix) The first protein contains at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence: DADTAASLFHP (SEQ ID NO: 2625), and the second protein contains at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence: GVTIHLWDGVTFTSREEFREWFERLFSTSKDAWREIVELRVRGDTVVVVVLHFTLNGQKHVVVLVHTFRFRGNRVTEVRVEIIPAPSLDEESIEARVAEARRLAEERLAELGDPPPSISEEQIRQFLRRFYEALDSG (SEQ ID NO: 2626). (x) The first protein contains at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence: GVTIHLW (SEQ ID NO: 2627), and the second protein contains at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence: DGVTFTSREEFREWFERLFSTSKDAWREIVELRVRGDTVVVVVLHFTLNGQKHVVVLVHTFRFRGNRVTEVRVEIIPAPSLDEESIEARVAEARRLAEERLAELGDPPPSISEEQIRQFLRRFYEALDSGDADTAASLFHP (SEQ ID NO: 2628). (xi) The first protein contains at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence: DGVTFT (SEQ ID NO: 2629), and the second protein contains at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence: SREEFREWFERLFSTSKDAWREIVELRVRGDTVVVVVLHFTLNGQKHVVVLVHTFRFRGNRVTEVRVEIIPAPSLDEESIEARVAEARRLAEERLAELGDPPPSISEEQIRQFLRRFYEALDSGDADTAASLFHPGVTIHLW (SEQ ID NO: 2630). (xii) The first protein contains at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence: SREEFREWFERLFSTSK (SEQ ID NO: 2631), and the second protein contains at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence: DAWREIVELRVRGDTVVVVVLHFTLNGQKHVVVLVHTFRFRGNRVTEVRVEIIPAPSLDEESIEARVAEARRLAEERLAELGDPPPSISEEQIRQFLRRFYEALDSGDADTAASLFHPGVTIHLWDGVTFT (SEQ ID NO: 2632). (xiii) The first protein contains at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence: DAWREIVELRVRG (SEQ ID NO: 2633), and the second protein contains at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence: DTVVVVVVLHFTLNGQKHVVVLVHTFRFRGNRVTEVRVEIIPAPSLDEESIEARVAEARRLAEERLAELGDPPPSISEEQIRQFLRRFYEALDSGDADTAASLFHPGVTIHLWDGVTFTSREEFREWFERLFSTSK (SEQ ID NO: 2634). (xiv) The first protein contains at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence: DTVVVVVVLHFTLN (SEQ ID NO: 2635), and the second protein contains the amino acid sequence: GQKHVVVLVHTFRFRGNRVTEVRVEIIPAPSLDEESIEARVAEARRLAEERLAELGDPPPSISEEQIRQFLRRFYEALDSGDADTAASLFHPGVTIHLWDGVTFTSREEFREWFERLFSTSKDAWREIVELRVRG (SEQ ID NO: 2635). At least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with 2636); or (xv) The first protein contains at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence: MSGMSEEQIRQNLRRFYEALDSGDAITAASLFDPGVTITLWDGTTFTSVEEFREWFESQFSTSKDALREISSLEVRGDTVEVTVRLSFTRNGQKHTVDLLQLFKFV (SEQ ID NO: 2733), and the second protein contains at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence: MSGGNRVVAVRVYVNPT (SEQ ID NO: 2734).

148. One or both of the first nucleic acid and the second nucleic acid according to claim 147, wherein the first protein is a fusion protein containing a first domain, and the second protein is a fusion protein containing a second domain, wherein the first domain and the second domain are capable of associating with each other.

149. One or both of the first nucleic acid and the second nucleic acid according to claim 148, wherein the first domain and the second domain are associated with each other in the presence of a molecule that binds to the first domain, the second domain, or both.

150. An expression vector comprising one or both of a first nucleic acid and a second nucleic acid according to any one of claims 146-149, or a first expression vector comprising a first nucleic acid according to any one of claims 146-149 and a second expression vector comprising a second nucleic acid according to any one of claims 146-149.

151. A kit comprising: (a) The protein according to any one of claims 1-85, 112-113, 117-135, 137-141, (b) the polypeptide according to any one of claims 86-93, (c) The fusion protein according to claim 94, 136 or 142 (d) The polypeptide component according to any one of claims 95-111, (e) The nucleic acid according to claims 114, 143, (f) The expression vector according to claims 115 and 144 (g) The host cell according to claims 116, 145, and / or (h) the first and second nucleic acids according to any one of claims 146-149, and / or (i) The first expression vector and the second expression vector according to claim 150.

152. The kit of claim 151, further comprising a substrate, optionally wherein the substrate is a luciferin analog, optionally wherein the luciferin analog is a compound of formula (I) or a stereoisomer, tautomer or salt thereof, a compound of formula (Ia), a compound of formula (Ib), a compound of formula (Ic), a compound of formula (Id), a compound of formula (Ie) or a compound of claim 194.

153. The kit according to claim 152, wherein the compound of formula (I) is: (I) Or its stereoisomers, tautomers, or salts, wherein: R 1 R 2 and R 3 Selected independently from: C 3-6 cycloalkyl; aryl; C 1-3 Alkyl, halogen, C 1-3 An aryl group substituted with at least one of a haloalkyl, hydroxyl, alkoxy, nitro, or amino alcohol; a 5- to 10-membered heteroaryl group having 1 to 3 independently selected cyclic heteroatoms chosen from O, S, Se, and N; and a heterocycle; in: If R 1 If it is aryl, then R 2 and R 3 Selected independently from: C 3-6 cycloalkyl; aryl; C 1-3 Alkyl, halogen, C 1-3 An aryl group substituted with at least one of haloalkyl, hydroxyl, alkoxy or nitro groups; and a 5- to 10-membered heteroaryl group having 1 to 3 cyclic heteroatoms independently selected from O, S, Se and N; If R 3 If it is aryl, then R 1 and R 2 Selected independently from: C 3-6 cycloalkyl; aryl; C 1-3 Alkyl, halogen, C 1-3 An aryl group substituted with at least one of a haloalkyl, hydroxyl, alkoxy, nitro, or amino alcohol; a 5-membered heteroaryl group having 1-3 independently selected cyclic heteroatoms chosen from O, S, Se, and N; a 10-membered heteroaryl group having 1-3 independently selected cyclic heteroatoms chosen from O, S, and Se; a 6-membered heteroaryl group having 1-3 independently selected cyclic heteroatoms chosen from O, S, Se, and N; a heterocycle, and If R 2 If it is aryl, then R 1 and R 3 Selected independently from: C 3-6 cycloalkyl; aryl; C 1-3 Alkyl, halogen, C 1-3 An aryl group substituted with at least one of alkyl, hydroxyl, alkoxy, nitro, or amino alcohols; a 5-membered heteroaryl group having 1-3 cyclic heteroatoms independently selected from O, S, Se, and N; a 10-membered heteroaryl group having 1-3 cyclic heteroatoms independently selected from O, S, Se, and N; and heterocycles.

154. A compound of formula (Ia): (him) Or its stereoisomers, tautomers, or salts, wherein: X 1 -X 2 Independently selected from: halogen, hydroxyl, haloalkyl, alkyl or nitro; The conditions are: When X 2 When it is hydrogen, then X 1 Selected from: haloalkyl, alkyl, or nitro groups; When X 1 When it is a hydroxyl group, then X 2 Selected from halogens.

155. The compound according to claim 153, wherein X 2 It is hydrogen and X 1 It is a haloalkyl group.

156. The compound according to claim 154, wherein X 2 It is hydrogen and X 1 It is trifluoromethyl.

157. The compound according to claim 153, wherein X 2 It is hydrogen and X 1 It is an alkyl group.

158. The compound according to claim 156, wherein X 2 It is hydrogen and X 1 It is a methyl group.

159. The compound according to claim 153, wherein X 2 It is hydrogen and X 1 It is a nitro group.

160. The compound according to claim 153, wherein X 1 It is a hydroxyl group and X 2 Selected from fluorine, chlorine, bromine or iodine.

161. The compound according to claim 159, wherein X 1 It is a hydroxyl group and X 2 It's fluorine.

162. A compound of formula (Ib): (One) Or its stereoisomers, tautomers, or salts, wherein: R 1 Selected from: cycloalkyl groups, 5-membered heteroaryl groups having 1-3 cyclic heteroatoms independently selected from O, S, Se and N; and 10-membered heteroaryl groups having 1-3 cyclic heteroatoms independently selected from O, S, Se and N; or R 1 Selected from: 。 163. The compound according to claim 161, wherein R 1 It is a cycloalkyl group.

164. The compound according to claim 162, wherein R 1 It is cyclopropyl.

165. The compound according to claim 161, wherein R 1 It is a 5-membered heteroaryl group having 1-3 independently selected cyclic heteroatoms chosen from O, S, Se and N.

166. The compound according to claim 164, wherein R 1 Selected from pyrrole, furan, thiophene, selenophene, imidazole, thiazole and oxazole.

167. The compound according to claim 161, wherein R 1 It is a 10-membered heteroaryl group having 1-3 independently selected cyclic heteroatoms chosen from O, S, Se and N.

168. The compound according to claim 166, wherein R 1 It is quinoline.

169. The compound according to claim 161, wherein R 1 yes .

170. The compound according to claim 161, wherein R 1 yes .

171. The compound according to claim 161, wherein R 1 yes .

172. The compound according to claim 161, wherein R 1 yes .

173. A compound of formula (Ic): (Ic) Or its stereoisomers, tautomers, or salts, wherein: R 3 Selected from: cycloalkyl groups, 5-membered heteroaryl groups having 1-3 cyclic heteroatoms independently selected from O, S, Se and N; and 10-membered heteroaryl groups having 1-3 cyclic heteroatoms independently selected from O, S, Se and N.

174. The compound according to claim 172, wherein R 3 It is a cycloalkyl group.

175. The compound according to claim 173, wherein R 3 It is cyclopropyl.

176. The compound according to claim 172, wherein R 3 It is a 5-membered heteroaryl group having 1-3 independently selected cyclic heteroatoms chosen from O, S, Se and N.

177. The compound according to claim 175, wherein R 3 Selected from pyrrole, furan, thiophene, selenophene, imidazole, thiazole and oxazole.

178. The compound according to claim 172, wherein R 3 It is a 10-membered heteroaryl group having 1-3 independently selected cyclic heteroatoms chosen from O, S, Se and N.

179. The compound according to claim 177, wherein R 3 It is quinoline.

180. A compound of formula (Id): (Id) Or its stereoisomers, tautomers, or salts, wherein: X 2 -X 3 Independently selected from: hydrogen, halogen, or hydroxyl. X 4 It is an alkoxy group; The condition is X 2 -X 3 One of them is hydrogen.

181. The compound according to claim 179, wherein X 2 It is hydrogen and X 3 It is halogen.

182. The compound according to claim 180, wherein X 2 It is hydrogen and X 3 Selected from fluorine, chlorine, bromine or iodine.

183. The compound according to claim 181, wherein X 2 It is hydrogen and X 3 It's fluorine.

184. The compound according to claim 179, wherein X 2 It is hydrogen and X 3 It is a hydroxyl group.

185. The compound according to claim 179, wherein X 3 It is hydrogen and X 2 It is halogen.

186. The compound according to claim 184, wherein X 3 It is hydrogen and X 2 Selected from fluorine, chlorine, bromine or iodine.

187. The compound according to claim 185, wherein X 3 It is hydrogen and X 2 It's fluorine.

188. The compound according to claim 179, wherein X 3 It is hydrogen and X 2 It is a hydroxyl group.

189. A compound of formula (Ie): (Ie) Or its stereoisomers, tautomers, or salts, wherein: R 1 Selected from: or R 2 Selected from: .

190. The compound according to claim 188, wherein R 1 yes .

191. The compound according to claim 188, wherein R 1 yes .

192. The compound according to claim 188, wherein R 1 yes .

193. The compound according to claim 188, wherein R 1 yes .

194. The compound according to claim 188, wherein R 1 yes .

195. A compound, said compound being selected from: ， ， ， ， ， ， ， ， ， 。 196. A method for detecting luminescence in a sample, the method comprising: The sample is contacted with a luciferin analogue, wherein the luciferin analogue is a compound of formula (I) or a stereoisomer, tautomer, or salt thereof, a compound of formula (Ia), a compound of formula (Ib), a compound of formula (Ic), a compound of formula (Id), a compound of formula (Ie), or a compound as described in claim 194; and Detecting luminescence.

197. The method of claim 195, wherein the sample comprises luciferase, optionally wherein the luciferase is a protein according to any one of claims 1-85, 94-113 or a polypeptide according to any one of claims 86-93.

198. The method according to claim 195 or 196, wherein the sample contains live cells.

199. A method for detecting luminescence in transgenic animals, the method comprising: The administration of a luciferin analog to a transgenic animal, wherein the luciferin analog is a compound of formula (I) or a stereoisomer, tautomer, or salt thereof, a compound of formula (Ia), a compound of formula (Ib), a compound of formula (Ic), a compound of formula (Id), a compound of formula (Ie), or a compound as described in claim 194; and Detecting luminescence; The transgenic animal expressed luciferase, optionally wherein the luciferase is a protein according to any one of claims 1-85, 94-113 or a polypeptide according to any one of claims 86-93.

200. A method for determining luciferase activity using a protein, polypeptide component, fusion protein, nucleic acid, expression vector, host cell, and / or kit according to any of the preceding claims.

201. The method of claim 199, wherein the method comprises performing luminescent reporter assays, diagnostic assays, cell localization of a target, cell imaging, gene editing, animal imaging, cancer labeling, CAR-T cell reporting, secretion assays, gene delivery, and / or tissue engineering.

202. A solution for measuring the luciferase activity of a protein, said solution containing 1 mM-1000 mM imidazole.

203. The solution according to claim 201, wherein the pH of the solution is pH 6-pH 9, for example pH 7-pH 9 or pH 7.5-8.

5.

204. The solution according to claim 201 or 202, wherein the solution further comprises phosphate-buffered saline.

205. The solution according to any one of claims 201-203, wherein the solution comprises 5 mM-500 mM imidazole, 10 mM-1000 mM imidazole, 50 mM-1000 mM imidazole, 10 mM-500 mM imidazole, 50 mM-500 mM imidazole, 75 mM-250 mM imidazole, 75 mM-150 mM imidazole, 10 mM-250 mM imidazole, 10 mM-200 mM imidazole, or 5 mM-300 mM imidazole.

206. The solution according to any one of claims 201-204, wherein the solution further comprises a stabilizer.

207. The solution according to claim 205, wherein the stabilizer is ascorbic acid, glycine and / or propylene glycol.

208. The solution of claim 205, wherein the stabilizer is glycine, and optionally the solution comprises 200 mM-500 mM glycine or 200 mM-400 mM glycine.

209. The solution according to claim 206 or claim 207, wherein the stabilizer is propylene glycol, and optionally the solution comprises 0.1%-10% propylene glycol, 0.1%-0.3% propylene glycol, 0.3%-0.5% propylene glycol, 0.5%-0.8% propylene glycol, 0.3%-0.8% propylene glycol, 0.8%-1% propylene glycol, 1%-2% propylene glycol, 2%-3% propylene glycol, or 3%-5% propylene glycol.

210. The solution according to any one of claims 201-208, the solution further comprising a luciferin substrate, or a kit comprising the solution according to any one of claims 201-208 and the luciferin substrate.

211. The solution of claim 209 or the kit of claim 209, wherein the luciferin substrate is DTZ, a compound of formula (I), a compound of formula (Ia), a compound of formula (Ib), a compound of formula (Ic), a compound of formula (Id), a compound of formula (Ie), or a compound of claim 194.

212. A kit comprising: a compound of formula (I), a compound of formula (Ia), a compound of formula (Ib), a compound of formula (Ic), a compound of formula (Id), a compound of formula (Ie), or a compound according to claim 194, wherein the kit optionally further comprises a luciferase and / or an assay buffer, wherein the luciferase is a multiparticulate protein comprising two or three self-complementary components, and / or the assay buffer is a solution according to any one of claims 201-210.

213. A kit comprising: a nucleic acid containing a nucleotide sequence encoding a protein according to any one of claims 1-82, 112-113, 117-136, 141-142, a first polypeptide component according to any one of claims 83-85, or a second polypeptide component according to any one of claims 83-85; and optionally an assay buffer.

214. The kit according to claim 212, wherein the nucleic acid is the nucleic acid according to claim 114, claim 143, or the expression vector according to claim 144.

215. The kit according to claim 212, wherein the nucleic acid is present in the expression vector.

216. The kit according to claim 212, wherein the nucleic acid is the first nucleic acid or the second nucleic acid according to any one of claims 146-149, optionally wherein the first nucleic acid or the second nucleic acid is in an expression vector.

217. The kit of claim 212, wherein the kit comprises a first nucleic acid and a second nucleic acid according to any one of claims 146-149, optionally wherein the first nucleic acid is in a first expression vector and the second nucleic acid is in a second expression vector.

218. The kit according to any one of claims 212-216, wherein the kit comprises the compound according to any one of claims 153-194.

219. The kit according to claim 217, wherein the compound is compound 1c.

220. The kit according to any one of claims 212-218, wherein the kit comprises an assay buffer.

221. The kit of claim 219, wherein the assay buffer comprises phosphate-buffered saline.

222. The kit according to claim 219 or claim 220, wherein the assay buffer comprises imidazole.

223. The kit of claim 221, wherein the assay buffer comprises 5 mM-500 mM imidazole, 10 mM-1000 mM imidazole, 50 mM-1000 mM imidazole, 10 mM-500 mM imidazole, 50 mM-500 mM imidazole, 75 mM-250 mM imidazole, 75 mM-150 mM imidazole, 10 mM-250 mM imidazole, or 5 mM-300 mM imidazole.

224. The kit according to any one of claims 219-222, wherein the assay buffer contains a stabilizer.

225. The kit according to claim 223, wherein the stabilizer is ascorbic acid, glycine and / or propylene glycol.

226. The kit according to claim 223 or 224, wherein the stabilizer is glycine, and optionally wherein the solution contains 200 mM-500 mM glycine.

227. The kit according to any one of claims 223-225, wherein the stabilizer is propylene glycol, and optionally the assay buffer comprises 0.1%-10% propylene glycol, 0.1%-0.3% propylene glycol, 0.3%-0.5% propylene glycol, 0.5%-0.8% propylene glycol, 0.8%-1% propylene glycol, 1%-2% propylene glycol, 2%-3% propylene glycol, or 3%-5% propylene glycol.

228. The kit according to any one of claims 212-226, wherein the nucleic acid is in lyophilized form; the kit according to any one of claims 217-218, wherein the compound is in lyophilized form; and / or the kit according to any one of claims 219-226, wherein the assay buffer is in lyophilized form.

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