De novo designed luciferase

Abstract

Disclosed is a protein having luciferase activity, which has a secondary structural arrangement of H1-L1-H2-L2-E1-L3-E2-L4-H3-L5-E3-L6-L7-E5-L8-E6 wherein "H" is a helical domain, "L" is a cyclic domain, and "E" is a beta chain domain; wherein: (a) the H1 domain has a length of at least 18 or 19 amino acids; the residue 14 of the H1 domain is Y, D or E, and the residue 18 of the H1 domain is D or E; (b) the E3 domain has a length of at least 6, 7, 8, 9 or 10 amino acids, and residue 2 of the E3 domain is R; and (c) the length of the E5 domain is at least 10, 11, 12, 13 or 14 amino acids, and the residue 9 of the E5 domain is H or M.

Description

[0001] Citation of Related Applications

[0002] This application claims priority to U.S. Provisional Patent Application Serial No. 63 / 300,171 filed on January 17, 2022 and U.S. Provisional Patent Application Serial No. 63 / 381,922 filed on November 1, 2022, each of which is incorporated herein by reference in its entirety.

[0003] Federal Funding Statement

[0004] This invention was made with government support under Grant No. K99EB031913 awarded by the National Institutes of Health. The U.S. Government has certain rights in this invention.

[0005] Sequence Listing Statement

[0006] A computer-readable form of the sequence listing is submitted electronically with this application and is incorporated by reference in its entirety. The sequence listing is contained in a file created on January 8, 2023, with the file name "21-1622-WO.xml" and a size of 638 kb. Background Art

[0007] Bioluminescence generated by enzymatic oxidation of luciferin substrates is widely used for bioassays and imaging in biomedical research. Since no excitation light source is required, luminescent photons are generated in the dark, which results in higher sensitivity than fluorescence imaging in live animal models and biological samples where autofluorescence or phototoxicity is a problem. However, the development of luciferases as molecular probes has lagged behind the mature fluorescent protein toolkit for a number of reasons: (i) very few natural luciferases have been identified; (ii) many of the identified luciferases require multiple disulfide bonds to stabilize their structure and are therefore prone to misfolding in mammalian cells; (iii) most natural luciferases do not recognize synthetic luciferins with more desirable photophysical properties; and (iv) multiplexed imaging using mutually orthogonal luciferase-luciferin pairs to track multiple processes in parallel is limited by the low substrate specificity of natural luciferases. Summary of the invention

[0008] In one aspect, the present disclosure provides a protein having luciferase activity comprising a secondary structure arrangement H1-L1-H2-L2-E1-L3-E2-L4-H3-L5-E3-L6-E4-L7-E5-L8-E6, wherein "H" is a helical domain, "L" is a loop domain, and "E" is a beta strand domain; wherein:

[0009] (a) 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 18 of the H1 domain is D or E;

[0010] (b) the E3 domain is at least 6, 7, 8, 9 or 10 amino acids in length and residue 2 of the E3 domain is R; and

[0011] (c) the E5 domain is at least 10, 11, 12, 13 or 14 amino acids in length, and residue 9 of the E5 domain is H or N.

[0012] In various embodiments, residue 7 of the E5 domain is M; the length of the E6 domain is at least 9, 10, 11, 12 or 13 amino acids, and wherein residue 5 of the E6 domain is V; residue 1 of the L5 domain is S; residue 7 of the E5 domain is M and residue 5 of the E6 domain is V; and / or residue 7 of the E5 domain is M, residue 5 of the E6 domain is V, and residue 1 of the L5 domain is S.

[0013] In other embodiments, the H2 domain is at least 5, 6 or 7 amino acids in length, the H3 domain is at least 9, 10, 11, 12, 13 or 14 amino acids in length, the E1 domain is at least 3 or 4 amino acids in length, the E2 domain is at least 3 or 4 amino acids in length, and / or the E4 domain is at least 8, 9, 10, 11 or 12 amino acids in length.

[0014] In one embodiment, 1, 2, 3, 4 or all 5 of the following conditions are met:

[0015] (a) Residue 13 of domain H1 is F;

[0016] (b) residue 1 of domain L3 is W;

[0017] (c) residue 5 of domain E5 is V or another hydrophobic residue;

[0018] (d) residue 8 of domain E5 is A or L or another hydrophobic residue; and / or

[0019] (e) Residue 11 of domain E5 is W.

[0020] In another embodiment, 1, 2, 3, 4, 5 or all 6 of the following conditions are met:

[0021] (a) residue 2 of domain E1 is I or another hydrophobic residue;

[0022] (b) residue 4 of domain H3 is F;

[0023] (c) residue 6 of domain E4 is V or another hydrophobic residue;

[0024] (d) residue 8 of domain E4 is L or another hydrophobic residue;

[0025] (e) residue 5 of domain E6 is M or V or another hydrophobic residue; and / or

[0026] (f) Residue 7 of domain E6 is V or another hydrophobic residue.

[0027] In additional embodiments, the protein 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 an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-181 or SEQ ID NOs: 1-3. In another embodiment, the protein comprises the amino acid sequence of SEQ ID NO: 4.

[0028] In one aspect, the disclosure provides 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%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 1, wherein:

[0029] residue 14 is Y, D or E, and residue 98 is H or N; and

[0030] Residue 18 is D or E, and residue 65 is R.

[0031] In various embodiments, the protein comprises one or both of A96M and M110V substitutions relative to SEQ ID NO: 1; comprises both of A96M and M110V substitutions relative to SEQ ID NO: 1; and / or comprises R60S substitution relative to SEQ ID NO: 1; comprises R60S, A96M and M110V substitutions relative to SEQ ID NO: 1. In other embodiments, the protein comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to an amino acid sequence selected from the group consisting of: SEQ ID NO: 1-3 or 1-181.

[0032] In another aspect, the present disclosure provides a protein comprising the formula X1-Z1-X2-Z2-X3-Z3-X4-Z4-X5-Z5-X6-Z6-X7-Z7-X8-Z8, wherein:

[0033] X1 has the same RRF as MSEEQIRQFL Y an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to EALD (SEQ ID NO: 182) wherein residue 14 is Y, D or E and residue 18 is D or E;

[0034] X2 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 of ADTAASLF (SEQ ID NO: 183);

[0035] X3 has an amino acid sequence that is at least 50%, 75%, or 100% identical to the amino acid sequence of TIHL (SEQ ID NO: 184);

[0036] X4 has an amino acid sequence that is at least 33%, 66%, or 100% identical to the amino acid sequence of VTF;

[0037] X5 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 of EEFR EWFERLFST (SEQ ID NO: 185);

[0038] X6 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 of QREIKSL EVR (SEQ ID NO: 186), wherein residue 2 is R;

[0039] X7 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 of VEVH VQLHATH (SEQ ID NO: 187);

[0040] X8 has KHTVDAT H an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to HW HFR (SEQ ID NO: 188) wherein residue 8 is H or N;

[0041] X9 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 of VTEM RVHINPTG (SEQ ID NO: 189); and

[0042] wherein Z1, Z2, Z3, Z4, Z5, Z6, Z7 and Z8 are independently present or absent, and when present may comprise any amino acid sequence.

[0043] In another embodiment, the disclosure provides a self-complementary multipart protein having luciferase activity, comprising at least a first polypeptide component and a second polypeptide component, wherein at least the first polypeptide component and the second polypeptide component are not covalently linked, wherein at least the first polypeptide component and the second polypeptide component collectively comprise the domains X1-Z1-X2-Z2-X3-Z3-X4-Z4-X5-Z5-X6-Z6-X7-Z7-X8-Z8-X9, wherein each domain is as defined herein;

[0044] wherein (a) each X domain is entirely present in one polypeptide component of at least the first polypeptide component and the second polypeptide component, and (b) at least the first polypeptide component and the second polypeptide component do not include all of X1, X2, X3, X4, X5, X6, X7, X8 and X9.

[0045] In additional embodiments, the present disclosure provides a self-complementary multi-part protein having luciferase activity, comprising at least a first polypeptide component and a second polypeptide component, wherein at least the first polypeptide component and the second polypeptide component are not covalently linked, wherein at least the first polypeptide component and the second polypeptide component collectively comprise a secondary structure arrangement H1-L1-H2-L2-E1-L3-E2-L4-H3-L5-E3-L6-E4-L7-E5-L8-E6, wherein each domain is as defined herein.

[0046] In additional embodiments, the present disclosure provides a protein having luciferase activity comprising a secondary structure arrangement H1-L1-H2-L2-E1-L3-E2-L4-H3-L5-E3-L6-E4-L7-E5-L8-E6, wherein "H" is a helical domain, "L" is a loop domain, and "E" is a beta strand domain; wherein:

[0047] (a) 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 18 of the H1 domain is D or E;

[0048] (b) the E3 domain is at least 6, 7, 8, 9 or 10 amino acids in length and residue 2 of the E3 domain is R; and

[0049] (c) the E5 domain is at least 10, 11, 12, 13 or 14 amino acids in length, and residue 9 of the E5 domain is H or N.

[0050] The present disclosure also provides a fusion protein comprising:

[0051] (a) the protein or polypeptide component of any embodiment; and

[0052] (b) one or more additional functional domains.

[0053] The present disclosure further provides nucleic acids encoding proteins, polypeptide components or fusion proteins of the present disclosure, expression vectors comprising nucleic acids operably linked to suitable control elements, host cells comprising proteins, polypeptide components, fusion proteins, nucleic acids and / or expression vectors of the present disclosure; and kits comprising proteins, polypeptide components, fusion proteins, nucleic acids, expression vectors and / or host cells of the present disclosure; and instructions for use thereof. The present disclosure also provides methods of using proteins, polypeptide components, fusion proteins, nucleic acids, expression vectors, host cells and / or kits of the present disclosure. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] Figure 1: Generation of idealized scaffolds and computational design of de novo luciferase. (a) Family-wide hallucination. Sequences encoding proteins with desired topology were optimized by Monte Carlo sampling with a multicomponent loss function. Structurally conserved regions were evaluated based on consistency with input residue-residue distance and orientation distributions obtained from experimental structures of 85 NTF2-like proteins, while variable non-ideal regions were evaluated based on the confidence of predicted inter-residue geometry calculated as the KL-divergence between network predictions and background distributions. Sequence-space MCMC sampling combined both sequence alterations and insertions / deletions (see Methods) to guide hallucination sequences to encode structures with desired folds. Hydrogen-bond networks were incorporated into the designed structures to increase structural specificity. (bd) Design of the luciferase active site. (b) Generation of DTZ conformers using AIMNet. (c) Generation of rotamer interaction fields (RIFs) to stabilize anionic DTZ and form hydrophobic packing interactions around DTZ conformers. (d) RIF was docked into the hallucination scaffolds and substrate-scaffold interactions were optimized using position-specific scoring matrix (PSSM) biased sequence design. (e) NTF2 topology selection. RIF was docked into 4000 natural small molecule binding proteins, and proteins that bind the fluorescein substrate using more than 5 loop residues were excluded. Most of the best hits came from the NTF2-like protein superfamily. Using the whole-family hallucination scaffold generation protocol, we generated 1615 scaffolds and found that these scaffolds yielded better predicted RIF binding energies than natural proteins. (f) Scaffolds generated using whole-family hallucination sampled more of the natural structure space than scaffolds generated from previous blueprints and (g) had stronger sequence-structure relationships than natural or blueprint de novo NTF2 scaffolds.

[0055] Figure 2. Biophysical characterization of LuxSit. (a) Coomassie-stained SDS-PAGE of purified recombinant LuxSit from E. coli. (b) Size exclusion chromatography of purified LuxSit showing monodispersity and monomeric properties. (c) Far-UV CD spectra at 25°C, 95°C, and cooling back to 25°C. Inset: CD melting curve of LuxSit at 220 nm. (d) Luminescence emission spectra of DTZ in the presence and absence of LuxSit. (e) Structural alignment of the designed model and the AlphaFold2 predicted model, which are highly consistent at both the backbone (left) and side chain levels (right). (fi) Site-saturation mutagenesis of substrate-interacting residues. A zoomed-in view of the designed and AlphaFold2 models at the side chain level (left) illustrates the designed enzyme-substrate interactions, including: (f), Tyr14-His98 core HBNet, (g), Asp18-Arg65 dyad, (h), π-stacking, and i, hydrophobic stacking residues. The sequence profile (right) is scaled according to the activity of different sequence variants: (activity of the specified amino acid) / (sum of activities of all tested amino acids at that position). Substitutions that increase activity (Ala96 and Met110) are highlighted.

[0056] Figure 3. Characterization of luciferase activity in vitro and in human cells. (a) Substrate concentration dependence of LuxSit, LuxSit-f, and LuxSit-i activities. Numbers indicate V max (b) Fluorescence and luminescence imaging of live HEK293T cells transiently expressing LuxSit-i-mTagBFP2; LuxSit-i activity can be detected at single-cell resolution. Left: Fluorescence channel representing the mTagBFP2 signal. Right: Total luminescence photons collected during a 10-second exposure. Inset: Negative control, untransfected cells with DTZ. Luminescence images were acquired immediately after the addition of 25 μM DTZ in the absence of excitation light. Scale bar: 20 μm. 40x.

[0057] Figure 4. High substrate specificity of designed luciferases allows multiplexed bioassays. (a) Chemical structures of coelenterazine substrate analogs. (b) Activity of LuxSit-i on selected luciferin substrates. Luminescence images (upper panel) and signal quantification (lower panel) of indicated substrates in the presence of 100 nM LuxSit-i. LuxSit-i has high specificity for the designed target substrate DTZ. (c) Heatmap visualization of substrate specificity of LuxSit-i, Renilla luciferase (RLuc), Gaussia luciferase (GLuc), and engineered NLuc from Oplophorus luciferase. The heatmap shows the luminescence of each enzyme on each substrate; on a per enzyme basis, values ​​are normalized to the highest signal of that enzyme on all substrates. (d) Luminescence emission spectra of LuxSit-i / DTZ and RLuc / PP-CTZ can be spectrally resolved by 528 / 20 and 390 / 35 filters (shown as dashed lines), and only the cognate substrates are recognized. (e) Schematic diagram of the multiplex luciferase assay. HEK293T cells transiently transfected with CRE-RLuc, NFkB-LuxSit-i and CMV-CyOFP plasmids were treated with forskolin or human tumor necrosis factor α (TNFα) to induce the expression of labeled luciferases. (fg) The luminescent signal from the cells can be measured by a microplate reader under substrate resolution or spectral resolution methods. (f) For the substrate resolution method, the luminescence intensity was recorded without a filter after adding PP-CTZ or DTZ. (g) For the spectral resolution method, both PP-CTZ and DTZ were added, and the signal was acquired using 528 / 20 and 390 / 35 filters at the same time. In (f) and (g), the lower figure indicates that forskolin or TNFα was added. From CelLytic TM Luminescence signals were obtained from lysates of 15,000 cells in reagent M, and CyOFP fluorescence signals were used to normalize cell number and transfection efficiency. All data were normalized to the corresponding non-stimulated control. Data are expressed as mean ± SD (n = 3).

[0058] Figure 5 .Proposed catalytic mechanism of luciferase using coelenterazine. Density functional theory (DFT) calculations show that the formation of the anionic state is a triplet oxygen ( 3 O 2 ) is the necessary electron source for activation. 26,27 and experimental evidence 28,29 Supported by both, the next oxidation process is likely to be via a single electron transfer (SET) mechanism, in which the surrounding reaction field can greatly affect the Gibbs free energy (ΔG SET) changes. Finally, the thermal decomposition of the dioxetane light emitter intermediate can produce photons via a gradually reversible charge transfer induced emission (GRCTIL) mechanism (which is usually exothermic). Because all historical evidence is based on calculations in virtual solvents or chemiluminescence in ideal organic solvents. The detailed mechanism of the luminescent reaction catalyzed by luciferase remains unclear. We propose that the key steps of the enzyme are to promote the formation of anionic states and create a suitable environment to promote efficient SET. Therefore, the goal of this study is to design an enzyme reaction field around the substrate to stabilize the anionic substrate state and change the local proton activity, solvent polarity and hydrophobicity to effectively activate 3 O 2 .

[0059] Figure 6 Schematic diagram of colony-based luciferase screening. Computationally designed DNA sequences are provided in oligo arrays, where fragments are amplified by PCR, assembled, and ligated into the pBAD bacterial expression vector. The plasmid library is used to transform DH10B cells. Each colony grown on an LB agar plate represents one luciferase design. The plate is sprayed with DTZ solution and the ChemiDoc TM Imager to identify active colonies. All active colonies are plated in 96-well plates, expressed and purified to confirm individual luciferase activity. Selected plasmids can then be sequenced to indicate active design models that provide insights into design principles and enzyme function, or can be subjected to random mutagenesis for further evolution. Inset: Three luciferases were identified from this screen. We call the most active and DTZ-specific luciferase "LuxSit."

[0060] Figure 7. Expression, purification and structural characterization of LuxSit variants. (ac) Recombinant expression of (a) LuxSit, (b) LuxSit-i and (c) LuxSit-f in E. coli. Annotation of each lane is as follows - 1: before IPTG; 2: after IPTG; 3: soluble lysate; 4: flow-through; 5: wash; 6: elution; 7: post-TEV lysis; 8: post-SEC. (df) Size exclusion chromatograms of purified (d) LuxSit; (e) LuxSit-i; and (f) LuxSit-f monomers. (gi) Deconvoluted mass spectra of (g) LuxSit, (h) LuxSit-i and (i) LuxSit-f. (jk) Far-UV circular dichroism (CD) spectra of (j) LuxSit-i; and (k) LuxSit-f at 25°C, 95°C and when cooled back to 25°C (left). CD melting curve at 220 nm (right). (l) When LuxSit-i was concentrated to high concentration (about 50 μM) in Tris pH 8.0 buffer, a dimer SEC peak was observed. Both the dimer and monomer SEC fractions showed the expected size on SDS PAGE, and in the presence of 25 μM DTZ, both peaks were catalytically active and luminescent.

[0061] Fig. 8. Random NNK library was screened at 60, 96 and 110 positions, and sequence alignment of LuxSit and its variants was performed. We generated a completely randomized library at 60, 96 and 110 positions to thoroughly screen all possible combinations. After colony-based screening, we identified many colonies with strong luciferase activity with DTZ. Each colony was expressed separately in each well (1 mL culture) of a 96-well plate and purified accordingly (see method). (a) The single luminescent activity of each selected mutant was plotted and compared with the parent LuxSit. Luminescent activity was measured in the presence of 25 μM DTZ. Luminescent activity (RLU) is shown as the integrated signal for the first 15 minutes. Statistical analysis of the amino acid frequency at residues (b) 60, (c) 96 and (d) 110 relative to luciferase activity. In all selected mutants, Arg60 was confirmed to be variable because Arg60 may be less well defined structurally because it is derived from a ring and has no hydrogen bond partner. Ala96 prefers larger side chains (Leu, Ile, Met and Cys), and Met110 prefers hydrophobic residues (Val, Ile and Ala). The newly discovered variant (R60S / A96L / M110V) has a photon flux more than 100 times higher than LuxSit and was named LuxSit-i for its high brightness.

[0062] Fig. 9.Sequence alignment of Lux-Sit (SEQ ID NO: 1), Lux-Sit-i (SEQ ID NO: 2) and Lux-Sit-f (SEQ ID NO: 3). In the sequence alignment, mutations are highlighted. The conserved catalytic dyad of Asp18-Arg65 and Tyr14-His98 is shown.

[0063] Figure 10. Additional features of LuxSit variants. (a) Normalized emission kinetics of 15,000 intact HeLa cells expressing LuxSit-i, 100nM purified LuxSit-i or 100nM purified LuxSit-f in the presence of 50μM DTZ. The more extensive emission kinetics in HeLa cells may be due to the diffusion rate of DTZ across the cell membrane. (b) Normalized luminescence decay curves of LuxSit-i in various pH buffers reveal a pH-dependent catalytic mechanism. (c) Luminescence quantum yields were estimated from the integrated luminescence signal until 125pmol substrate was completely converted into photons in the presence of 50nM corresponding luciferase (see Methods). All data points are plotted as the average of three measurements.

[0064] Figure 11. Expression, localization and luminescent activity of LuxSit-i in live HEK293T and HeLa cells. (ab) Fluorescence imaging of live (a) HEK293T and (b) HeLa cells expressing LuxSit-i-mTagBFP2, which is non-targeted or localized to the nuclear (histone 2B), plasma membrane (KRasCAAX) or mitochondrial (DAKAP) cell compartments. Scale bar: 10 μm. (cd) Luminescent signals were measured with 15,000 intact (c) HEK293T or (d) HeLa cells in the presence of 25 μM DTZ in DPBS. Transfection efficiency ranged from 60%-70% for HEK293T cells and from 5%-10% for HeLa cells. (e) The luminescence emission spectrum obtained from HEK293T cells expressing LuxSit-i is consistent with the emission spectrum of recombinant LuxSit-i purified from E. coli. (fg) The luminescence signal was measured with 15,000 (f) HEK293T cells expressing complete LuxSit-i or (g) cell lysates in the presence of 25 μM of the indicated substrate in DPBS. The luminescence intensity was normalized to the DTZ signal, showing higher DTZ specificity than other substrates in cell-based assays. The data are shown as the total luminescence signal for the first 20 minutes and are technically repeated three times. (h) Normalized luminescence intensity distribution diagram of the line of different cells (n=10) passing through the main luminescence image of Figure 3b; the line represents untransfected cells. The error bars represent ±SEM.

[0065] Figure 12. Substrate specificity of LuxSit-i and spectrally resolved luciferase-luciferin pairs allows multiplexed bioassays. (a) Orthogonal relationship between LuxSit-i-DTZ and RLuc-PP-CTZ (Prolume Purple, methoxy-coelenterazine) luminescent pairs. Indicated amounts of each luciferase were mixed in different ratios, totaling 100%. (b) After adding 25 μM of both substrates DTZ and PP-CTZ, filtered light from 528 / 20 and 390 / 35 was measured simultaneously. Data are expressed as mean ± SD (n = 3). The heat map shows the luminescent signal of a single luciferase (100 nM) or a 1:1 mixture in the presence of homologous or non-homologous (DTZ or PP-CTZ or both) substrates. The response signal is obtained by Neo2 with 528 / 20 and 390 / 35 filters. TM The microplate reader was used to acquire the data simultaneously. (c) Multiplex luciferase assay in live HEK293T cells after co-transfection of CRE-RLuc, NFκB-LuxSit-i and CMV-CyOFP plasmids and stimulation with forskolin (FSK) or human tumor necrosis factor α (TNFα). (d, e) After addition of DTZ, PP-CTZ or both DTZ and PP-CTZ in DPBS, 15,000 intact cells were assayed by (d) substrate resolution or (e) spectral resolution mode without cell lysis (see Methods). Area scans of CyOFP fluorescence signals were used to estimate cell number and transfection efficiency. The reported units are RLU / au; Ex. / Em. = relative light units / fluorescence intensity measurement at 480 / 580nm. All data were normalized to the corresponding non-stimulated control. Data are expressed as mean ± SD (n = 3).

[0066] Fig.13 . Shows the secondary structure mapped onto an exemplary protein of the present disclosure (SEQ ID NO: 1). DETAILED DESCRIPTION

[0067] All references cited are incorporated herein by reference in their entirety.

[0068] As used herein, the unused articles "the" and "the" include plural referents unless the context clearly dictates otherwise.

[0069] As used herein, amino acid residues are abbreviated 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 (Ile; 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).

[0070] In all embodiments of the polypeptides disclosed herein, any N-terminal methionine residue is optional (ie, the N-terminal methionine residue may be present or may be deleted).

[0071] Unless the context clearly dictates otherwise, all embodiments of any aspect of the disclosure can be used in combination.

[0072] Unless the context clearly requires otherwise, throughout the specification and claims, the words "comprising," "including," and the like are to be interpreted in an inclusive sense and not in an exclusive or exhaustive sense; that is, in the sense of "including but not limited to." Words using the singular or plural number also include the plural and singular, respectively. In addition, the words "herein," "above," and "below," and words of similar meaning, when used in this application, shall refer to this application as a whole and not to any particular portions of this application.

[0073] In a first aspect, the present disclosure provides a protein having luciferase activity, comprising a secondary structure arrangement H1-L1-H2-L2-E1-L3-E2-L4-H3-L5-E3-L6-E4-L7-E5-L8-E6, wherein "H" is a helical domain, "L" is a loop domain, and "E" is a beta strand domain; wherein:

[0074] (a) 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 18 of the H1 domain is D or E;

[0075] (b) the E3 domain is at least 6, 7, 8, 9 or 10 amino acids in length and residue 2 of the E3 domain is R; and

[0076] (c) the E5 domain is at least 10, 11, 12, 13 or 14 amino acids in length, and residue 9 of the E5 domain is H or N.

[0077] As disclosed in the Examples below, the proteins of the present disclosure are non-naturally occurring, have luciferase activity, and share this enumerated secondary structure arrangement. Fig.13 The alignment of the amino acid sequence with respect to SEQ ID NO: 1 is shown. The inventors have conducted extensive studies to evaluate the key residues in the polypeptide for maintaining luciferase activity and have prepared a number of modified forms of the polypeptide, as detailed in SEQ ID NO: 4 and in the Examples below. As described below and in the Examples, the required amino acids mentioned above are those involved in the catalytic dyad.

[0078] Unless otherwise specified, the various domains may be any suitable length.

[0079] In one embodiment, residue 7 of the E5 domain is M. In another embodiment, the length of the E6 domain is at least 9, 10, 11, 12 or 13 amino acids, and residue 5 of the E6 domain is V. In another embodiment, residue 1 of the L5 domain is S. In one embodiment, residue 7 of the E5 domain is M and residue 5 of the E6 domain is V. In another domain, residue 7 of the E5 domain is M, residue 5 of the E6 domain is V, and residue 1 of the L5 domain is S. In another embodiment, 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 E1 domain is at least 3 or 4 amino acids, the length of the E2 domain is at least 3 or 4 amino acids, and the length of the E4 domain is at least 8, 9, 10, 11 or 12 amino acids. In all of these embodiments, one or more of the listed domains may independently include amino acid residues. In one embodiment, one or more of the domains may independently include an additional 1, 2, 3, 4 or 5 residues.

[0080] In one embodiment:

[0081] The H1 domain is 19 amino acids long;

[0082] The H2 domain is 7 amino acids long;

[0083] The E1 domain is 4 amino acids in length;

[0084] The E2 domain is 4 amino acids in length;

[0085] The H3 domain is 14 amino acids in length;

[0086] The E3 domain is 10 amino acids in length;

[0087] The E4 domain is 12 amino acids in length;

[0088] The E5 domain is 14 amino acids in length; and

[0089] The E6 domain is 12 or 13 amino acids in length.

[0090] The loop domain may be of any length and may include insertion of any 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.

[0091] In another embodiment, the protein 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 an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-3, wherein residues in brackets are optional and may be present or deleted.

[0092] SEQ ID NO: 1 is the Lux-Sit construct disclosed herein. Fig.13 The domain structure mapped onto the amino acid sequence of SEQ ID NO: 1 is shown.

[0093]

[0094] SEQ ID NO: 2 is the Lux-Sit-i construct disclosed herein.

[0095]

[0096] SEQ ID NO: 3 is the Lux-Sit-f construct disclosed herein.

[0097]

[0098] In each of the annotated sequences shown in SEQ ID NOs: 1-3:

[0099] (a) Positions in bold and underlined with increased font size are dyad 1 (catalytic residues) Y14 (H1 domain residue 14) + H98 (E5 domain residue 9);

[0100] (b) Positions in bold and increased font size are dyad 2 (catalytic residues) D18 (H1 domain residue 9) + R65 (E3 domain residue 2);

[0101] (c) positions in increased font and non-bold are core packing (recognition residues) F13 (residue 13 of domain H1), I35 (residue 2 of domain E1), W38 (residue 1 of domain L3), F49 (residue 4 of domain H3), V81 (residue 6 of domain E4), L83 (residue 8 of domain E4), V94 (residue 5 of domain E5), A / L 97 ​​(residue 8 of domain E5), W100 (residue 11 of domain E5), M / V110 (residue 5 of domain E6), V112 (residue 7 of domain E6); and

[0102] (d) Underlined and non-bold positions are regions for cleavage enzymes or insertion of other functional domains (loop domains or immediately adjacent regions).

[0103] In some embodiments of the protein, 1, 2, 3, 4 or all 5 of the following conditions are met:

[0104] (a) Residue 13 of domain H1 is F;

[0105] (b) residue 1 of domain L3 is W;

[0106] (c) residue 5 of domain E5 is V or another hydrophobic residue;

[0107] (d) residue 8 of domain E5 is A or L or another hydrophobic residue; and / or

[0108] (e) Residue 11 of domain E5 is W.

[0109] In other embodiments of the protein, the length of the H2 domain is 7 amino acids, the length of the H3 domain is 14 amino acids, the length of the E1 domain is 4 amino acids, the length of the E2 domain is 4 amino acids, and / or the length of the E4 domain is 12 amino acids. In further embodiments, 1, 2, 3, 4, 5 or all 6 of the following conditions are met:

[0110] (a) residue 2 of domain E1 is I or another hydrophobic residue;

[0111] (b) residue 4 of domain H3 is F;

[0112] (c) residue 6 of domain E4 is V or another hydrophobic residue;

[0113] (d) residue 8 of domain E4 is L or another hydrophobic residue;

[0114] (e) residue 5 of domain E6 is M or V or another hydrophobic residue; and / or

[0115] (f) Residue 7 of domain E6 is V or another hydrophobic residue.

[0116] In another embodiment, the protein comprises the amino acid sequence of SEQ ID NO:4.

[0117] SEQ ID NO:4

[0118]

[0119]

[0120]

[0121]

[0122] In another embodiment, the protein comprises an amino acid sequence having 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% identity to an amino acid selected from the group consisting of SEQ ID NOs: 1-181, as shown in Table 1. SEQ ID NOs: 5-181 in Table 1 are amino acid sequences redesigned based on LuxSit-i (SEQ ID NO: 2), showing their luciferase activity.

[0123] Table 1

[0124]

[0125]

[0126]

[0127]

[0128]

[0129]

[0130]

[0131]

[0132]

[0133]

[0134]

[0135]

[0136]

[0137]

[0138]

[0139]

[0140]

[0141]

[0142]

[0143]

[0144]

[0145]

[0146]

[0147]

[0148]

[0149]

[0150]

[0151]

[0152]

[0153]

[0154]

[0155]

[0156]

[0157]

[0158]

[0159]

[0160]

[0161]

[0162]

[0163]

[0164]

[0165]

[0166]

[0167]

[0168]

[0169]

[0170]

[0171]

[0172]

[0173]

[0174]

[0175]

[0176]

[0177]

[0178]

[0179]

[0180]

[0181]

[0182]

[0183]

[0184]

[0185]

[0186]

[0187]

[0188]

[0189] In another aspect, the disclosure provides a protein having luciferase activity, comprising an amino acid sequence having 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% identity to SEQ ID NO: 1, wherein:

[0190] residue 14 is Y, D or E and residue 98 is H or N;

[0191] Residue 18 is D or E and residue 65 is R.

[0192] Proteins in this regard are non-naturally occurring. In one embodiment, the percent identity relative to a reference sequence is achieved by aligning the sequences using the Needleman-Wunsch algorithm (a sequence alignment tool commonly used by those skilled in the art that allows for insertions and deletions).

[0193] In one embodiment, the protein comprises one or both of A96M and M110V substitutions relative to SEQ ID NO: 1. In another embodiment, the protein comprises an R60S substitution relative to SEQ ID NO: 1. In an additional embodiment, the protein comprises an R60S, A96M and M110V substitution relative to SEQ ID NO: 1. In another embodiment, any substitution at residues F12, I35, W38, F49, V81, L83, V94, A97, W100, M110, V112 relative to SEQ ID NO: 1 is a conservative amino acid substitution. In one embodiment, the protein comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to an amino acid sequence selected from SEQ ID NO: 1-3. In another embodiment, the protein comprises an amino acid sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to an amino acid sequence selected from SEQ ID NOs: 1-181.

[0194] In another embodiment, the protein comprises the formula X1-Z1-X2-Z2-X3-Z3-X4-Z4-X5-Z5-X6-Z6-X7-Z7-X8-Z8, wherein:

[0195] X1 has the same Y an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to EALD (SEQ ID NO: 182) wherein residue 14 is Y, D or E and residue 18 is D or E;

[0196] X2 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 of ADTAASLF (SEQ ID NO: 183);

[0197] X3 has an amino acid sequence that is at least 50%, 75%, or 100% identical to the amino acid sequence of TIHL (SEQ ID NO: 184);

[0198] X4 has an amino acid sequence that is at least 33%, 66%, or 100% identical to the amino acid sequence of VTF;

[0199] X5 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 of EEFREWFERLFST (SEQ ID NO: 185);

[0200] X6 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 of QREIKSLEVR (SEQ ID NO: 186), wherein residue 2 is R;

[0201] X7 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 of VEVHVQLHATH (SEQ ID NO: 187);

[0202] X8 has KHTVDAT H an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to HWHFR (SEQ ID NO: 188) wherein residue 8 is H or N;

[0203] X9 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 of VTEMRVHINPTG (SEQ ID NO: 189); and

[0204] wherein Z1, Z2, Z3, Z4, Z5, Z6, Z7 and Z8 are independently present or absent, and when present may comprise any amino acid sequence.

[0205] In one embodiment, 1, 2, 3, 4, 5, 6, 7 or all 8 of the following conditions are met

[0206] Z1 includes SGD;

[0207] Z2 comprises HPGV (SEQ ID NO: 190);

[0208] Z3 includes WDG;

[0209] The Z4 includes TSR;

[0210] Z5 comprises RKDA (SEQ ID NO: 191);

[0211] Z6 contains GDT;

[0212] Z7 includes NGQ;

[0213] Z8 contains GNR; and

[0214] wherein 0, 1, 2, 3, 4, 5, 6, 7 or all 8 of Z1, Z2, Z3, Z4, Z5, Z6, Z7 and Z8 further comprise additional polypeptide domains.

[0215] In one embodiment of all proteins disclosed herein, the amino acid substitutions relative to the reference protein are conservative amino acid substitutions. As used herein, "conservative amino acid substitutions" means that a given amino acid can be replaced by a residue with similar physiochemical characteristics, such as replacing another with an aliphatic residue (such as replacing another with Ile, Val, Leu or Ala), or replacing another with a polar residue (such as between Lys and Arg; between Glu and Asp; or between Gln and Asn). Other such conservative substitutions (e.g., substitutions of entire regions with similar hydrophobic characteristics) are known. Proteins containing conservative amino acid substitutions can be tested in any of the assays described herein to confirm that the desired activity is retained. Amino acids can be grouped according to the similarity of their side chain properties (A Lehninger, Biochemistry, 2nd edition, pp. 73-75, Worth Publishers, New York (1975)): (1) non-polar: Ala (A), Val (V), Leu (L), Ile (I), Pro (P), Phe (F), Trp (W), Met (M); (2) uncharged polar: Gly (G), Ser (S), Thr (T), Cys (C), Tyr (Y), Asn (N), Gln (Q); (3) acidic: Asp (D), Glu (E); (4) basic: Lys (K), Arg (R), His (H). Alternatively, naturally occurring residues can be divided into groups based on common side chain properties: (1) hydrophobic: norleucine, Met, Ala, Val, Leu, Ile; (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues affecting chain orientation: Gly, Pro; (6) aromatic: Trp, Tyr, Phe. Non-conservative substitutions would entail exchanging a member of one of these classes for another. Specific conservative substitutions include, for example: Ala to Gly or to Ser; Arg to Lys; Asn to Gln or to His; Asp to Glu; Cys to Ser; Gln to Asn; Glu to Asp; Gly to Ala or to Pro; His to Asn or to Gln; Ile to Leu or to Val; Leu to Ile or to Val; Lys to Arg, to Gln or to Glu; Met to Leu, to Tyr or to Ile; Phe to Met, to Leu or to Tyr; Ser to Thr; Thr to Ser; Trp to Tyr; Tyr to Trp; and / or Phe to Val, to Ile or to Leu.

[0216] In another embodiment, the disclosure provides a self-complementary multipart protein having luciferase activity, 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, wherein the at least first polypeptide component and the second polypeptide component collectively comprise the domains X1-Z1-X2-Z2-X3-Z3-X4-Z4-X5-Z5-X6-Z6-X7-Z7-X8-Z8-X9, wherein each domain is as defined above; and

[0217] wherein (a) each X domain is entirely present in one polypeptide component of at least the first polypeptide component and the second polypeptide component, and (b) at least the first polypeptide component and the second polypeptide component do not include all of X1, X2, X3, X4, X5, X6, X7, X8 and X9.

[0218] Split protein is non-naturally occurring. Split protein comprises at least the first polypeptide component and the second polypeptide component, wherein the X domain is retained, and the split point is obtained only in the Z domain. In other words, each X chain or (X1, X2, X3, X4, X5, X6, X7, X8 and X9) is completely present in a polypeptide component of at least the first polypeptide component and the second polypeptide component, and the protein is split into separate components at the Z domain (Z1, Z2, Z3, Z4, Z5, Z6, Z7 and Z8), wherein the Z domain that splits may not exist, or may be partially present in one or both of the first and second polypeptide components. As a non-limiting example, in various embodiments of split luciferase protein, the first polypeptide component and the second polypeptide component may include components as illustrated in Table 2.

[0219] Table 2

[0220]

[0221]

[0222] In various embodiments, cleavage may occur at Z4, Z5, Z6 or Z7.

[0223] In another embodiment, the disclosure provides a self-complementary multipartite protein having luciferase activity, comprising at least a first polypeptide component and a second polypeptide component, wherein at least the first polypeptide component and the second polypeptide component are not covalently linked, wherein at least the first polypeptide component and the second polypeptide component collectively comprise a secondary structure arrangement H1-L1-H2-L2-E1-L3-E2-L4-H3-L5-E3-L6-E4-L7-E5-L8-E6, wherein each domain is as defined above;

[0224] wherein (a) each of the H and E domains is completely present in at least one of the first and second polypeptide components, and (b) at least the first and second polypeptide components do not include all of the H and E domains.

[0225] In this embodiment, the split protein comprises at least a first polypeptide component and a second polypeptide component, wherein the H and E domains are retained, and the cleavage point is only in the L domain. In various embodiments, the cleavage occurs at L4, L5, L6, L7 or L8.

[0226] The split proteins of these embodiments are active only when they are bound together and are therefore conditionally active.

[0227] In another embodiment, the present disclosure provides a fusion protein comprising:

[0228] (a) a protein or polypeptide component of any embodiment or combination of embodiments herein; and

[0229] (b) one or more additional functional domains.

[0230] As used herein, a "functional domain" is any polypeptide that can be effectively fused to a luciferase protein or split protein component of the present disclosure. As non-limiting examples, the one or more additional functional domains can include a diagnostic polypeptide, any protein that is desired to be localized in a cell, tissue, or organism; etc.

[0231] On the other hand, the present disclosure provides nucleic acids encoding proteins, protein components or fusion proteins of any embodiment or combination of embodiments of the present disclosure. The nucleic acid sequence may comprise single-stranded or double-stranded RNA (such as mRNA) or DNA or DNA-RNA hybrids in the form of genomic or cDNA, each of which may comprise chemically or biochemically modified, non-natural or derived nucleotide bases. Such nucleic acid sequences may comprise additional sequences for promoting expression and / or purification of the encoded polypeptide, including but not limited to polyA sequences, modified Kozak sequences, and sequences encoding epitope tags, export signals and secretion signals, nuclear localization signals, and plasma membrane localization signals. Based on the teachings herein, it is clear to those skilled in the art what kind of nucleic acid sequence will encode the polypeptide of the present disclosure. In various non-limiting embodiments, the nucleic acid may comprise the nucleotide sequence of any one of SEQ ID NO:200-380, wherein the residues in brackets are optional and may or may not be present.

[0232] On the other hand, the present disclosure provides expression vectors, which include nucleic acids of any aspect of the present disclosure operably connected to suitable control sequences. "Expression vector" includes a vector that is operably connected to any control sequence that can achieve expression of a gene product by a nucleic acid coding region or a gene. "Control sequence" operably connected to a nucleic acid sequence of the present disclosure is a nucleic acid sequence that can affect the expression of a nucleic acid molecule. Control sequences do not have to be adjacent to nucleic acid sequences, as long as the control sequences work to guide the expression of the nucleic acid sequences. Therefore, for example, there may be an intermediate untranslated but transcribed sequence between a promoter sequence and a nucleic acid sequence, and it may still be considered that the promoter sequence is "operably connected" to the coding sequence. Other such control sequences include, but are not limited to, polyadenylation signals, termination signals, and ribosome binding sites. Such expression vectors may be of any type, including but not limited to plasmids and viral-based expression vectors. The control sequences used to drive expression of the disclosed nucleic acid sequences in mammalian systems can be constitutive (driven by any of a variety of promoters, including but not limited to CMV, SV40, RSV, actin, EF) or inducible (driven by any of a number of inducible promoters, including but not limited to tetracycline, ecdysone, steroid responsiveness). The expression vector must be replicable in the host organism as an episome or by integration into the host chromosomal DNA. In various embodiments, the expression vector can include a plasmid, a virus-based vector, or any other suitable expression vector.

[0233] In another aspect, the present disclosure provides a host cell comprising a nucleic acid disclosed herein, an expression vector (i.e., attached or chromosomally integrated), a non-naturally occurring polypeptide, a fusion protein, or a composition, wherein the host cell can be prokaryotic or eukaryotic. Cells can be temporarily or stably engineered to incorporate a nucleic acid or expression vector disclosed herein using techniques including, but not limited to, bacterial transformation, calcium phosphate coprecipitation, electroporation, or liposome-mediated, DEAE dextran-mediated, polycation-mediated, or viral-mediated transfection.

[0234] The present disclosure also provides a kit, the kit comprising:

[0235] (a) the protein, polypeptide component, fusion protein, nucleic acid, expression vector and / or host cell of any embodiment or combination of embodiments herein; and

[0236] (b) Instructions for their use.

[0237] In one embodiment, the kit further comprises diphenyltetrazine (DTZ).

[0238] On the other hand, the present disclosure provides methods for using the protein, polypeptide component, fusion protein, nucleic acid, expression vector, host cell and / or kit as described in any preceding claim for any suitable purpose, including but not limited to using luminescent reporter assays, diagnostic assays, cellular localization of targets of interest, cell imaging, gene editing, live animal imaging, cancer markers, CART cell reporters, secretion assays, gene delivery, tissue engineering, etc. Additional details can be found in the Examples.

[0239] On the other hand, the present disclosure provides a method for preparing luciferase, which includes designing from scratch using the method of any embodiment disclosed herein, starting from a protein comprising the amino acid sequence of SEQ ID NO:381. The embodiment provides a detailed method for designing the luciferase of DTZ from scratch. As described in the embodiment, the method includes designing a catalytic site of complementary shape to stabilize the anionic state of DTZ and reduce the SET energy barrier, assuming that the downstream dioxetane light emitter thermal decomposition step is spontaneous. In order to stabilize the anionic species of DTZ, we focus on the placement position of the positively charged guanidine group of the arginine residue to interact with the anionic imidazopyrazinone core. In order to calculate and design such an active site, the inventor first uses AIMNet to generate a set of anionic DTZ conformers (Figure 1b). Next, around each conformer, the inventor uses the RIFgen method to enumerate the rotational isomer interaction field (RIF) on a 3D grid, which is composed of millions of amino acid side chain positions, forming hydrogen bonds and non-polar interactions with DTZ (Figure 1c). In addition, the inventors included an arginine guanidine group near the deprotonation site at the nitrogen (N1 atom) of the imidazopyrazinone in the RIF. Each DTZ conformer and the associated RIF were then docked in the central cavity of each scaffold using RIFdock to maximize protein-DTZ interactions. An average of 8 side chain rotational isomers (including arginine for stabilizing the anionic imidazopyrazinone core) were located in each pocket (Figure 1d). For the top 50,000 dockings with the most favorable side chain-DTZ interactions, the inventors optimized the remainder of the sequence (Figure 1d) for high affinity binding to DTZ, favoring naturally observed sequence variations to ensure foldability. During the design process, the predefined hydrogen bond network (HBNet) in the scaffold remained intact to ensure structural specificity and stability, and these HBNet side chain interactions with DTZ were explicitly required in the RIFdock step to ensure pre-organization of residues essential for catalysis. In the first sequence design stage, the identities of all RIF and HBNet residues were kept fixed, and the surrounding residues were optimized to preserve side chain-DTZ interactions and maintain structural specificity. In the second sequence design step, RIF residue identities (except arginine) were also allowed to vary to identify nonpolar and aromatic stacking interactions missed in RIF due to binning effects. During the sequence design process, the scaffold backbone, side chains, and DTZ substrate were allowed to relax in Cartesian space.

[0240] Example

[0241] Overview

[0242] De novo enzyme design has sought to introduce active sites and substrate binding pockets predicted to catalyze reactions of interest into geometrically compatible natural scaffolds. 1,2, but is limited by the lack of suitable protein structures and the complexity of natural protein sequence-structure relationships. Here, we describe a deep learning-based “whole-family hallucination” approach that generates a large number of idealized protein structures containing different pocket shapes and the designed sequences encoding them. We use these scaffolds to design artificial luciferases that selectively catalyze the oxidative chemiluminescence of the synthetic luciferin substrate diphenyltetrazine (DTZ) by placing the arginine guanidinium group in the highly shape-complementary binding pocket close to the anionic species formed during the reaction. 3 We obtained engineered luciferases with high selectivity for the two luciferin substrates; the most active was a small (13.9 kDa) and thermostable (T M >95℃) enzyme, its catalytic efficiency (k cat / K M =10 6 M -1 s -1 ) is comparable to native luciferase, but has much higher substrate specificity. Designing from scratch highly active and specific biocatalysts with broad applications in biomedicine is an important milestone in computational enzyme design, and our approach should enable the design of a wide range of new and useful luciferases and other enzymes.

[0243] Research

[0244] Bioluminescence, produced by the enzymatic oxidation of luciferin substrates, is widely used for imaging in bioassays and biomedical research. Because no excitation light source is required, luminescent photons are generated in the dark, which results in higher sensitivity than fluorescence imaging in live animal models and biological samples where autofluorescence or phototoxicity issues exist. 4,5 However, the development of luciferases as molecular probes has lagged behind the mature fluorescent protein toolkit for a number of reasons: (i) very few natural luciferases have been identified; (ii) many of the identified luciferases require multiple disulfide bonds to stabilize their structure and are therefore prone to misfolding in mammalian cells; (iii) most natural luciferases do not recognize synthetic luciferins with more desirable photophysical properties; and (iv) multiplexed imaging using mutually orthogonal luciferase-luciferin pairs to track multiple processes in parallel is limited by the low substrate specificity of natural luciferases.

[0245] We sought to use de novo protein design to create new luciferases that were small, highly stable, expressed well in cells, specific for one substrate, and did not require cofactors to function. Since we were limited to natural luciferase substrates, we chose the synthetic luciferin diphenyltetrazine (DTZ) as a target substrate because of its good quantum yield, red-shifted emission, and 3, good in vivo pharmacokinetics 14,15 , and lacks the cofactor required for emission. Previous computational enzyme design studies have mainly reused natural protein scaffolds from the PDB, but few natural structures have binding pockets suitable for DTZ, and the effects of sequence changes on natural proteins are unpredictable. To circumvent these limitations, we set out to generate a large number of ideal protein scaffolds that have pockets of suitable size and shape for DTZ and have clear sequence-structure relationships to facilitate subsequent active site integration. To identify protein folds that can accommodate such pockets, we first docked DTZ into 4000 natural small molecule binding proteins. We found that many NTF2 (nuclear transport factor 2)-like folds have binding pockets of complementary shape and size suitable for DTZ placement (Figure 1e), so the NTF2-like superfamily was selected as the target topology.

[0246] Whole family hallucination

[0247] Natural NTF2 structures have a range of pocket sizes and shapes, but also contain undesirable features, such as long loops that compromise stability. To create a large number of desirable NTF2-like structures, we developed a deep learning-based "whole-family hallucination" approach that integrates unconstrained de novo design. 19,20 and fixed backbone sequence design methods 21 , thus enabling the generation of an essentially unlimited number of proteins with a desired fold (Figure 1a). The whole family hallucination approach exploits unconstrained protein hallucinations 19,20 We used trRosetta's de novo sequence and structure discovery capabilities for loops and variable regions, and structure-guided sequence optimization for core regions. TM Structure Prediction Neural Network 22 , which efficiently identifies experimentally successful de novo designed proteins and imagines new globular proteins of different topologies. Starting with the sequences and predicted structures of 2,000 naturally occurring NTF2s, we used trRosetta TM Optimization of amino sequences in conserved core and variable loop regions. Protein core idealization was performed using a topologically specific loss function for geometry (see Methods) and variable loop optimization using core residues by optimizing sequence length and identity to maximize the neural network's confidence in the predicted structure. To further encode structural specificity, we integrated a buried long-range hydrogen bond network. The resulting 1615 full-family hallucinated NTF2 scaffolds provided DTZ with more shape-complementary binding pockets than natural small molecule protein binding proteins (Fig. 1e). This approach sampled protein backbones that were closer to natural NTF2-like proteins (Fig. 1f) and were more robust than previous non-deep learning approaches. 23 It has better scaffold quality indicators (Figure 1g).

[0248] We selected the NFT2 scaffold of SEQ ID NO: 381 from which to design the luciferase of DTZ, as described in detail below.

[0249] >2692_0_0.35_5_19_1806_2_0.45_5_Y14_H98_W100d1o7nb__clean_0001_D18_R65.rd1.pdb

[0250] MSEEEIRQFLRRFYEAFDKGDVDTFASLFHPGVTIHVWQGITFTSREELR EWVERFLRNFKDMQREILSLEVRGDTVEVHVQVHTTHNGQKYTFDVTHHW HFRGHRVTEIRVHVNPT(SEQ ID NO:381)

[0251] De novo design of DTZ luciferase

[0252] Standard computational enzyme design usually starts with an ideal active site or an enzyme consisting of protein functional groups around a reaction transition state and then extrapolates to a set of existing scaffolds. 1,2 However, the detailed mechanism of native marine luciferase has not been well determined, as only a few apo structures and no full structure have been solved. 24,25 (excluding calcium-regulated photoproteins). Quantum chemical calculations 26,27 And experimental data 28,29 All indicate that the chemiluminescent reaction proceeds through anionic species and that the polarity of the environment can significantly change the subsequent triplet molecular oxygen ( 3 O 2 ) of the single electron transfer (SET) process. In these data ( Figure 5 ), we sought to design a shape-complementary catalytic site that stabilizes the anionic state of DTZ and lowers the SET barrier, assuming that the downstream dioxetane photoemitter thermolysis step is spontaneous. To stabilize the anionic species of DTZ, we focused on the placement of the positively charged guanidinium group of the arginine residue to interact with the anionic imidazopyrazinone core.

[0253] To computationally engineer such active sites into a large set of hallucinated NTF2 scaffolds, we first used AIMNet 30 A set of anionic DTZ conformers was generated (Figure 1b). Next, around each conformer, we used the RIFgen method 31,32The rotamer interaction field (RIF) was enumerated on a 3D grid consisting of millions of amino acid side chain positions that form hydrogen bonds and non-polar interactions with DTZ (Figure 1c). In addition, we included the arginine guanidinium group near the deprotonation site at the nitrogen (N1 atom) of the imidazopyrazinone in the RIF. Each DTZ conformer and the associated RIF were then docked in the central cavity of each scaffold using RIFdock to maximize protein-DTZ interactions. An average of 8 side chain rotamers, including the arginine for stabilization of the anionic imidazopyrazinone core, were located in each pocket (Figure 1d). For the top 50,000 dockings with the most favorable side chain-DTZ interactions, we used RosettaDesign TM The remainder of the sequence (Fig. 1d) was optimized for high affinity binding to DTZ, with a bias towards naturally observed sequence variations to ensure foldability. During the design process, the predefined hydrogen bond network (HBNet) in the scaffold remained intact to ensure structural specificity and stability, and these HBNet side chain interactions with DTZ were explicitly required in the RIFdock step to ensure pre-organization of residues essential for catalysis. In the first sequence design stage, the identities of all RIF and HBNet residues remained fixed, and the surrounding residues were optimized to preserve side chain-DTZ interactions and maintain structural specificity. In the second sequence design step, RIF residue identities (except arginine) were also allowed to vary to identify non-polar and aromatic stacking interactions that were missed in RIF due to binning effects. During the sequence design process, the scaffold backbone, side chains, and DTZ substrate were allowed to relax in Cartesian space. After sequence optimization, the designs were screened based on ligand binding energy, protein-ligand hydrogen bonding, shape complementarity, and contact molecular surfaces, and 7982 designs were selected and ranked as pooled oligonucleotides for experimental screening.

[0254] Screening and characterization of DTZ-specific luciferase

[0255] Oligonucleotides encoding the two halves of each design were assembled into full-length genes and cloned into an E. coli expression vector (see Methods). Active luciferase colonies from the library were imaged directly using a colony-based screening method, and activity of selected clones was confirmed using 96-well plate expression ( Figure 6). Three active designs were identified; we called the most active of these LuxSit (Latin: let light exist); LuxSit is the smallest known luciferase, with 117 residues (13.9 kDa). Biochemical analyses, including SDS-PAGE and size exclusion chromatography (Figures 2ab and 7), showed that LuxSit was highly expressed, soluble, and monomeric from Escherichia coli expression. Circular dichroism (CD) spectroscopy showed a strong far-UV CD signature, indicating an organized α-β structure. CD melting experiments showed that the protein was not fully unfolded at 95°C and that the intact structure was restored when the temperature was lowered (Figure 2c). Incubation of LuxSit with DTZ resulted in luminescence with an emission peak at approximately 480 nm (Figure 2d), which is consistent with the DTZ chemiluminescence spectrum. Although we were unable to determine the crystal structure of LuxSit, AlphaFold2 33 Predicted structures at the skeleton level The side chains that interact with the substrate are very close to the designed model (Figure 2e). The designed LuxSit active site contains a Tyr14-His98 and Asp18-Arg65 dyad; the imidazole nitrogen atom of His98 forms hydrogen bond interactions with Tyr14 and the O1 atom of DTZ (Figure 2f). The center of the Arg65 guanidine cation is 1.3 m from the N1 atom of DTZ. Furthermore, Asp18 forms a bidentate hydrogen bond with the guanidinium group and backbone NH of Arg65 ( Figure 2g ).

[0256] Activity Optimization

[0257] To better understand the contribution of LuxSit (the most active of our luciferase designs) to catalysis, we constructed a site-saturation mutagenesis (SSM) library in which each mutation was performed one at a time at each pocket residue (see Methods). Figure 2f-i shows the amino acid preferences at key positions. Arg65 is highly conserved, and its dyad partner Asp18 can only be mutated to Glu (which reduces activity), indicating that the carboxylate-Arg65 hydrogen bond is important for luciferase activity. In the Tyr14-His98 dyad, Tyr14 can be replaced by Asp and Glu, while His98 can be replaced by Asn. Because all active variants have hydrogen bond donors and acceptors at these positions, the dyad may help mediate the electron and proton transfer required for luminescence. Hydrophobic (Figure 2h) and π stacking (Figure 2i) residues at the binding interface allow other aromatic or aliphatic substitutions, and generally prefer amino acids in the original design, which is consistent with the model-based affinity prediction of the mutation effect. Compared to LuxSit, the A96M and M110V mutants showed 16-fold and 19-fold increased activity, respectively (Table 4). Optimization guided by these results resulted in LuxSit-f (A96M / M110V) with strong initial flash emission and LuxSit-i (R60S / A96L / M110V) with a photon flux more than 100-fold higher than LuxSit ( Fig. 9 ). Overall, the active site saturation mutagenesis results support the design model, in which the Tyr14-His98 and Asp18-Arg65 dyads play key roles in catalysis and the substrate binding pocket is largely preserved.

[0258] The most active catalysts, LuxSit-f and LuxSit-i, were both expressed at high levels of solubility in E. coli and were monomeric (some dimerization was observed at high protein concentrations, Figure 7l) and thermostable (Figure 7j-k). Similar to the native CTZ-utilizing luciferase, the apparent Michaelis constants K for both LuxSit-f and LuxSit-i were M The luminescence signal decayed over time due to the rapid catalytic turnover (Figure 10a). LuxSit-i is a very efficient enzyme with a k cat / K M For 10 6 M -1 s -1 This luminescent signal is easily visible to the naked eye, and the photon flux (photons s -1 ) was 38% higher than that of native Renilla reniformis luciferase (RLuc) (Table 3). The DTZ luminescence reaction catalyzed by LuxSit-i was pH-dependent (Figure 10b), consistent with the proposed mechanism.

[0259] Cellular imaging and multiplexed bioassays

[0260] Since luciferase is a commonly used genetic tag and reporter gene in cellular function studies, we evaluated the expression and function of LuxSit-i in living mammalian cells. HEK293T cells expressing LuxSit-i-mTagBFP2 had DTZ-specific luminescence (Figure 3b), which was maintained after LuxSit-i was targeted to the nucleus, membrane, and mitochondria (Figure 11). The activities of natural and previously engineered luciferases on many luciferin substrates were rather promiscuous (Figure 4ac), probably due to their large and open pockets (luciferases with high specificity for one luciferin substrate are difficult to control even with extensive directed evolution). 35 In contrast, LuxSit-i exhibited excellent specificity for its target luciferin, with 50-fold selectivity for DTZ over di-CTZ (they differ only at the benzylic carbon). Overall, the specificity of our engineered luciferase is superior to that of the native luciferase. 36,37 or previously engineered luciferase 38 Much stronger.

[0261] The high substrate specificity of LuxSit-i may allow multiplexing of luminescent reporters with substrate-specific or spectrally resolved luminescent signals (Fig. 4d and Fig. 12ab). To explore this possibility, we tracked two independent signaling pathways (cAMP / PKA and NF-κB) by placing the expression of RLuc or LuxSit-i downstream of the promoter of the NF-κB or cAMP response element, respectively (Fig. 4e). Imaging one at a time in the presence of two luciferase substrates (PP-CTZ for RLuc and DTZ for LuxSit-i) (Fig. 4f) clearly distinguished known activators of the two pathways. Because luminescence of the two reactions occurs at different wavelengths, we were also able (Fig. 4g) to simultaneously assess the activation of two signaling pathways in the same sample in intact HEK293T cells or cell lysates (Fig. 12c-e) by providing the two substrates together and monitoring luminescence at different wavelengths.

[0262] in conclusion

[0263] Computational enzyme design has so far been constrained by the number of available scaffolds, which limits the extent to which catalytic configurations and enzyme-substrate shape complementarity can be achieved. 16-18 The use of deep learning to generate a large number of de novo designed scaffolds removes this limitation here; looking forward, more accurate RoseTTAfold TM39 and AlphaFold2 TM33Still more efficient protein scaffold generation should be possible by exploiting the full family hallucination capabilities. The diversity of scaffold pocket shapes and sizes enables exploration of a range of catalytic geometries and maximizes substrate-enzyme shape complementarity; to our knowledge, no natural luciferase has a fold similar to LuxSit, and both enzymes have high specificity for fully synthetic luciferin substrates that do not occur in nature. By incorporating 2-3 substitutions, providing a more complementary pocket to stabilize the transition state, LuxSit-i has higher activity than any previously de novo designed enzyme;10 6 M -1 s -1 K cat / K M This is a significant advance in computational enzyme design, as it took dozens of rounds of directed evolution to obtain catalytic capabilities in this range for designed retroaldolases with considerable structural modification. 40 ; in contrast, the predicted differences in ligand-side chain interactions between LuxSit and LuxSit-i are very subtle. Achieving such high activity directly from the computer remains an outstanding goal for computational enzyme design. The small size of LuxSit makes it ideal as a genetic tag for capacity-limited viral vectors, biosensor development, and fusion to proteins of interest. In basic science, the small size, simplicity, and high activity make LuxSit-i an excellent model system for computational and experimental studies aimed at improving the understanding of the catalytic mechanism of luciferases. Extending the methods used here to create new luciferases with similar specificity for synthetic luciferin substrates beyond DTZ and h-CTZ would greatly expand the capabilities of the luciferases described in Figure 4 or with microscopy phase quantification. 41 The multiple opportunities shown here will lead to a broadly useful multiplexed luminescence toolkit. More generally, our family-wide hallucination approach opens up a nearly unlimited number of new scaffolding possibilities for substrate binding and catalytic residue placement, which is particularly important in situations where the reaction mechanism and how the reaction is facilitated are not fully understood: many structural and catalytic hypotheses can be readily enumerated by different catalytic residue placements in binding pockets that are complementary in shape and chemistry. While luciferases are unique in catalyzing the emission of light, the chemical transformation of substrate to product is common to all enzymes, and the methods developed here should be readily extended to a wide variety of chemical reactions.

[0264] Table 3. Photoluminescence properties of selected LuxSit variants

[0265]

[0266] a. Mean ± sd, n = 3 (technical triplicate).

[0267] LQY (luminescence quantum yield) measurements were performed with LuxSit-i, LuxSit-f, and CTZ (w / RLuc) and 50 nM of the corresponding recombinant luciferase. b. All LQY values ​​were estimated relative to the reported quantum yield of RLuc 42 .

[0268] All values ​​were calculated using the simplest Michaelis-Menten kinetic model, excluding potential substrate / product inhibition or enzyme modification during the reaction. 43,44 .

[0269] method

[0270] 1. Materials and General Methods

[0271] Synthetic genes and oligonucleotides were purchased from Integrated DNA Technologies or GenScript. The synthetic genes were inserted between the NdeI and XhoI sites of the pET29b+ vector, which contains an N-terminal hexahistidine tag, followed by a TEV protease cleavage site and a C-terminal stop codon. Restriction endonucleases, Q5PCR polymerase, and T4 ligase were purchased from NEB. Plasmid DNA, PCR products, or digested fragments were purified by Qiagen DNA purification kit. DNA sequences were analyzed by Genewiz. Coelenterazine (CTZ) was purchased from Gold Biotechnology. Diphenyltetrazine (DTZ), pyridyldiphenyltetrazine (8pyDTZ), and Furimazine (FRZ) were purchased from MedChemExpress. All other coelenterazine analogs (bis-CTZ: dideoxy coelenterazine; f-CTZ: f-coelenterazine; e-CTZ: e-coelenterazine-F; PP-CTZ: methoxy e-coelenterazine; v-CTZ: v-coelenterazine. All other chemicals were purchased from Sigma-Aldrich or Fisher Scientific and used without further purification. To identify the molecular mass of each protein, intact mass spectra were acquired by reversed-phase LC / MS on an AdvanceBio RP-desalting column via an Agilent 6230B TOF and subsequently deconvoluted using the total entropy algorithm by Bioconfirm software. The ELISA kit was controlled by UNICORN 6.3.2 workstation (GE Healthcare). pure M and Superdex TM 75Increase 10 / 300GL column was used for size exclusion chromatography. TMDNA and protein concentrations were determined using a low-volume 8-channel UV / vis spectrometer. CD spectroscopy and CD melting experiments were performed using the default settings of a J-1500 circular dichroism spectropolarimeter (Jasco). All luminescence measurements were performed using a Biotek Synergy Neo2 TM To convert relative arbitrary units (RLU) to photon numbers, the 45 , in K 2 CO 3 The Neo2 microplate reader is calibrated by measuring the chemiluminescence of luminol with a known quantum yield in the presence of horseradish peroxidase and hydrogen peroxide in aqueous solution. TM XRS+ captures SDS PAGE and luminescence images. Images are analyzed using Fiji image analysis software.

[0272] 2. General Procedures for Protein Production and Purification

[0273] The Lemo21 (DE3) strain was used for transformation with the pET29b+ plasmid encoding the gene of interest. The transformed cells were grown in TB medium supplemented with kanamycin for 12 h. The cells were inoculated in 100 mL of fresh TB medium at a ratio of 1:50, grown for 4 h at 37 °C, and then induced with IPTG for another 18 h at 16 °C. The cells were harvested by centrifugation at 4,000 g for 10 min and resuspended in 30 mL of lysis buffer (20 mM Tris-HCl pH 8.0, 300 mM NaCl, 30 mM imidazole and Pierce TM Protease inhibitor tablets). The cell resuspension was lysed by sonication for 5 min (10 s per cycle). The lysate was clarified by centrifugation at 24,000g for 40 min at 12°C and pre-equilibrated with 1 mL Ni-NTA nickel agarose at 4°C for 1 h. The resin was washed twice with 10 mL wash buffer and then eluted in 1 mL elution buffer (20 mM Tris-HCl pH 8.0, 300 mM NaCl, 300 mM imidazole). The eluted protein was purified by size exclusion chromatography in PBS. Fractions were collected based on A280 tracking, flash frozen in liquid nitrogen, and stored at -80°C.

[0274] 3. Computational design of idealized stent

[0275] Our generation of the idealized NTF2 scaffold can be divided into four parts: (3.1) generating the seed structure, (3.2) using the trRosetta-based TM Hallucination optimizes the backbone geometry, (3.3) generates a structurally conditional sequence model to bias the design, (3.4) designs and filters.

[0276] 3.1 Generation of seed structure

[0277] We envision using trRosetta TM22 The generated high-precision models complement the experimentally solved structures from the PDB to increase the collection of NTF2 structures. To achieve this, we first collected 85 NTF2-like protein structures from the PDB based on SCOPe annotations (d.17.4SCOPe v2.05). Then, using the corresponding sequences as queries, we searched for NTF2-like protein structures from UniProt by performing 8 iterations of hhblits against the uniclust30_2018_08 database with a 1e-20e value cutoff. TM Collect sequence homologs; relax the default filtering cutoffs (-maxfilt100000000 -neffmax 20 -nodiff-realign_max 10000000) to maximize the number of output hits. All hits use cd-hit 46 Redundancy reduction was performed at a 60% sequence identity cutoff, generating a set of 7,573 candidates for modeling.

[0278] To use trRosetta TM To generate input for structural modeling, we constructed a multiple sequence alignment (MSA) for each of the 7,573 selected sequences using hhblits, using a more conservative e-value cutoff of 1e-50; the resulting MSA was also supplemented with hits from uniref100 (release-2019_11) via hmmsearch, with a bit score threshold of 115 (i.e., approximately 1 bit per position). The above two sets of alignments were merged, and after filtering with a 90% sequence identity and 75% coverage cutoff, only sequences with more than 50 homologs in the corresponding MSA were retained for modeling (2,005 sequences). The filtered MSA and information on the top 25 putative structural homologs as determined by hhsearch against the PDB100 template database were used as trRosetta TM47 The template-aware version of is used as input to predict residue pair distances and orientations. Then, the Rosetta-based TM Folding protocol, using network prediction to reconstruct the complete atomic 3D structure model 22 .

[0279] 3.2 The illusion of idealized NTF2

[0280] To idealize the natural structural seeds, we infer that trRosetta TM(a convolutional residual neural network that predicts residue-residue orientations and distances from the sequence) can serve as a key component of the protein idealizer. Previously, this network has been used to generate different proteins that resemble the "ideal" structure of a de novo designed protein by changing the protein sequence to optimize the contrast (KL-divergence) between the predicted geometry and the randomly generated sequence. 19 .

[0281] For our purposes, the desired folding space is not diverse but rather focused on NTF2-like topologies. To guarantee that ideal structures are generated within this folding space, we implemented a new fold-specific loss function that biases towards hallucinations based on the geometry observed in native crystal structures. Since many experimentally characterized NTF2s contain non-ideal regions, we first created a set (χ) of pruned but ideal NTF2s by manually removing non-ideal structural elements such as twisted helices and long or rarely observed loops. For each seed structure, we then used a structure-based sequence alignment method (see 3.3) to find equivalent positions between the seed structure and χ. If there are 5 or more equivalent positions in χ, the residue pair is considered to be in a conserved tertiary motif (TERM). A smoothed probability distribution based on the geometry observed in χ is then calculated. For distances, we used a Gaussian distribution with a mean equal to the true distance denoted by D and a standard deviation denoted by . The probability density function of distance d is given by:

[0282]

[0283] Using this density function, a categorical distribution of bin distances can be constructed by evaluating the function at the center of the bin and then normalizing by the sum of all values ​​in different bins. Similarly, the von Mises distribution is used for ω-angle smoothing, and its probability density function is given by f(ω; Ω, κ) = N(κ)exp[κcos(ω-Ω)], where N(κ) is the normalization constant, Ω is the crystal value, κ is the inverse variance chosen to be 100, and ω is the angle of smoothing. For and angle θ, the von Mises-Fisher blur is given by f(x;μ,κ)=N(κ)exp[κμTx], where N(κ) is a normalization constant and μ is the value corresponding to the crystal structure and θ, x is a unit vector on a 3D sphere with angles θ, and κ is the inverse variance chosen to be 100.

[0284] Next, we transform these probability distributions into energy landscapes (i.e., negative log-likelihoods) and seek to minimize the expected energy. This soft constraint encourages the network to find a consensus structure while still allowing for necessary deviations. Specifically, we formulate the fold-specific loss as:

[0285]

[0286] m ij = 1 if i and j are in TERM; otherwise 0

[0287] where p is the network prediction and s is the smoothed probability distribution of conserved residue pairs. For the second part of the loss function, similar to previous work 19 , we attempt to maximize the Kullback-Leibler (KL) divergence between the predicted probability distribution and the background distribution for all i, j residue pairs not in TERM.

[0288]

[0289] where b is the background distribution, and N x is the number of bins in each probability distribution (N d =37, N ω，θ， =25, ). In short, b is obtained by training trRosetta on the same training data. TM The final loss is given by:

[0290] L=L 折叠 +L 幻觉

[0291] We use the Markov Chain Monte Carlo (MCMC) procedure to search for trRosetta TMPredict sequences that fold into structures that minimize this loss function. We allow four types of moves with different sampling probabilities: mutations (p=0.55), insertions (p=0.15), deletions (p=0.15), and moving fragments (p=0.15). Mutations randomly change one amino acid to another, with equal probability of transition for all 20 amino acids. Insertions insert a new amino acid (all with equal probability) into a random position, resulting in a KL-divergence loss. Deletions remove random residues from the same position. Finally, we also allow "fragment" moves that cut and paste themselves from one part of the sequence to another while maintaining the same overall fragment order. Here, a "fragment" is a stretch of consecutive amino acids that have all undergone folding-specific loss, typically consisting of a single strand or helix. Starting with a random sequence of an initial length (typically 120 amino acids), we use the standard Metropolis criterion to accept or reject moves:

[0292] A i =min[1,exp(-(L i -L i-1 ) / T)]

[0293] Among them A i is the chance of accepting the move at step i, L i is the loss at the current step, L i-1 is the loss at the previous step, and T is the temperature. The temperature starts at 0.2 and is reduced by half every 5k steps. Typically, it took 30k steps to converge.

[0294] 3.3 Structure-conditional multiple sequence alignment

[0295] Given the complexity of the NTF2-like protein fold, we hypothesized that it would be necessary to impose sequence design rules to suppress alternative states (negative design). To this end, we computed a structure-conditional multiple sequence alignment based on native NTF2-like proteins. Specifically, we used TMalign 48 Each of the 2005 predicted native structures (from 3.1) was superimposed onto each hallucinated skeleton (from 3.2). Next, to find the structurally corresponding positions, we implemented a structure-based dynamic programming algorithm similar to the Needleman-Wunsch algorithm. 49 However, instead of using amino acid similarity as a scoring metric, we used a tunable structure-based scoring function. After aligning two structures, we scored the structural similarity of any two residues by empirically weighting several metrics: (1) the distance between the Ca atoms, (2) the backbone torsion angle ( and ψ), and (3) the angle (in degrees) between the vectors pointing from Cα to Cβ in each residue. To calculate the unweighted score for each component, we used the maximum possible value (angle 180 degrees and distance ) normalizes each component and includes a "set point" that roughly depicts the metric at which we judge two residues to be more similar than to be dissimilar. Values ​​above this set point are positive, indicating that the two residues are similar, and values ​​below the set point indicate that the two residues are dissimilar.

[0296] Score 未加权 = (set point - value) / maximum value

[0297] Each value is scaled by its normalized weight and summed to give an overall similarity score between any two amino acids.

[0298] These similarity scores were used as similarity metrics in our dynamic programming algorithm, replacing the typical BLOSUM62 similarity metric. We used a gap penalty of 0.1 and an extension penalty of 0.0. Finally, after concatenating all structure-conditional aligned sequences, we used PSI-BLAST-exB 50,51 To calculate the sequence redundancy weighted log-odds score for each amino acid at each position (position-specific scoring matrix, PSSM).

[0299] 3.4 Design

[0300] To design the final backbone, we sought to further define the backbone conformation and functionalize the pockets by fitting, in addition to the sequence patterns captured in PSSM (3.3), a complete hydrogen-bonding network from a native NTF2-like protein. We compiled two sets of hydrogen-bonding networks: one for the cavity containing 85 networks, and another set of networks connecting the C-terminal region of the first helix to the third β-strand containing 25 networks. In 20 independent attempts for each backbone, we randomly grafted a network from each set, fixed the identities of the hydrogen-bonding residues, and designed sequences at all other positions under PSSM constraints. The resulting models were screened for various backbone quality metrics and maintenance of hydrogen-bonding networks in the absence of constraints, resulting in a total of 1615 idealized scaffolds.

[0301] 4RIFdock Adjustment File

[0302] RifDock's hierarchical search framework is a powerful way to search for rigid-body orientations in 6 dimensions. While originally designed for use with physics-based force fields, the scoring mechanism can be easily modified to do other things. A system called "tuning files" has been added that allows one to tune the rifdock energetics by "requiring" specific interactions. Specific interactions can range from specific hydrogen bonds to specific bidentates, or even specific hydrophobic interactions. Specifically, during the RifGen phase, each stored rotamer is compared to a list of definitions in the tuning file. If the rotamer meets the definitions, it is stored in the Rif along with a "requirement number." Subsequently during RifDock, these requirement numbers are available at scoring time, and the presence or absence of certain rotamer interactions can be used to penalize or even completely discard the dock solution. In this work, tuning files were used to require the presence of specific hydrogen bonding interactions between the arginine and secondary amines in the pyrazine ring of the enteroid substrate.

[0303] 5 Designing the enzyme architecture into the de novo NTF2 scaffold

[0304] The de novo design of luciferase can be divided into three major steps - scaffold construction, substrate placement with desired interactions, and sequence design. With an idealized NTF2-like scaffold, we selected 5 different rotamers from AIMNet and used the rotamer interaction field (RIF) docking method. 31 A wide range of interacting side chains with the anionic form of DTZ was thoroughly searched. Chemically, deprotonation of the N1 hydrogen is the first step in the formation of the anionic species ( Figure 5 ). We first use RifGen 31 Generate a RIF to guide placement in the protein scaffold. We need to place the positively charged arginine side chain via an adjustment file (see below) to stabilize the formation of the negatively charged N1 atom where deprotonation initially occurs and to enumerate a large number of possible side chain interactions with the rest of the DTZ.

[0305] HBOND_DEFINITION

[0306] N1 1ARG

[0307] END_HBOND_DEFINITION

[0308] REQUIREMENT_DEFINITION

[0309] 1HBOND N1 1

[0310] END_REQUIREMENT_DEFINITION

[0311] Rifdock was then used to hierarchically search for the best combination of RIFs to place on the input scaffold. While the negative charge can be moved to another negatively charged atom O1 via resonance of the imidazopyrazinone core, it is unclear which anionic species is more critical for luciferase-catalyzed luminescence emission. Therefore, we let RIFdock place the polar rotamer at O1 based on the hydrogen bonding geometry and the non-polar rotamer at DTZ without special requirements. In the next docking step, we parsed the -scaffold_res parameter with a series of residue numbers as scaffold scaffold positions, which were annotated as pocket residues to allow a hierarchical search for RIF positions. We only allowed the placement of RIFs in pocket residues and kept the predefined hydrogen bond network (HBNet) intact. After RIFdock, we proceeded with Rosetta TM Sequential design where the scoring function is reweighted to obtain a higher buried_unsat_penalty 52 , and bias the amino acid selection by giving it a pre-generated PSSM file via the SeqprofConsensus task operation. This will minimize buried unsatisfied residues and increase pre-organized architecture in the core, which is known to be beneficial for the catalytic pocket 53 Two rounds of FastDesign calculations were included: we restricted RIF rotamers and core HBNet to restacking in the first round, while we allowed other residues to be redesigned based on PSSM in a Monte Carlo simulated annealing procedure. After optimizing the surrounding residues to preserve RIF interactions, we allowed the RIF rotamers to be redesigned to find efficient aromatic and hydrophobic packing around DTZ, while the catalytic residues (N1 requirement) remained restricted to restacking. The final set of designs was obtained after screening by ligand binding interface energy, shape complementarity, contacting molecular surface, number of HbondsToResidue and presence of N1_hbond.

[0312] 6. AlphaFold2’s structural prediction of LuxSit and its comparison with the design model

[0313] To computationally evaluate the accuracy of the LuxSit design models, we performed single sequence structure prediction using AlphaFold2. All models were run for 12 cycles and the resulting models were relaxed using AMBER 54 The model with the highest pLDDT was used for comparison with Rosetta TM The design model was compared and the structure was superimposed using the Theseus alignment tool to determine the backbone RMSD between the design model and the AlphaFold2 model. 33 .

[0314] 8. Computational SSM Experiments to Estimate Mutation Binding Free Energy

[0315] Will Rosetta TM cartesian_ddg application 55,56 Used to computationally estimate enzyme and substrate binding free energies. The LuxSit design model was pre-relaxed in Cartesian space with substrate binding. For the 21 positions experimentally screened for single mutation effects on luciferase activity, each residue was computationally mutated to other amino acid types and stacking and Cartesian relaxation were performed to evaluate the final score for REU. The procedure was applied three times in parallel for both substrate binding and apo states. The average of the three calculations was used to calculate the relative binding free energy (ddG) by subtracting the total score for the apo state from the composite state. bind ).

[0316] 9. Design of luciferase library construction and screening

[0317] The construction of the assembled gene library has been described in detail previously 57 . In short, first the amino acid sequence of all designed luciferases is reverse transcribed into a DNA sequence optimized for Escherichia coli codons. According to the gene length, all DNA sequences are classified into multiple sub-libraries (about 500 designs for each sub-library). Each gene is subsequently divided into two fragments (fragment A and fragment B), and external and internal primer sequences (e.g., Outer_oligoA_5primer+design_half_A+Inner_oligoA_3primer and Inner_oligoB_5primer+design_half_B+Outer_oligoB_3primer) are added to the 5' and 3' ends. All oligomers are sorted in a torsion 250nt oligonucleotide library. In order to construct the library of each sub-library, the polymerase chain reaction (PCR) using oligoA_5primer / oligoA_3primer or oligoB_5primer / oligoB_3primer oligonucleotide pairs is used to amplify single fragment A or fragment B from each sub-library. Library-specific sequences were removed using uracil-specific excision reagent (USER) followed by NEB end repair kit. External primers (oligoA_5primer and oligoB_3primer) were then used for fragment A and fragment B assembly and amplification. The assembled full-length fragments were digested with XhoI / HindIII and ligated into the pre-digested pBAD / His B vector. All ligation products were used to transform ElectroMAX TMDH10B cells, which were then plated on 150mm×15mmLB agar plates supplemented with carbenicillin and L-arabinose. We sequenced 30 random colonies, and 11 of the sequences were in the library we designed. The plates (about 2000 colonies per plate) were incubated overnight at 37°C to form bacterial colonies and placed at 4°C for another 24h. In order to directly image the luminescent activity of bacterial colonies, we sprayed a PBS solution containing 30 μM DTZ onto each agar plate, waited for 2min, and acquired luminescent images and processed with Bio-Rad ChemiDoc XRS+. After screening 15 plates, active colonies were collected for sequencing, protein expression, and other downstream characterizations, where LuxSit was selected from three active designs showing catalytic signals above background.

[0318] 10. Construction and evaluation of LuxSit site-saturation mutagenesis library

[0319] To create a library of each single amino acid substitution at residues 13, 14, 17, 18, 35, 37, 38, 49, 52, 53, 56, 60, 65, 81, 83, 94, 96, 98, 100, 110, and 112, a plasmid of LuxSit was amplified using a forward oligo mix with degenerate codons (NDT, VHG, and TGG = 1:1:0.1 ratio) and overlapping reverse oligos. The resulting PCR products were circularized by the Gibson assembly protocol and subsequently used to transform ElectroMAX TMDH10B cells. The cells were plated on 150 mm × 15 mm LB agar plates supplemented with carbenicillin and L-arabinose, incubated overnight at 37 ° C, and placed at 4 ° C for another 24 h. As described in the luciferase library screening, colony-based screening by spraying DTZ solution was used to identify active colonies. Inactive colonies were also randomly picked. As a result, a total of 32 colonies were picked for each residue library. In a 96-well deep well culture plate, 32 × 21 individual colonies were grown in 1 mL TB supplemented with carbenicillin and L-arabinose. The plate was shaken overnight (about 16-18 h) at 37 ° C at 1,100 rpm on a 96-well plate shaker. The cells were precipitated by centrifugation at 4,000 g for 15 min in a tabletop centrifuge. The culture medium was discarded, and the cell pellet was resuspended in 0.2 mL BugBuster HT protein extraction buffer. The plate was transferred back to the 96-well plate shaker and incubated for another 30 min at 1,100 rpm. Cell debris was reprecipitated by centrifugation at 4,000 g for 15 min, and the soluble lysate was transferred to a new half-depth 96-well plate and incubated with 10 μL of magnetic Ni-NTA beads for 30 min to allow binding. A magnetic extractor was first used to transfer the beads from the binding plate to a wash plate containing 200 μL of IMAC wash buffer in each well, and then the beads were transferred to an elution plate containing 30 μL of IMAC elution buffer in each well. The concentration of all proteins in each well was determined directly by Bradford assay. The elution solution in each well was used to prepare 25 μL of a protein solution of the indicated concentration and mixed with 25 μL of a 50 μM DTZ PBS solution. Luminescent signals were acquired over the course of 15 min while the actual point mutations were identified by sequencing. Thus, the relationship between mutations and activity can be mapped. To evaluate whether these beneficial mutations are synergistic, we sequenced individual mutants with combined mutations at residues 14, 60, 96, 98, and 110 (see Table 4), expressed and purified these LuxSit variants to measure kinetics, emission spectra, and luminescence intensity. We identified four mutants that could produce 47 to 77 times more photons than the parent LuxSit. We designated one of them, LuxSit-f(A96M / M110V), because of its strong initial flash emission. Because the mutations at residues 96 and 110 are potent and the mutation at residue 60 is multifaceted, we generated a completely randomized library at positions 60, 96, and 110 to thoroughly screen all possible combinations. After colony-based screening, we identified many colonies with strong luciferase activity using DTZ ( Fig. 9). Among all the selected mutants, Arg60 was confirmed to be variable, Ala96 preferred larger hydrophobic side chains (Leu, Ile, Met, and Cys), and Met110 preferred hydrophobic residues (Val, Ile, and Ala). The newly discovered mutant R60S / A96L / M110V had a photon flux more than 100 times higher than LuxSit and was named LuxSit-i for its high brightness.

[0320] 11. In vitro characterization of photoluminescence properties

[0321] For Michaelis-Menten kinetic measurements, 25 μL of serially diluted DTZ substrate in Tris pH 8.0 buffer was added to the wells of a white 96-well half-area microplate containing 25 μL of purified luciferase (final enzyme concentration: 100 nM; substrate concentration: 0.78 to 50 μM). Measurements were performed every 1 min (with 0.1 s integration and 10 s shaking each time) for a total of 20 min. The initial velocity was estimated as the average of the light intensity from the first three data points to fit the Michaelis-Menten equation. The luminol-H 2 O 2 -HRP calibration method 45 , convert all relative arbitrary units (RLU) / second values ​​to photons / s. Follow the formula: max =LQY×k cat ×[E],I max is the maximum photon flux (photons s -1 ), [E] is the total enzyme concentration, and V max is the maximum photon flux per molecule obtained by fitting the Michaelis equation (photons s -1 molecular -1 ). To determine the luminescence quantum yield, 25 μL of 5 μM individual substrates in PBS were injected into 25 μL PBS containing 100 nM of the corresponding luciferase. DTZ was used for all LuxSit variants, while CTZ was used as a substrate for native RLuc. The luminescence signal was monitored until the reaction was complete (0.1 s integration and measurements were taken every 5 s for a total of 40 min). The sum of luminescence photon counts was normalized to the RLuc / CTZ pair (LQY = 5.3 ± 0.1%) 58 The total photon counts were used to derive the relative luminescence quantum yields of the LuxSit variants (Figure 10c). The k of each individual enzyme was calculated using the following formula cat Value: k cat =V max / LQY. To record the emission spectrum, 25 μL of 50 μM DTZ in PBS was injected into 25 μL of 200 nM pure luciferase, and the emission spectrum was collected from 300 to 700 nm with 0.1 s integration and 2 nm increments. In vitro luminescent activity measurements of HEK293T or HeLa cells expressing LuxSit-i were performed similarly to using 15,000 whole cells or lysates instead of purified luciferase in the assay. To evaluate substrate specificity, 25 μL of 50 μM substrate analogs in PBS were added to 25 μL of 200 nM indicated luciferase, and the signal was recorded within 20 min. The data are shown as the total luminescent signal within the first 10 min. We normalized the data by setting the highest emission substrate to 100%.

[0322] 12. Circular Dichroism (CD)

[0323] Purified protein samples were prepared at 15 μM in 10 mM phosphate buffer at pH 7.4. Spectra were recorded from 190 nm to 260 nm at 25°C, 50°C, 75°C, 95°C and after cooling back to 25°C. Thermal denaturation was monitored at 220 nm from 25°C to 95°C (1°C increment per min). Tm values ​​were not reported because the melting curves had no distinct inflection point.

[0324] 13. Mammalian Cell Culture and Transfection

[0325] HEK293T and HeLa cell lines were maintained at 37 °C and humidified 5% CO 2 The cells were cultured in Dulbecco's modified Eagle's medium (DMEM, GIBDO) supplemented with 10% fetal bovine serum (FBS, Sigma) under an atmosphere of 40 °C. Turbofectin containing 500 μg of plasmid DNA was used. TM 8.0 (Origene) to transfect cells. 2 After 24 h in an incubator at 37°C, the culture medium was removed and the cells were collected and resuspended in Dulbecco's phosphate-buffered saline (DPBS).

[0326] 14. Fluorescence Microscopy and Image Analysis

[0327] Cells were washed twice with HBSS and then imaged in HBSS at 37°C in the dark. Just before imaging, cells were incubated with 25 μM DTZ. Radiofluorescence imaging was performed on a Yokogawa CSU-X1 microscope equipped with a Hamamatsu ORCA-Fusion scientific CMOS camera and a Lumencor Celesta light engine. The objective lens used was: 10 times, NA0.45, WD 4.0mm, 20 times, NA1.4, WD 0.13mm, and 40 times, NA 0.95, WD 0.17-0.25mm, with a correction ring (Plan Apochromat Lambda) for cover glass thickness (0.11mm to 0.23mm). The imaging of BFP uses a 408nm laser, 432 / 36nm dichroic and 440 / 40nm emission filter (Semrock). The exposure time is 200ms for BFP and 10s for luminescence. All radiofluorescence experiments were subsequently analyzed using NIS Elements software.

[0328] 15. Multiplex dual-luciferase reporter assays for cAMP / PKA and NF-κB pathways

[0329] HEK293T cells were grown in tissue culture grade white 96-well plates and transfected with specified CRE-RLuc, NFκB-LuxSit-i and CMV-CyOFP plasmids. 24h after transfection, the culture medium was replaced with 2μM forskolin (FSK) or 300ng / mL human tumor necrosis factor α (TNFα) in conventional cell culture medium. 23h after stimulation, the cells were resuspended in DPBS by pipette mixing. 25μL DPBS containing 30,000 intact cells was mixed with 25μL CelLytic M for 15min to prepare cell lysates. For intact cell assays, 25μL DPBS containing 15,000 intact cells was mixed with 25μL PP-CTZ (2μM) or / and DTZ (10μM) in DPBS. For cell lysate assay, 25 μL of cell lysate was added to 25 μL of PP-CTZ (2 μM) or / and DTZ (10 μM) to trigger a luminescent reaction. Signals were recorded every 1 min for a total of 10 min. Light signals were collected in substrate resolution mode without filters, and 528 / 20 and 390 / 35 filters were used in spectral resolution mode. The fluorescence intensity of the regional scan CyOFP at 480 nm (excitation wavelength) and 580 nm (emission wavelength) was used to estimate the total cell number and transfection efficiency. The reported unit is the average value of the first 10 min luminescence (RLU) relative to the relative fluorescence unit (au). In order to derive the activation multiple, all data were normalized to the corresponding non-stimulated control.

[0330] Statistical analysis

[0331] No statistical methods were used to predetermine sample size. No samples were excluded from data analysis. Results were reproduced on different days using different batches of pure protein. Unless otherwise stated, data are shown as mean ± SD, and error bars in figures represent SD of three technical replicates. Data were analyzed and plotted using GraphPad Prism 8, seaborn, and matplotlib.

[0332] Additional information

[0333] Table 4. Enzymatic and photoluminescent properties of LuxSit and its mutants

[0334]

[0335]

[0336] a. Mean (95% CI), n=3 (technical triplicate); b. Integrated luminescence intensity during the first 20 min. All values ​​were normalized to the LuxSit signal containing 25 μM DTZ in Tris pH 8.0 buffer.

[0337] Table 5. Amino acid and DNA sequences of de novo luciferases in this study

[0338] The following sequences (underlined) contain PolyHis-TEV or PolyHis tags for protein purification (they are optional and may be present or absent)

[0339]

[0340]

[0341] References

[0342] 1. Jiang, L et al. De novo computational design of retro-aldolenzymes. Science319,1387-1391(2008).

[0343] 2.Rothlisberger, D et al. Kemp elimination catalysts by computationalenzyme design. Nature 453,190-195(2008).

[0344] 3. Yeh, H. W. et al. Red-shifted luciferase-luciferin pairs for enhanced bioluminescence imaging. Nat. Methods 14, 971-974 (2017).

[0345] 4. Love, A. C. & Prescher, J. A. Seeing (and Using) the Light: Recent Developments in Bioluminescence Technology. Cell Chemical Biology 27, 904-920 (2020).

[0346] 5. Syed, A. J. & Anderson, J. C. Applications of bioluminescence in biotechnology and beyond. Chem. Soc. Rev. 50, 5668-5705 (2021).

[0347] 6. Yeh, H.-W. & Ai, H.-W. Development and Applications of Bioluminescent and Chemiluminescent Reporters and Biosensors. Annu. Rev. Anal. Chem. 12, 129-150 (2019).

[0348] 7. Zambito, G., Chawda, C. & Mezzanotte, L. Emerging tools for bioluminescence imaging. Curr. Opin. Chem. Biol. 63, 86-94 (2021).

[0349] 8. Markova, S. V., Larionova, M. D. & Vysotski, E. S. Shining Light on the Secreted Luciferases of Marine Copepods: Current Knowledge and Applications. Photochem. Photobiol. 95, 705-721 (2019).

[0350] 9. Wu, N et al. Solution structure of Gaussia Luciferase with five disulfide bonds and identification of a putative coelenterazine binding cavity by heteronuclear NMR. Sci. Rep. 10, (2020).

[0351] 10. Jiang, T.Y., Du, L.P. & Li, M.Y. Lighting up bioluminescence with coelenterazine: strategies and applications. Photochem. Photobiol. Sci. 15, 466 - 480 (2016).

[0352] 11. Shakhmin, A et al. Coelenterazine analogues emit red - shifted bioluminescence with NanoLuc. Org. Biomol. Chem. 15, 8559 - 8567 (2017).

[0353] 12. Michelini, E et al. Spectral - resolved gene technology for multiplexed bioluminescence and high - content screening. Anal. Chem. 80, 260 - 267 (2008).

[0354] 13. Rathbun, C.M et al. Parallel screening for rapid identification of orthogonal bioluminescent tools. ACS Cent. Sci. 3, 1254 - 1261 (2017).

[0355] 14. Yeh, H.-W., Wu, T., Chen, M. & Ai, H.-W. Identification of Factors Complicating Bioluminescence Imaging. Biochemistry 58, 1689 - 1697 (2019).

[0356] 15. Su, Y. C. et al. Novel NanoLuc substrates enable bright two-population bioluminescence imaging in animals. Nat. Methods 17, 852 - 860 (2020).

[0357] 16. Lombardi, A., Pirro, F., Maglio, O., Chino, M. & DeGrado, W. F. De Novo design of four-helix bundle metalloproteins: One scaffold, diverse reactivities. Acc. Chem. Res. 52, 1148 - 1159 (2019).

[0358] 17. Chino, M. et al. Artificial diiron enzymes with a DE Novo designed four-helix bundle structure. Eur. J. Inorg. Chem. 2015, 3352 - 3352 (2015).

[0359] 18. Basler, S. et al. Efficient Lewis acid catalysis of an abiological reaction in a de novo protein scaffold. Nat. Chem. 13, 231 - 235 (2021).

[0360] 19. Anishchenko, I. et al. De novo protein design by deep network hallucination. Nature (2021) doi:10.1038 / s41586 - 021 - 04184 - w.

[0361] 20. Wang, J. et al. Scaffolding protein functional sites using deep learning. Science 377, 387 - 394 (2022).

[0362] 21. Norn, C et al. Protein sequence design by conformational landscape optimization. Proc. Natl. Acad. Sci. U.S.A. 118, (2021).

[0363] 22. Yang, J.Y et al. Improved protein structure prediction using predicted interresidue orientations. Proc. Natl. Acad. Sci. U.S.A. 117, 1496 - 1503 (2020).

[0364] 23. Basanta, B et al. An enumerative algorithm for de novo design of proteins with diverse pocket structures. Proc. Natl. Acad. Sci. U.S.A. 117, 22135 - 22145 (2020).

[0365] 24. Loening, A.M., Fenn, T.D. & Gambhir, S.S. Crystal structures of the luciferase and green fluorescent protein from Renilla reniformis. J. Mol. Biol. 374, 1017 - 1028 (2007).

[0366] 25. Tomabechi, Y et al. Crystal structure of nanoKAZ: The mutated 19kDa component of Oplophorus luciferase catalyzing the bioluminescent reaction with coelenterazine. Biochem. Biophys. Res. Commun. 470, 88 - 93 (2016).

[0367] 26. Ding, B. W. & Liu, Y. J. Bioluminescence of Firefly Squid via Mechanism of Single Electron-Transfer Oxygenation and Charge-Transfer-Induced Luminescence. J. Am. Chem. Soc. 139, 1106 - 1119 (2017).

[0368] 27. Isobe, H., Yamanaka, S., Kuramitsu, S. & Yamaguchi, K. Regulation mechanism of spin-orbit coupling in charge-transfer-induced luminescence of imidazopyrazinone derivatives. J. Am. Chem. Soc. 130, 132 - 149 (2008).

[0369] 28. Kondo, H et al. Substituent effects on the kinetics for the chemiluminescence reaction of 6-arylimidazo[1,2-a]pyrazin-3(7H)-ones (Cypridina luciferin analogues): support for the single electron transfer (SET)-oxygenation mechanism with triplet molecular oxygen. Tetrahedron Lett. 46, 7701 - 7704 (2005).

[0370] 29. Branchini, B. R et al. Experimental Support for a Single Electron-Transfer Oxidation Mechanism in Firefly Bioluminescence. J. Am. Chem. Soc. 137, 7592 - 7595 (2015).

[0371] 30. Zubatyuk, R., Smith, J. S., Leszczynski, J. & Isayev, O. Accurate and transferable multitask prediction of chemical properties with an atoms-in-molecules neural network. Science Advances 5, (2019).

[0372] 31. Dou, J. Y et al. De novo design of a fluorescence-activating beta-barrel. Nature 561, 485 - 491 (2018).

[0373] 32. Cao, L et al. Design of protein-binding proteins from the target structure alone. Nature 605, 551 - 560 (2022).

[0374] 33. Jumper, J et al. Highly accurate protein structure prediction with AlphaFold. Nature 596, 583 - +(2021).

[0375] 34. Dauparas, J et al. Robust deep learning based protein sequence design using ProteinMPNN. bioRxiv (2022) doi:10.1101 / 2022.06.03.494563.

[0376] 35. Yeh, H.-W et al. ATP-Independent Bioluminescent Reporter Variants To Improve in Vivo Imaging. ACS Chem. Biol. 14, 959 - 965 (2019).

[0377] 36. Bhaumik, S. & Gambhir, S. S. Optical imaging of Renilla luciferase reporter gene expression in living mice. Proc. Natl. Acad. Sci. U.S.A. 99, 377 - 382 (2002).

[0378] 37. Szent-Gyorgyi, C., Ballou, B. T., Dagnal, E. & Bryan, B. Cloning and characterization of new bioluminescent proteins. in Biomedical Imaging: Reporters, Dyes, and Instrumentation (eds. Bornhop, D. J., Contag, C. H. & Sevick-Muraca, E. M.) (SPIE, 1999). doi:10.1117 / 12.351015.

[0379] 38. Hall, M. P etc. Engineered luciferase reporter from a deep sea shrimp utilizing a novel imidazopyrazinone substrate. ACS Chem. Biol. 7, 1848 - 1857 (2012).

[0380] 39. Baek, M. etc. Accurate prediction of protein structures and interactions using a three-track neural network. Science 373, 871 - +(2021).

[0381] 40. Giger, L. etc. Evolution of a designed retro-aldolase leads to complete active site remodeling. Nat. Chem. Biol. 9, 494 - 498 (2013).

[0382] 41. Yao, Z et al. Multiplexed bioluminescence microscopy via phasor analysis. Nat. Methods 19, 893 - 898 (2022).

[0383] 42. Loening, A.M., Dragulescu - Andrasi, A. & Gambhir, S.S. A red - shifted Renilla luciferase for transient reporter - gene expression. Nat. Methods 7, 5 - 6 (2010).

[0384] 43. Dijkema, F.M et al. Flash properties of Gaussia luciferase are the result of covalent inhibition after a limited number of cycles. Protein Sci. 30, 638 - 649 (2021).

[0385] 44. Schenkmayerova, A et al. Engineering the protein dynamics of an ancestral luciferase. Nat. Commun. 12, (2021).

[0386] 45. Ando, Y et al. Development of a quantitative bio / chemiluminescence spectrometer determining quantum yields: Re - examination of the aqueous luminol chemiluminescence standard. Photochem. Photobiol. 83, 1205 - 1210 (2007).

[0387] 46. Li, W. & Godzik, A. Cd - hit: a fast program for clustering and comparing large sets of protein or nucleotide sequences. Bioinformatics 22, 1658 - 1659 (2006).

[0388] 47. Farrell, D.P et al. Deep learning enables the atomic structure determination of the Fanconi Anemia core complex from cryoEM. IUCrJ 7, 881 - 892 (2020).

[0389] 48. Zhang, Y. & Skolnick, J. TM-align: a protein structure alignment algorithm based on the TM-score. Nucleic Acids Res. 33, 2302 - 2309 (2005).

[0390] 49. Needleman, S.B. & Wunsch, C.D. A general method applicable to the search for similarities in the amino acid sequence of two proteins. J. Mol. Biol. 48, 443 - 453 (1970).

[0391] 50. Oda, T., Lim, K. & Tomii, K. Simple adjustment of the sequence weight algorithm remarkably enhances PSI-BLAST performance. BMC Bioinformatics 18, 288 (2017).

[0392] 51. Altschul, S.F et al. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25, 3389 - 3402 (1997).

[0393] 52. Coventry, B. & Baker, D. Protein sequence optimization with a pairwise decomposable penalty for buried unsatisfied hydrogen bonds. PLoS Comput. Biol. 17, (2021).

[0394] 53. Smith, A. J. T et al. Structural Reorganization and Preorganization in Enzyme Active Sites: Comparisons of Experimental and Theoretically Ideal Active Site Geometries in the Multistep Serine Esterase Reaction Cycle. J. Am. Chem. Soc. 130, 15361 - 15373 (2008).

[0395] 54. Salomon-Ferrer, R., Case, D. A. & Walker, R. C. An overview of the Amber biomolecular simulation package. Wiley Interdiscip. Rev. Comput. Mol. Sci. 3, 198 - 210 (2013).

[0396] 55. Kellogg, E. H., Leaver-Fay, A. & Baker, D. Role of conformational sampling in computing mutation-induced changes in protein structure and stability. Proteins 79, 830 - 838 (2011).

[0397] 56. Park, H et al. Simultaneous optimization of biomolecular energy functions on features from small molecules and macromolecules. J. Chem. Theory Comput. 12, 6201 - 6212 (2016).

[0398] 57. Klein, J. C., et al. Multiplex pairwise assembly of array-derived DNA oligonucleotides. Nucleic Acids Res. 44, (2016).

[0399] 58. Loening, A. M., Wu, A. M., & Gambhir, S. S. Red-shifted Renilla reniformis luciferase variants for imaging in living subjects. Nat. Methods 4, 641 - 643 (2007).

[0400] 59. Liang, J., Feng, X., Hait, D., & Head-Gordon, M. Revisiting the performance of time-dependent density functional theory for electronic excitations: Assessment of 43 popular and recently developed functionals from rungs one to four. J. Chem. Theory Comput. 18, 3460 - 3473 (2022).

[0401] 60. Chai, J.-D., & Head-Gordon, M. Long-range corrected hybrid density functionals with damped atom-atom dispersion corrections. Phys. Chem. Chem. Phys. 10, 6615 - 6620 (2008).

[0402] 61. Ditchfield, R., Hehre, W. J., & Pople, J. A. Self-consistent molecular-orbital methods. IX. An extended Gaussian-type basis for molecular-orbital studies of organic molecules. J. Chem. Phys. 54, 724 - 728 (1971).

[0403] 62.Grimme,S.Exploration of chemical compound,conformer,and reactionspace with meta-dynamics simulations based on tight-binding quantum chemicalcalculations.J.Chem.Theory Comput.15,2847-2862(2019).

[0404] 63.Pracht,P.,Bohle,F.&Grimme,S.Automated exploration of the low-energy chemical space with fast quantum chemicalmethods.Phys.Chem.Chem.Phys.22,7169–7192(2020).

[0405] 64.Luchini,G.,Alegre-Requena,J.V.,Funes-Ardoiz,I.&Paton,R.S.GoodVibes:automated thermochemistry for heterogeneous computationalchemistry data.F1000Res.9,291(2020).

[0406] 65.Li,Y.-P.,Gomes,J.,Mallikarjun Sharada,S.,Bell,A.T.&Head-Gordon,M.Improved force-field parameters for QM / MM simulations of the energies ofadsorption for molecules in zeolites and a free rotor correction to the rigidrotor harmonic oscillator model for adsorption enthalpies.J.Phys.Chem.CNanomater.Interfaces 119,1840-1850(2015).

[0407] 66. A.W. et al. Routine microsecond molecular dynamics simulations with AMBER on GPUs. 1. Generalized Born. J. Chem. Theory Comput. 8, 1542 - 1555 (2012).

[0408] 67. Becke, A.D. Density-functional thermochemistry. III. The role of exact exchange. J. Chem. Phys. 98, 5648 - 5652 (1993).

[0409] 68. Grimme, S., Antony, J., Ehrlich, S. & Krieg, H. A consistent and accurate ab initio parametrization of density functional dispersion correction (DFT-D) for the 94 elements H - Pu. J. Chem. Phys. 132, 154104 (2010).

[0410] 69. Grimme, S., Ehrlich, S. & Goerigk, L. Effect of the damping function in dispersion corrected density functional theory. J. Comput. Chem. 32, 1456 - 1465 (2011).

[0411] 70. Meiler, J. & Baker, D. ROSETTALIGAND: Protein-small molecule docking with full side-chain flexibility. Proteins 65, 538 - 548 (2006).

[0412] 71. Davis, I.W. & Baker, D. RosettaLigand docking with full ligand and receptor flexibility. J. Mol. Biol. 385, 381 - 392 (2009).

[0413] 72.Davis,I.W.,Raha,K.,Head,M.S.&Baker,D.Blind docking ofpharmaceutically relevant compounds using RosettaLigand.Protein Sci.18,1998-2002(2009).

[0414] 73.Wang,J.,Wolf,R.M.,Caldwell,J.W.,Kollman,P.A.&Case,D.A.Developmentand testing of a general amber force field.J.Comput.Chem.25,1157-1174(2004).

[0415] 74.Bayly,C.I.,Cieplak,P.,Cornell,W.&Kollman,P.A.A well-behavedelectrostatic potential based method using charge restraints for derivingatomic charges:the RESP model.J.Phys.Chem.97,10269-10280(1993).

[0416] 75.Besler,B.H.,Merz,K.M.&Kollman,P.A.Atomic charges derived fromsemiempirical methods.J.Comput.Chem.11,431-439(1990).

[0417] 76.Singh,U.C.&Kollman,P.A.An approach to computing electrostaticcharges for molecules.J.Comput.Chem.5,129-145(1984).

[0418] 77. Jorgensen, W.L., Chandrasekhar, J., Madura, J.D., Impey, R.W. & Klein, M.L. Comparison of simple potential functions for simulating liquid water. J. Chem. Phys. 79, 926 - 935 (1983).

[0419] 78. Maier, J.A etc. Ff14SB: Improving the accuracy of protein side chain and backbone parameters from ff99SB. J. Chem. Theory Comput. 11, 3696 - 3713 (2015).

[0420] 79. Darden, T., York, D. & Pedersen, L. Particle mesh Ewald: An N·log(N) method for Ewald sums in large systems. J. Chem. Phys. 98, 10089 - 10092 (1993).

[0421] 80. Roe, D.R. & Cheatham, T.E., III. PTRAJ and CPPTRAJ: Software for processing and analysis of molecular dynamics trajectory data. J. Chem. Theory Comput. 9, 3084 - 3095 (2013).

Claims

1. A protein having luciferase activity, comprising a secondary structure arrangement H1-L1-H2-L2-E1-L3-E2-L4-H3-L5-E3-L6-E4-L7-E5-L8-E6, wherein "H" is a helical domain, "L" is a loop domain, and "E" is a beta strand domain; wherein: (a) 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 18 of the H1 domain is D or E; (b) the E3 domain is at least 6, 7, 8, 9 or 10 amino acids in length and residue 2 of the E3 domain is R; and (c) the E5 domain is at least 10, 11, 12, 13 or 14 amino acids in length, and residue 9 of the E5 domain is H or N.

2. The protein of claim 1, wherein residue 7 of the E5 domain is M.

3. The protein of claim 1 or 2, wherein the E6 domain is at least 9, 10, 11, 12 or 13 amino acids in length, and wherein residue 5 of the E6 domain is V.

4. The protein of any one of claims 1-3, wherein residue 1 of the L5 domain is S.

5. The protein of any one of claims 3-4, wherein residue 7 of the E5 domain is M and residue 5 of the E6 domain is V.

6. The protein of any one of claims 3-4, wherein residue 7 of the E5 domain is M, residue 5 of the E6 domain is V, and residue 1 of the L5 domain is S.

7. The protein of any one of claims 1-6, wherein the H2 domain is at least 5, 6 or 7 amino acids in length, the H3 domain is at least 9, 10, 11, 12, 13 or 14 amino acids in length, the E1 domain is at least 3 or 4 amino acids in length, the E2 domain is at least 3 or 4 amino acids in length, and / or the E4 domain is at least 8, 9, 10, 11 or 12 amino acids in length.

8. The protein according to any one of claims 1 to 7, wherein: The H1 domain is 19 amino acids long; The H2 domain is 7 amino acids long; The E1 domain is 4 amino acids in length; The E2 domain is 4 amino acids in length; The H3 domain is 14 amino acids in length; The E3 domain is 10 amino acids in length; The E4 domain is 12 amino acids in length; The E5 domain is 14 amino acids in length; and The E6 domain is 12 or 13 amino acids in length.

9. The protein of any one of claims 1-8, wherein 1, 2, 3, 4 or all 5 of the following conditions are met: (a) Residue 13 of the H1 domain is F; (b) Residue 1 of the L3 domain is W; (c) residue 5 of the E5 domain is V or another hydrophobic residue; (d) residue 8 of the E5 domain is A or L or another hydrophobic residue; and / or (e) Residue 11 of the E5 domain is W.

10. The protein of claim 8 or 9, wherein 1, 2, 3, 4, 5 or all 6 of the following conditions are met: (a) residue 2 of the E1 domain is I or another hydrophobic residue; (b) residue 4 of the H3 domain is F; (c) residue 6 of the E4 domain is V or another hydrophobic residue; (d) residue 8 of the E4 domain is L or another hydrophobic residue; (e) residue 5 of the E6 domain is M or V or another hydrophobic residue; and / or (f) Residue 7 of the E6 domain is V or another hydrophobic residue.

11. The protein of any one of claims 1-10, comprising an amino acid sequence 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 an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-181 or SEQ ID NOs: 1-3.

12. The protein of claim 11, comprising the amino acid sequence of SEQ ID NO:

4.

13. A protein having luciferase activity, comprising an amino acid sequence having 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% identity to the amino acid sequence of SEQ ID NO: 1, wherein: residue 14 is Y, D or E and residue 98 is H or N; and Residue 18 is D or E and residue 65 is R.

14. The protein of claim 13, comprising one or both of A96M and M110V substitutions relative to SEQ ID NO:

1.

15. The protein of claim 13, comprising both of A96M and M110V substitutions relative to SEQ ID NO:

1.

16. The protein of any one of claims 13-15, comprising a R60S substitution relative to SEQ ID NO:

1.

17. The protein of claim 16, comprising R60S, A96M and M110V substitutions relative to SEQ ID NO:

1.

18. The protein of any one of claims 10-14 and 16, wherein any substitutions at residues F12, I35, W38, F49, V81, L83, V94, A97, W100, M110 and / or V112 relative to SEQ ID NO: 1 are conservative amino acid substitutions.

19. The protein of any one of claims 13-18, comprising an amino acid sequence at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 1-3 or 1-181.

20. The protein of any one of claims 11 and 13-19, wherein any substitution relative to a reference sequence is a conservative amino acid substitution.

21. A protein comprising the formula X1-Z1-X2-Z2-X3-Z3-X4-Z4-X5-Z5-X6-Z6-X7-Z7-X8-Z8, wherein: X1 has the same RRF as MSEEQIRQFL Y an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to EALD (SEQ ID NO: 182) wherein residue 14 is Y, D or E and residue 18 is D or E; X2 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 of ADTAASLF (SEQ ID NO: 183); X3 has an amino acid sequence that is at least 50%, 75%, or 100% identical to the amino acid sequence of TIHL (SEQ ID NO: 184); X4 has an amino acid sequence that is at least 33%, 66%, or 100% identical to the amino acid sequence of VTF; X5 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 of EEFREWFERLFST (SEQ ID NO: 185); X6 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 of QREIKSLEVR (SEQ ID NO: 186), wherein residue 2 is R; X7 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 of VEVHVQLHATH (SEQ ID NO: 187); X8 has KHTVDAT H an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to HWHFR (SEQ ID NO: 188) wherein residue 8 is H or N; X9 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 of VTEMRVHINPTG (SEQ ID NO: 189); and wherein Z1, Z2, Z3, Z4, Z5, Z6, Z7 and Z8 are independently present or absent, and when present may comprise any amino acid sequence.

22. The protein of claim 21, wherein the following 1, 2, 3, 4, 5, 6, 7 or all 8 conditions are met: Z1 includes SGD; Z2 comprises HPGV (SEQ ID NO: 190); Z3 includes WDG; The Z4 includes TSR; Z5 comprises RKDA (SEQ ID NO: 191); Z6 contains GDT; Z7 contains NGQ; and / or Z8 contains GNR; and wherein 0, 1, 2, 3, 4, 5, 6, 7 or all 8 of Z1, Z2, Z3, Z4, Z5, Z6, Z7 and Z8 further comprise additional polypeptide domains.

23. A self-complementary multipart protein having luciferase activity, 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, wherein the at least first polypeptide component and the second polypeptide component together comprise the domains X1-Z1-X2-Z2-X3-Z3-X4-Z4-X5-Z5-X6-Z6-X7-Z7-X8-Z8-X9, wherein each domain is as defined in claims 21-22; wherein (a) each X domain is entirely present in one polypeptide component of the at least first polypeptide component and the second polypeptide component, and (b) the at least first polypeptide component and the second polypeptide component do not include all of X1, X2, X3, X4, X5, X6, X7, X8 and X9.

24. A self-complementary multipart protein as claimed in claim 23, wherein cleavage occurs at Z4, Z5, Z6 or Z7.

25. A self-complementary multipart protein having luciferase activity, 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, wherein the at least first polypeptide component and the second polypeptide component together comprise a secondary structure arrangement H1-L1-H2-L2-E1-L3-E2-L4-H3-L5-E3-L6-E4-L7-E5-L8-E6, wherein each domain is as defined in any one of claims 1-9; wherein (a) each of the H and E domains is completely present in one polypeptide component of the at least first polypeptide component and the second polypeptide component, and (b) both the at least first polypeptide component and the second polypeptide component do not include all of the H and E domains.

26. A self-complementary multipart protein as claimed in claim 25, wherein cleavage occurs at L4, L5, L6, L7 or L8.

27. A fusion protein comprising: (a) a protein or polypeptide component as claimed in any preceding claim; and (b) one or more additional functional domains.

28. A nucleic acid encoding a protein, polypeptide component or fusion protein as claimed in any preceding claim.

29. The nucleic acid of claim 28, comprising the nucleotide sequence of any one of SEQ ID NOs: 200-380.

30. An expression vector comprising the nucleic acid of claim 28 or 29 operably linked to suitable control elements.

31. A recombinant host cell comprising the protein, polypeptide component, fusion protein, nucleic acid and / or expression vector of any preceding claim.

32. A kit comprising: (a) a protein, polypeptide component, fusion protein, nucleic acid, expression vector and / or host cell as claimed in any preceding claim; as well as (b) Instructions for their use.

33. The kit of claim 32, further comprising diphenyltetrazine (DTZ).

34. A method of using a protein, polypeptide component, fusion protein, nucleic acid, expression vector, host cell and / or kit as described in any preceding claim for any suitable purpose, including but not limited to luminescent reporter assays, diagnostic assays, cellular localization of targets of interest, cell imaging, gene editing, live animal imaging, cancer markers, CART cell reporters, secretion assays, gene delivery and tissue engineering.

35. A method for preparing luciferase, comprising de novo design starting with a protein comprising the amino acid sequence of SEQ ID NO: 381 using the method of any embodiment disclosed herein.

Images