Reagent kit containing polypeptides for use in detecting intermolecular interactions

Abstract

To provide a novel probe which has a small molecular mass and can be used for intermolecular interaction detection.SOLUTION: There is provided a reagent kit comprising: a first polypeptide including a part of any amino acid sequence of A-C: and a second polypeptide including a part of any amino acid sequence of A-C, having a different sequence different from that of the first polypeptide, and generating light emission enzyme activity by contact with the first polypeptide. In the reagent kit, (A) is an amino acid sequence in which, in a specific amino acid sequence, amino acid sequences on a first position to 69-th position and 204-th position to 221-th position are absent, (B) is an amino acid sequence in which, in a specific amino acid sequence, amino acid sequences on a first position to 69-th position are absent, and at least one out of amino acid residues on 146-th position to 156-th position is absent or substituted, and (C) is an amino acid sequence in which, in the amino acid sequence in the (A) or (B), at least one out of amino acid residues on 70-th position to 74-th position is absent.SELECTED DRAWING: Figure 2

Description

[Technical Field]

[0001] The present invention relates to a reagent kit containing polypeptides used for detecting intermolecular interactions. [Background technology]

[0002] In basic biology, diagnostics, and testing, luciferases are used as reporter proteins to detect target proteins. Commonly used reporter proteins include luciferases, fluorescent proteins, fluorescent dyes, quantum dots, and peroxidases. While fluorescent proteins, fluorescent dyes, and quantum dots have high fluorescence intensity, they require excitation light, which has several drawbacks: (1) phototoxicity to cells; (2) the excitation light spectrum overlaps with the fluorescence spectrum, resulting in a low signal-to-background ratio and making them unsuitable for trace detection; and (3) the detector must incorporate an excitation light irradiator and spectral filters. Luciferases, however, do not require excitation light and thus avoid these drawbacks. Furthermore, luciferase detection is generally more suitable for trace detection than colorimetric methods using peroxidases and other enzymes.

[0003] To date, various luciferases have been reported, including wild-type firefly luciferase (FLuc), NanoLuc, TurboLuc, copepod (Gaussia princeps) luciferase (GLuc), sea pansy (Renilla reniformis) luciferase (), and copepod (Metridia longa) luciferase (MLuc). Patent documents 1 and 2 disclose an artificial luciferase (Aluc) that was created by selecting frequently occurring amino acids from the amino acid sequence of copepod luciferase.

[0004] Luminescent enzymes can be split and used as probes for detecting intermolecular interactions. For example, in PCA (Protein-Fragment Complementation Assay), split luminescent enzymes are attached to molecules whose interactions are to be detected, and intermolecular interactions can be detected by luminescence signals. Patent Document 1, Patent Document 2, and Non-Patent Document 1 disclose probes used for detecting intermolecular interactions. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Japanese Patent Application Laid-Open No. 2014-100137 [Patent Document 2] International Publication No. 2017 / 057752 [Non-patent literature]

[0006] [Non-Patent Document 1] Remy and Michnick, Nat Methods. 2006 Dec;3(12):977-9. Summary of the Invention [Problem to be solved by the invention]

[0007] When analyzing molecular interactions, if the probe size is large, the fusion protein of the probe and the target protein may not be expressed properly in cells, or the target protein may not function properly due to steric hindrance.

[0008] The small size of the probe is useful in analyzing intermolecular interactions. An object of the present invention is to provide a probe with a small molecular weight that can be used to detect intermolecular interactions. [Means for solving the problem]

[0009] The present invention provides a first polypeptide containing a part of any one of the amino acid sequences (A) to (C); and a second polypeptide that contains a portion of the amino acid sequence of any one of (A) to (C), has a sequence different from that of the first polypeptide, and produces luciferase activity upon contact with the first polypeptide. (A) an amino acid sequence in which the amino acid sequences at positions 1 to 69 and 204 to 221 in the amino acid sequence shown in SEQ ID NO: 1 are deleted; (B) an amino acid sequence in which the amino acid sequence at positions 1 to 69 is deleted and at least one amino acid residue at positions 146 to 156 is deleted or substituted in the amino acid sequence shown in SEQ ID NO: 1; (C) An amino acid sequence in which at least one of the amino acid residues at positions 70 to 74 in the amino acid sequence shown in (A) or (B) is further deleted.

[0010] The present invention provides A first polypeptide comprising a part of any one of the amino acid sequences (A) to (C), a luciferase that produces luciferase activity upon contact with a second polypeptide containing a part of the amino acid sequence of any one of (A) to (C), It also relates to a first polypeptide having a sequence that differs from said second polypeptide. (A) an amino acid sequence in which the amino acid sequences at positions 1 to 69 and 204 to 221 in the amino acid sequence shown in SEQ ID NO: 1 are deleted; (B) an amino acid sequence in which the amino acid sequence at positions 1 to 69 is deleted and at least one amino acid residue at positions 146 to 156 is deleted or substituted in the amino acid sequence shown in SEQ ID NO: 1; (C) An amino acid sequence in which at least one of the amino acid residues at positions 70 to 74 in the amino acid sequence shown in (A) or (B) is further deleted. [Effects of the Invention]

[0011] According to the present invention, intermolecular interactions can be detected using a new probe with a small molecular weight. [Brief explanation of the drawings]

[0012] [Figure 1] FIG. 1 shows an example of luminescence due to contact between a first polypeptide and a second polypeptide. [Figure 2] FIG. 1 is a schematic diagram showing an example of detection of an intermolecular interaction using a first polypeptide and a second polypeptide. [Figure 3] FIG. 1 is a schematic diagram showing an example of detection of an intermolecular interaction using a first polypeptide and a second polypeptide. [Figure 4] FIG. 1 is a schematic diagram showing the structure of an ALuc30 mutant prepared in an example. [Figure 5] FIG. 1 shows the amino acid sequence identity between miniALuc30 and miniALuc16. [Figure 6] FIG. 1 shows the amino acid sequence identity between miniALuc30 and miniALuc48. [Figure 7] FIG. 1 shows the amino acid sequence identity between miniALuc48 and miniALuc16. [Figure 8] 10 is a graph showing the luminescence value of miniALuc in Experiment 2. [Figure 9] FIG. 1 shows the three-dimensional structure of miniALuc30. [Figure 10] 10 is a graph showing the luminescence value of ΔloopN1 in Experiment 4. [Figure 11] 10 is a graph showing luminescence values when coelenterazine (0.5 μM) was used as a substrate in Experiment 5. [Figure 12] 10 is a graph showing luminescence values when coelenterazine (5 μM) was used as a substrate in Experiment 5. [Figure 13] 10 is a graph showing luminescence values when coelenterazine h (5 μM) was used as a substrate in Experiment 5. [Figure 14] 10 is a graph showing luminescence values when coelenterazine h (25 μM) was used as a substrate in Experiment 5. [Figure 15] 10 is a graph showing luminescence values when furimazine was used as a substrate in Experiment 5. [Figure 16] 10 is a graph showing luminescence values when furimazine was used as a substrate in Experiment 5. [Figure 17] This shows the results of Western blotting to detect enzyme proteins in the supernatant in Experiment 6. The left panel detects flag-tag, and the right panel detects His-tag. [Figure 18] 10 is a graph showing the specific activity when coelenterazine was used as a substrate in Experiment 7. [Figure 19] 10 is a graph showing the specific activity when coelenterazine h is used as a substrate in Experiment 7. [Figure 20] FIG. 10 shows the emission spectrum when coelenterazine was used as a substrate in Experiment 8. [Figure 21] FIG. 10 shows the emission spectrum when coelenterazine h was used as a substrate in Experiment 8. [Figure 22] 10 is a graph showing the luminescence value of secreted and expressed miniALuc after heat treatment in Experiment 9. [Figure 23] 10 is a graph showing the luminescence values of secreted and expressed miniALuc and miniALuc expressed in Escherichia coli in Experiment 10. [Figure 24] 10 is a graph showing the luminescence values of miniALuc expressed in E. coli after heat treatment in Experiment 10. [Figure 25] 11 is a graph showing the luminescence values of divided miniALuc and combinations thereof in Experiment 11-1. [Figure 26] 11 is a graph showing the luminescence values of divided miniALuc and combinations thereof in Experiment 11-2. [Figure 27] 11 is a graph showing the luminescence values of divided miniALuc and combinations thereof in Experiment 11-2. [Figure 28] 11 is a graph showing the luminescence values of divided miniALuc and combinations thereof in Experiment 11-2. [Figure 29] 10 is a graph showing the results of detecting bimolecular interactions using split miniALuc in Experiment 12. [Figure 30] 10 is a graph showing the results of detecting bimolecular interactions using split miniALuc in Experiment 12. [Figure 31] 10 is a graph showing the results of detecting bimolecular interactions using split miniALuc in Experiment 12. [Figure 32] 10 is a graph showing the results of detecting bimolecular interactions using split miniALuc in Experiment 12. [Figure 33] 10 is a graph showing the results of detecting bimolecular interactions using split miniALuc in Experiment 12. [Figure 34] 10 is a graph showing the results of detecting bimolecular interactions using split miniALuc in Experiment 12. [Figure 35] 10 is a graph showing the results of detecting bimolecular interactions using split miniALuc in Experiment 12. [Figure 36] 10 is a graph showing the results of detecting bimolecular interactions using split miniALuc in Experiment 12. [Figure 37] 13 is a graph showing the results of detecting intermolecular interactions using circular permutants containing split miniALuc in Experiment 13. DETAILED DESCRIPTION OF THE INVENTION

[0013] <Luminescent enzyme A> The reagent kit according to the present invention comprises a first polypeptide and a second polypeptide, which are parts of luciferase A having luciferase activity.

[0014] Luminescent enzymes (luciferases) are enzymes that oxidize luminescent substrates (luciferins), producing luminescence during the oxidation process. Luminescent enzyme activity, as used herein, refers to the activity of an enzymatic reaction between a luminescent enzyme and a substrate, and is measured by detecting the light (emission spectrum) emitted when the substrate returns to its ground state after becoming excited by the enzymatic reaction with the luminescent enzyme. The light emitted when returning to the ground state can be detected using a known luminometer (e.g., the "GloMax" series manufactured by Promega) or a spectrophotometer (e.g., the "Infinite200PRO" manufactured by TECAN). By measuring the intensity at a specific wavelength every minute, the time-dependent change in luminescence and its stability can be detected. A shift to longer wavelengths can be detected by measuring all wavelengths.

[0015] The optimum pH and temperature for luciferase activity may be the same as those of known luciferases (e.g., copepod luciferases or artificial luciferases). The luciferase activity preferably has the luciferase activity of copepods. The optimum pH for luciferase activity is 5.0 to 8.0, preferably pH 7.0, and the optimum temperature is 4°C to 30°C, preferably 25°C.

[0016] The luminescent substrate is not particularly limited and may be appropriately selected depending on the luminescent enzyme. The luminescent substrate may be a known substrate such as a coelenterazine-based substrate, a firefly luciferin-based substrate, a Cypridina luciferin-based substrate, or furimazine, but is preferably a coelenterazine-based substrate. Examples of coelenterazine-based substrates include natural coelenterazine, coelenterazine IP, coelenterazine I, coelenterazine HCP, coelenterazine 400A, coelenterazine, coelenterazine CP, coelenterazine F, coelenterazine H, and coelenterazine N, and preferably includes coelenterazine or coelenterazine H.

[0017] One embodiment of luciferase A is (A) The amino acid sequence shown in SEQ ID NO: 1 includes an amino acid sequence in which the amino acid sequences at positions 1 to 69 and 204 to 221 are deleted.

[0018] One embodiment of luciferase A is (A1) The luciferase A may contain the amino acid sequence of positions 75 to 203 of the amino acid sequence shown in SEQ ID NO: 1 and may have 140 or fewer amino acid residues. The luciferase A may consist of the amino acid sequence of positions 75 to 203 of the amino acid sequence shown in SEQ ID NO: 1.

[0019] (A2) Luciferase A may contain the amino acid sequence from positions 75 to 203 of the amino acid sequence shown in SEQ ID NO: 1 and have a molecular weight of 20 kDa or less.

[0020] One embodiment of luciferase A is (B) The amino acid sequence shown in SEQ ID NO: 1 includes an amino acid sequence in which the amino acid sequence at positions 1 to 69 is deleted and at least one amino acid residue at positions 146 to 156 is deleted or substituted.

[0021] One embodiment of luciferase A is (B1) It may be an amino acid sequence comprising the amino acid sequence of positions 70 to 221 of the amino acid sequence shown in SEQ ID NO: 1, in which at least one amino acid residue from positions 146 to 156 has been deleted or substituted. Luciferase A may consist of the amino acid sequence of positions 70 to 221 of the amino acid sequence shown in SEQ ID NO: 1, in which at least one amino acid residue from positions 146 to 156 has been deleted or substituted.

[0022] Luminescent enzyme A is (C) In the amino acid sequence of (A) or (B) above, at least one of the amino acid residues at positions 70 to 74 of the amino acid sequence shown in SEQ ID NO: 1 may be further deleted, or all of the amino acid residues at positions 70 to 74 may be deleted.

[0023] Luciferase A is preferably missing 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, or 7 or more amino acid residues at positions 146 to 156 of the amino acid sequence shown in SEQ ID NO: 1. A linker sequence of one to several bases may be inserted at the deleted sites. Luciferase A may have 6 or less, 5 or less, 4 or less, 3 or less, 2 or less, 1 or less, or zero amino acid residues between positions 146 and 156 of the amino acid sequence shown in SEQ ID NO: 1.

[0024] The molecular weight of luciferase A is preferably 20 kDa or less, more preferably 18 kDa or less, even more preferably 15 kDa or less, still more preferably 14 kDa or less, and particularly preferably 13 kDa or less. The molecular weight of luciferase A is, for example, 10 kDa or more.

[0025] The number of amino acid residues in luciferase A is, for example, 160 or less, and preferably 155 or less, 150 or less, 146 or less, 143 or less, 140 or less, 136 or less, 133 or less, 130 or less, 126 or less, 123 or less, 122 or less, 121 or less, 120 or less, 119 or less, 118 or less, or 117 or less. The number of amino acid residues in luciferase A is, for example, 100 or more.

[0026] Among the amino acids represented by Xaa in SEQ ID NO: 1, any amino acids may be used at positions 3, 20-29, 31, 32, 35, 37, 64-66, 69, 76-77, 85-86, 89-90, 129, 140-144, 148-151, 159, 161, 188, 191, 202, and 206. Among these, positions 22-23, 39-40, 76-77, 140, and 148-151 may be deleted. Preferably, position 3 is E or G, positions 20-29 are PTENKDDI sequence (2 residues deleted, SEQ ID NO: 2), ATINENFEDI sequence (2 residues deleted, SEQ ID NO: 3), ATINENFEDI sequence (SEQ ID NO: 4), HHHHHHHH sequence (2 residues deleted, SEQ ID NO: 5), EKLISEE sequence (2 residues deleted, SEQ ID NO: 6), MMYPYDVP sequence (2 residues deleted, SEQ ID NO: 7) or MMDYKDDD sequence (2 residues deleted, SEQ ID NO: 8), position 31 is I, L, Y or K, position 32 is V or A, position 35 is E or G, position 37 is K or S, positions 64-66 are ANS sequence or DAN sequence, position 69 is D or G, and positions 76-77 are GG sequence or K (1 residue deleted) or deleted. positions 85-86 are the LE, KA or KE sequence, positions 89-90 are the KE, IE, LE or KI sequence, position 129 is E, G or A, positions 140-144 are the TEEET sequence (SEQ ID NO: 9), the GEAI sequence (one residue deleted, SEQ ID NO: 10) or the VGAI sequence (one residue deleted, SEQ ID NO: 11), positions 148-151 are the GVLG sequence (SEQ ID NO: 12) or I (three residues deleted) or may be completely deleted, position 159 is D, E, N, F, Y or W, position 161 is E, A or L, position 188 is K, F, Y or W, position 191 is D, A, N, F, Y or W, position 202 is A or K, and position 206 is S, D, N, F, Y or W.

[0027] The amino acids at positions 13, 16, 174, and 218 of SEQ ID NO: 1 are hydrophobic amino acids (e.g., V, F, A, L, I, G), and preferably, position 13 is V or F, position 16 is V or A, position 174 is V or A, and position 218 is A or L.

[0028] Positions 5, 67, 75, 101, 119, and 214 of SEQ ID NO: 1 are hydrophilic amino acids (e.g., Q, K, D, R, H, E, and T), and preferably, position 5 is Q or K, position 67 is D or R, position 75 is K, H, R, or E, position 101 is T or H, position 119 is K, E, or Q, and position 211 is K or T.

[0029] In SEQ ID NO: 1, positions 4, 6, 7, 10, 11, 15, 33, 34, 39-41, 63, 68, 74, 78, 83, 137, 160, and 203 are aliphatic amino acids. However, positions 39, 40, and 70 may be deleted. Preferably, positions 4, 6, 7, 10, 11, 15, 34, 63, 78, 83, and 160 are high-molecular-weight aliphatic amino acids (e.g., I, V, L, and M), but rare low-molecular-weight aliphatic amino acids may also be used. More preferably, I or V is at position 4, V or L at position 6, L or I at position 7, L or V at position 10, I or L at position 11, L or V at position 15, I or V at position 34, L or V at position 63, L or M at position 78, L or M at position 83, and L or M at position 160. Preferably, low-molecular-weight aliphatic amino acids (e.g., A, G, T) are used at positions 33, 39-41, 68, 74, 137, and 203, although rare high-molecular-weight aliphatic amino acids may also be used. More preferably, position 33 is G, L, or A, position 39 is G or A or may be deleted, or is S or F, position 40 is T or may be deleted, position 41 is T or A, position 68 is A or G, position 74 is G or may be deleted, position 137 is G or A, and position 203 is T or G.

[0030] Positions 72, 73, 97, and 110 of SEQ ID NO: 1 are positively charged amino acids (basic amino acids, e.g., K, R, and H). However, positions 72 and 73 may be deleted. Preferably, positions 72 and 73 are R or may be deleted, position 97 is K or R, and position 110 is H or K.

[0031] Positions 62 and 211 of SEQ ID NO: 1 are negatively charged amino acids (acidic amino acids, such as N, D, Q, and E), and preferably, position 62 is N or D and position 211 is Q or E.

[0032] Specific examples of luciferases having the amino acid sequence shown in SEQ ID NO: 1 include ALuc10 (SEQ ID NO: 13), ALuc15 (SEQ ID NO: 14), ALuc16 (SEQ ID NO: 15), ALuc17 (SEQ ID NO: 16), ALuc18 (SEQ ID NO: 17), ALuc19 (SEQ ID NO: 18), ALuc21 (SEQ ID NO: 19), ALuc22 (SEQ ID NO: 20), ALuc23 (SEQ ID NO: 21), ALuc24 (SEQ ID NO: 22), ALuc25 (SEQ ID NO: 23), ALuc26 (SEQ ID NO: 24), ALuc27 (SEQ ID NO: 25), ALuc28 (SEQ ID NO: 26), ALuc29 (SEQ ID NO: 27), ALuc30 (SEQ ID NO: 28), ALuc31 (SEQ ID NO: 29), ALuc32 (SEQ ID NO: 30), ALuc33 (SEQ ID NO: 31), ALuc34 (SEQ ID NO: 32), ALuc35 (SEQ ID NO: 33), ALuc36 (SEQ ID NO: 34), ALuc37 (SEQ ID NO: 35), ALuc38 (SEQ ID NO: 36), ALuc39 (SEQ ID NO: 37), ALuc40 (SEQ ID NO: 40), ALuc41 (SEQ ID NO: 41), ALuc42 (SEQ ID NO: 42), ALuc43 (SEQ ID NO: 43), ALuc44 (SEQ ID NO: 44), ALuc45 (SEQ ID NO: 45), ALuc46 (SEQ ID NO: 46), ALuc47 (S Examples of such ALuc32 (SEQ ID NO: 30), ALuc33 (SEQ ID NO: 31), ALuc34 (SEQ ID NO: 32), ALuc41 (SEQ ID NO: 33), Aluc42 (SEQ ID NO: 34), ALuc43 (SEQ ID NO: 35), Aluc44 (SEQ ID NO: 36), ALuc45 (SEQ ID NO: 37), Aluc46 (SEQ ID NO: 38), ALuc47 (SEQ ID NO: 39), ALuc48 (SEQ ID NO: 40), ALuc49 (SEQ ID NO: 41), Aluc50 (SEQ ID NO: 42), ALuc51 (SEQ ID NO: 43), Aluc52 (SEQ ID NO: 44), ALuc53 (SEQ ID NO: 45), ALuc55 (SEQ ID NO: 46), Aluc56 (SEQ ID NO: 47), ALuc57 (SEQ ID NO: 48), and the like. The luciferase having the amino acid sequence shown in SEQ ID NO: 1 may be deleted in part or in whole from positions 1 to 19 (secretory signal), positions 20 to 31 (antigen recognition site, etc.), and positions 217 to 221 (GS linker sequence).

[0033] The region from positions 1 to 71 of the amino acid sequence shown in SEQ ID NO: 1 may have the amino acid sequence shown in SEQ ID NO: 49. Typical examples of luciferases having this sequence include ALuc15, ALuc16, ALuc17, ALuc18, and ALuc24.

[0034] The region from positions 1 to 157 of the amino acid sequence shown in SEQ ID NO: 1 may have the amino acid sequence shown in SEQ ID NO: 50. Typical examples of luciferases having this sequence include ALuc22, ALuc25, ALuc26, ALuc27, ALuc28, and ALuc29.

[0035] Luciferase A allows the size of the luciferase to be reduced. When a fusion protein of a luciferase with a target protein, antibody, or the like is expressed intracellularly, a small luciferase facilitates normal expression of the fusion protein and reduces the likelihood of the target protein malfunctioning. Because the luminescence intensity of small luciferases is less affected by low-molecular-weight compounds, they can be suitably used as reporter proteins for drug or ligand screening. When analyzing intermolecular interactions using bioluminescence resonance energy transfer (BRET), small luciferases can detect strong signals. Small luciferases can be used as secreted luciferases. Secreted luciferases eliminate the need to lyse cells to measure luminescence intensity, allowing for the measurement of changes in gene expression over time. Small luciferases are easily expressed intracellularly, allowing for large-scale expression and purification. Small luciferases can be expressed in a variety of expression systems. Small luciferases also have excellent structural stability.

[0036] Luminescent enzyme A preferably has a high luminescence value. The peak luminescence value of luciferase A is preferably at least as high as that of known luciferases, such as NanoLuc and ALuc. Luciferases with a high luminescence value enable highly sensitive detection of luminescence, and the detection limit concentration can be lowered.

[0037] Luminescent enzyme A preferably has high thermal stability, and retains 80% or more of its activity after heat treatment for 10 minutes at 50° C., and preferably retains 80% or more of its activity after heat treatment for 10 minutes at 60° C. Luminescent enzymes with high thermal stability are less likely to be inactivated by temperature increases during transportation, and are highly practical in diagnostic and testing settings.

[0038] The enzymatic activity of luciferase A preferably has a broad base on the long-wavelength side of the emission spectrum, and the emission spectrum is shifted to the long-wavelength side compared to, for example, conventional copepod luciferases. Luciferases with an emission spectrum with a broad base on the long-wavelength side are suitable for live imaging because long wavelengths have excellent biological permeability. When coelenterazine is used as a substrate, luciferase A preferably has an emission wavelength peak of 470 nm or more and 490 nm or less, more preferably about 482 nm. When coelenterazine h is used as a substrate, luciferase A preferably has an emission wavelength peak of 470 nm or more and 490 nm or less, more preferably about 488 nm.

[0039] The C-terminus of ALuc was thought to be essential for binding to a substrate. One embodiment of luciferase A lacks the C-terminus of ALuc and retains luciferase activity. Therefore, the substrate-binding site of a polypeptide lacking the C-terminus of ALuc is thought to have a structure different from that of ALuc.

[0040] Luciferase A may have an antibody recognition site inside or at its end. Examples of antibody recognition sites include, but are not limited to, His-tag (HHHHHH) (SEQ ID NO: 67), FLAG-tag (DYKDDDDK) (SEQ ID NO: 68), Myc-tag (EQKLISEEDL) (SEQ ID NO: 69), and HA-tag (YPYDVPDYA) (SEQ ID NO: 70).

[0041] Luminescent enzyme A may have a functional peptide added to its N-terminus or C-terminus. For example, the luciferase can be localized to the cell membrane by adding a membrane localization signal (MLS) to its N-terminus or C-terminus. Even if not otherwise specified herein, when two or more peptides, including a signal peptide, are linked, the length, reading frame, etc., may be adjusted using a known linker as appropriate. The localization of the luciferase to the cell membrane facilitates the supply of substrates and oxygen from the outside, and in the case of luciferase-based luminescent probes (e.g., luminescent capsules), this has the advantage of allowing them to respond quickly to external signals.

[0042] Luciferase A preferably comprises (a) any of the amino acid sequences set forth in SEQ ID NOs: 51 to 56, and may consist of any of the amino acid sequences set forth in SEQ ID NOs: 51 to 56. The amino acid sequence set forth in SEQ ID NO: 51 is the amino acid sequence of ALuc30, an artificial luciferase, with the N-terminus and C-terminus removed (miniALuc30). The amino acid sequence set forth in SEQ ID NO: 54 is the amino acid sequence of ALuc30 with the N-terminus and intermediate sequence removed (ALuc30Δloop2N1). Similarly, the amino acid sequences set forth in SEQ ID NOs: 52 and 53 are the amino acid sequences of ALuc16 and ALuc48, artificial luciferases, with the N-terminus and C-terminus removed, respectively, and the amino acid sequences of SEQ ID NOs: 55 and 56 are the amino acid sequences of ALuc16 and ALuc48, with the N-terminus and intermediate sequence removed, respectively.

[0043] Luminescent enzyme A preferably comprises (b) an amino acid sequence that has 85% or more identity to the amino acid sequence set forth in any one of SEQ ID NOs: 51 to 56, and may consist of an amino acid sequence that has 85% or more identity to the amino acid sequence set forth in any one of SEQ ID NOs: 51 to 56. Luminescent enzyme A preferably has 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 99.5% or more identity to the amino acid sequence set forth in any one of SEQ ID NOs: 51 to 56.

[0044] Luciferase A preferably comprises (c) an amino acid sequence in which one or several amino acid residues have been deleted, substituted, inserted or added relative to the amino acid sequence set forth in any of SEQ ID NOs: 51 to 56, and may be an amino acid sequence in which one or several amino acid residues have been deleted, substituted, inserted or added relative to the amino acid sequence set forth in any of SEQ ID NOs: 51 to 56. In the present specification, "several" may mean, for example, 2 to 20, 2 to 10, 2 to 5, or 2 to 3 amino acid residues.

[0045] Luciferase A may contain an amino acid (often methionine) corresponding to the initiation codon before the amino acid sequence at position 1. SEQ ID NOs: 57 to 62 show the amino acid sequences of SEQ ID NOs: 51 to 56 with methionine added at position 1. One embodiment of the luciferase A may comprise or consist of the following (a1) to (c1): (a1) an amino acid sequence set forth in any one of SEQ ID NOs: 57 to 62; (b1) an amino acid sequence having 85% or more identity to any of the amino acid sequences set forth in SEQ ID NOs: 57 to 62, or (c1) An amino acid sequence in which one or several amino acid residues have been deleted, substituted, inserted or added to the amino acid sequence set forth in any one of SEQ ID NOs: 57 to 62. Luciferase A preferably has 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 99.5% or more identity to the amino acid sequence set forth in any one of SEQ ID NOs: 57 to 62.

[0046] <Polypeptide> The first and second polypeptides are portions of luciferase A. The first and second polypeptides comprise, for example, a portion of any of the amino acid sequences (A) to (C) above, and preferably a portion of any of the amino acid sequences (a) to (c) above. The first and second polypeptides do not generally have luciferase activity by themselves. As shown in FIG. 1 , the first polypeptide 11 develops luciferase activity upon contact with the second polypeptide 12. That is, the first polypeptide can emit light in the presence of the second polypeptide and a substrate. The first and second polypeptides can be brought into contact with or close to each other by, for example, mixing a solution containing the first polypeptide with a solution containing the second polypeptide.

[0047] As used herein, "not having luminescent enzyme activity" means that a sample is judged to have no luminescent enzyme activity when, for example, in the method of Experiment 11-1 described below, the luminescence value is similar to that of samples ranked 4th to 120th. "Having luminescent enzyme activity" means that a sample is judged to have luminescent enzyme activity when, for example, in the method of Experiment 11-1 described below, the luminescence value is sufficiently higher, for example, at least two times, preferably at least three times, than the luminescence value of samples judged to have no luminescent activity.

[0048] The first polypeptide and the second polypeptide are small in size because they are further portions of small luciferase A. Using small polypeptides as probes makes it easier to normally express fusion proteins with target proteins. Furthermore, small polypeptides are less likely to inhibit the function of the target protein. Small polypeptides are less likely to inhibit the interaction of the target protein with other molecules. The luciferase activity generated by contact or proximity between the first polypeptide and the second polypeptide preferably has the same properties as the activity of luciferase A. Each polypeptide may have a functional peptide or antibody recognition site attached, as with luciferase A.

[0049] The amino acid sequence constituting the first polypeptide is usually different from the amino acid sequence constituting the second polypeptide. The amino acid sequence constituting the first polypeptide and the amino acid sequence constituting the second polypeptide may or may not overlap partially. If they do not overlap, the amino acid sequence constituting the first polypeptide and the amino acid sequence constituting the second polypeptide may be contiguous in the amino acid sequence constituting luciferase A (the amino acid sequence set forth in SEQ ID NO: 1), or one or several amino acid residues may be missing. An example of a combination of the first polypeptide and the second polypeptide is a combination of the N-terminal polypeptide and the C-terminal polypeptide when luciferase A is divided into two parts.

[0050] At least one of the first polypeptide and the second polypeptide is selected from the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NOs: 51 to 53, and is selected from the amino acid sequence set forth in SEQ ID NO: 51, and is selected from the amino acid sequence set forth in SEQ ID NO: 52, and is selected from the amino acid sequence set forth in SEQ ID NO: 53, and is selected from the amino acid sequence set forth in SEQ ID NO: 52, and is selected from the amino acid sequence set forth in SEQ ID NO: 53, and is selected from the amino acid sequence set forth in SEQ ID NO: 52, and is selected from the amino acid sequence set forth in SEQ ID NO: 53, and is selected from the amino acid sequence set forth in SEQ ID NO: 51, and is selected from the amino acid sequence set forth in SEQ ID NO: 52, and is selected from the amino acid sequence set forth in SEQ ID NO: 53, and is selected from the amino acid sequence set forth in SEQ ID NO: 51, and is selected from the amino acid sequence set forth in SEQ ID NO: 52, and is selected from the amino acid sequence set forth in SEQ ID NO: 53, and is selected from the amino acid sequence set forth in SEQ ID NO: 51, and is selected from the amino acid sequence set forth in SEQ ID NO: 51, and is selected from the amino acid sequence set forth in SEQ ID NO: 52, and is selected from the amino acid sequence set forth in SEQ ID NO: 51 ... positions 4 to 65, 4 to 66, 4 to 67, 4 to 70, 4 to 71, 4 to 72, 4 to 73, 4 to 74, 4 to 75, 4 to 76, 4 to 77, 4 to 78, 4 to 79, 4 to 80, 4 to 84, 4 to 88, 4 to 89, 4 to 90, 4 to 91, 4 to 92, 4 to 93, 4 to 101, 4 to 102, 4 to 103, 4 to 104, and 4 to 105, or may consist of the amino acid sequence. At least one of the first polypeptide and the second polypeptide may have, in addition to the above sequence, one or more amino acid residues corresponding to positions 1 to 3 of the amino acid sequence set forth in SEQ ID NO:51 at its N-terminus. At least one of the first polypeptide and the second polypeptide is selected from the group consisting of positions 21 to 120, 22 to 120, 23 to 120, 24 to 120, 25 to 120, 34 to 120, 35 to 120, 36 to 120, 37 to 120, 38 to 120, 39 to 120, 40 to 120, 41 to 120, 42 to 120, 43 to 120, 44 to 120, 45 to 120, 46 to 120, 47 to 120, 48 to 120, 49 to 120, 50 to 120, 51 to 52, 52 to 120, 53 to 120, 54 to 120, 55 0th place, 37th place - 120th place, 38th place - 120th place, 46th place - 120th place, 47th place - 120th place, 48th place - 120th place, 49th place - 120th place, 50th place - 120th place, 57th place - 120th place , 58th to 120th, 59th to 120th, 60th to 120th, 61st to 120th, 63rd to 120th, 64th to 120th, 65th to 120th, 66th to 120th, 6 7th to 120th, 68th to 120th, 69th to 120th, 70th to 120th, 71st to 120th, 72nd to 120th, 73rd to 120th, 74th to 120th, 75th 1st to 120th, 76th to 120th, 77th to 120th, 78th to 120th, 79th to 120th, 80th to 120th, 81st to 120th, 85th to 120th, 89th to The polypeptide may have or consist of an amino acid sequence corresponding to any one selected from positions 120, 90 to 120, 91 to 120, 92 to 120, 93 to 120, 94 to 120, 102 to 120, 103 to 120, 104 to 120, 105 to 120, and 106 to 120. At least one of the first polypeptide and the second polypeptide may have, in addition to the above sequence, one or more amino acid residues corresponding to positions 121 and 122 of the amino acid sequence set forth in SEQ ID NO:51 at its C-terminus.

[0051] At least one of the first polypeptide and the second polypeptide is preferably (1) any of the amino acid sequences of positions 4 to 77 (SEQ ID NO: 76), 4 to 22 (SEQ ID NO: 77), 4 to 58 (SEQ ID NO: 78), 4 to 64 (SEQ ID NO: 79), 4 to 72 (SEQ ID NO: 80), 23 to 120 (SEQ ID NO: 81), 78 to 120 (SEQ ID NO: 82), 65 to 120 (SEQ ID NO: 83), 73 to 120 (SEQ ID NO: 84), and 104 to 120 (SEQ ID NO: 85) in the amino acid sequence set forth in SEQ ID NO: 51; (2) an amino acid sequence having 85% or more identity to any of the amino acid sequences set forth in SEQ ID NOs: 76 to 85, or (3) The amino acid sequence has one or several amino acid residues deleted, substituted, inserted or added to the amino acid sequence set forth in any one of SEQ ID NOs: 76 to 85. At least one of the first polypeptide and the second polypeptide preferably has an identity of 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 99.5% or more to the amino acid sequence set forth in any of SEQ ID NOs: 76 to 85.

[0052] When the first polypeptide has the amino acid sequence set forth in SEQ ID NO:76 or SEQ ID NO:77, the second polypeptide may have the amino acid sequence set forth in SEQ ID NO:81 or SEQ ID NO:82. The combinations of the first and second polypeptides may be interchanged. A preferred combination of the first and second polypeptides is SEQ ID NO:77 with SEQ ID NO:81, or SEQ ID NO:76 with SEQ ID NO:81. Other preferred combinations of the first and second polypeptides are SEQ ID NO:78 with SEQ ID NO:81, SEQ ID NO:79 with SEQ ID NO:83 or SEQ ID NO:84, and SEQ ID NO:80 with SEQ ID NO:81, SEQ ID NO:84, or SEQ ID NO:85.

[0053] As shown in FIG. 2 , the first polypeptide 11 may be connected to another molecule, for example, a first target protein 21. In a fusion protein comprising the polypeptide 11 and the first target protein 21, the first polypeptide 11 and the first target protein 21 may be connected by a linker sequence. The first target protein may be a target whose interaction with another target molecule, for example, a second target protein 22, is to be detected. The first target protein 21 and the second target protein 22 may form a dimer, or may form a dimer only in the presence of a small molecule 30. The second target protein 22 is connected to a second polypeptide 12. The second target protein 22 and the second polypeptide 12 may be connected by a linker sequence. When the first target protein 21 and the second target protein 22 interact, the first polypeptide 11 and the second polypeptide 12 are brought into close proximity or contact with each other, and luminescence is detected in the presence of a substrate.

[0054] The first polypeptide 11 and the second polypeptide 12 may not be connected. The first polypeptide 11 and the second polypeptide 12 may be connected, for example, by a linker sequence. As shown in FIG. 3, the first target protein 21, the first polypeptide 11, the second polypeptide 12, and the second target protein 22 may be connected. Such a fusion protein is also referred to as a circular permutant. A fusion protein comprising the first target protein 21, the first polypeptide 11, the second polypeptide 12, and the second target protein 22 may be connected by a linker sequence. When the first target protein 21 and the second target protein 22 interact, the first polypeptide 11 and the second polypeptide 12 come into close proximity or contact, and luminescence is detected in the presence of a substrate. Compared to when the first target protein 21 and the second target protein 22 are separated, the circular permutant makes it easier to detect the interaction between the first target protein 21 and the second target protein 22.

[0055] <Nucleic acid encoding a polypeptide> A nucleic acid according to one embodiment of the present invention encodes the above-described polypeptide or fusion protein. The above-described polypeptide or fusion protein can be produced from the nucleic acid. The nucleic acid is preferably DNA or RNA. The nucleic acid encoding the polypeptide may contain an initiation codon at the 5' end of the base sequence corresponding to the above-described polypeptide and a stop codon at the 3' end of the base sequence. The nucleic acid may also contain an intron sequence.

[0056] The nucleic acid according to one embodiment includes a nucleic acid comprising a nucleotide sequence in which a codon encoding each amino acid in the coding region is substituted with another codon encoding the same amino acid. From the viewpoint of improving the expression of the polypeptide, the nucleic acid according to one embodiment may be a nucleic acid comprising a nucleotide sequence in which the codon usage has been changed to suit the host organism or the type of transformed cell.

[0057] The nucleic acid according to this embodiment can be obtained by chemical synthesis, PCR, or the like.

[0058] <Vector> A vector according to one embodiment of the present invention comprises the nucleic acid described above. A vector is a nucleic acid molecule capable of amplifying and maintaining DNA, and examples thereof include expression vectors and cloning vectors. In one example, the nucleic acid described above is inserted into an expression vector and introduced into a host cell, etc., to express the polypeptide or fusion protein described above. The expression vector may have a promoter sequence and a terminator sequence for expressing the incorporated gene. The vector according to this embodiment can be obtained by inserting the nucleic acid described above into an appropriate vector.

[0059] The vector may be, for example, a bacterial plasmid-derived vector, a yeast plasmid-derived vector, a viral vector, a cosmid vector, a phagemid vector, an artificial chromosome vector, or the like. Examples of the vector include pBR322, pUC plasmid vectors, and pET-based plasmid vectors. Specific examples of the vector include pUC19, pUC18, pUC119, pBluescriptII, and pET32 when Escherichia coli is used as the host cell. When mammalian cells are used as the host cell, examples of the vector include pRc / RSV, pRc / CMV, retroviral vectors, adenoviral vectors, and adeno-associated viral vectors.

[0060] The nucleic acid encoding the second polypeptide or a fusion protein comprising the second polypeptide and a second target protein may be contained in the same vector as the vector comprising the nucleic acid encoding the first polypeptide, or may be contained in a different vector.

[0061] <Transformed cells> A transformed cell according to one embodiment of the present invention is a cell into which the above-described nucleic acid has been introduced. The nucleic acid may be introduced into the cell in the form of a vector. The transformed cell can express a first polypeptide or a fusion protein containing the first polypeptide. Preferably, the same transformed cell also expresses a second polypeptide or a fusion protein containing the second polypeptide and a second target protein. These may be secreted into the supernatant. Examples of methods for introducing nucleic acids into cells include chemical methods such as the calcium phosphate method, the DEAE-dextran method, and the cationic liposome method; biological methods such as adenovirus vectors, vaccinia virus vectors, retrovirus vectors, and HVJ liposomes; and physical methods such as electroporation, direct DNA injection, and gene guns. An appropriate introduction method can be selected depending on the cells to be introduced.

[0062] The cells into which the nucleic acid is introduced may be eukaryotic or prokaryotic cells, including bacteria, fungi, plant cells, animal cells, and insect cells. The cells may be yeast, Escherichia coli, or mammalian cells, including mammalian cells such as humans, cows, horses, sheep, monkeys, pigs, mice, rats, hamsters, guinea pigs, rabbits, and dogs.

[0063] <Protein interaction analysis method> A protein interaction analysis method according to one embodiment of the present invention utilizes the above-described reagent kit. This method allows the detection of an interaction between two proteins by luminescence. The interaction between the two proteins may be mediated by a small molecule (ligand). This method allows the detection of the presence of the small molecule by luminescence. Because the polypeptide used in this method has a small molecular weight, it is easy to successfully express a fusion protein with the target protein. Furthermore, the polypeptide used in this method is less likely to inhibit the function of the target protein.

[0064] An example of a protein interaction analysis method includes a step of mixing the first polypeptide or a fusion protein comprising the first polypeptide with the second polypeptide or a fusion protein comprising the second polypeptide in the presence of a luminescent substrate. The protein interaction analysis method may further include a step of preparing a plasmid that expresses the fusion protein, a step of expressing the first polypeptide or the fusion protein comprising the first polypeptide in a cell, a step of expressing the second polypeptide or the fusion protein comprising the second polypeptide in a cell, a step of recovering the first polypeptide or the fusion protein comprising the first polypeptide from the cell or its culture supernatant, a step of recovering the second polypeptide or the fusion protein comprising the second polypeptide from the cell or its culture supernatant, and a step of detecting luminescence.

[0065] <Reporter analysis> The above-mentioned polypeptides can also be used as probes to perform various reporter analyses. The first polypeptide, in combination with the second polypeptide, can be used as a substitute for luminescent or fluorescent substances in reporter analyses using conventional luciferases or various fluorescent proteins.

[0066] As used herein, reporter analysis refers to an analytical method that uses a first polypeptide or a second polypeptide as a reporter protein to observe the behavior of a target protein or target gene in cells in response to an external stimulus by measuring the presence or absence of luminescence, the amount of luminescence, the timing of luminescence, or the location of luminescence. Specifically, reporter analysis can be said to be a method that qualitatively or quantitatively measures the expression location, timing, or amount of expression of a target gene as the location, timing, or amount of luminescence. Reporter analysis may also be used in multiplexing with multiple enzymes or proteins that emit light of different wavelengths.

[0067] Reporter analysis can be performed in vivo, such as in mammals, in cultured cells, or in test tubes. When used in vivo, such as in a living organism, a reporter gene consisting of a nucleic acid encoding the amino acid sequence constituting the above-mentioned polypeptide is linked to a target gene, inserted into a vector, and then introduced into the target cells. Cultured cells include mammalian cells commonly used in genetic engineering, such as COS cells, CHO-K1 cells, HeLa cells, HEK293 cells, and NIH3T3 cells, as well as yeast, bacteria such as E. coli, and insect cells.

[0068] Below, we will explain the application of the above polypeptides to each of the three analytical methods, dividing the reporter analysis methods of the present invention into three types: "basic," "inducible," and "activatable," which are the three classifications presented in Niu et al., Theranostics, 2, 2012, 413.

[0069] (1)Basic method The basic method is the simplest reporter analysis system, in which a probe is attached to and labeled with a target protein whose behavior is to be investigated. When applying the basic method using a first polypeptide as a probe, a fusion protein containing the first polypeptide and the target protein or a protein that binds to the target protein can be created. This method differs from other reporter analysis methods in that the fusion protein is expressed under a non-regulated promoter. Fusion proteins can also be used for in vivo imaging of target proteins.

[0070] Fusion proteins include (i) proteins expressed as a single unit from nucleic acids encoding a fusion protein containing a first polypeptide and a target protein or a protein (including peptides) that recognizes the target protein, and (ii) proteins in which the first polypeptide and the target protein or the protein that recognizes the target protein are expressed separately and then linked by a chemical reaction. Examples of means for linking separately expressed proteins by chemical reaction include linkage using a crosslinker, linkage utilizing the avidin-biotin binding ability, and linkage utilizing the chemical reactivity of amino acid residues.

[0071] Fusion proteins include probe-labeled antibodies, in which a probe is linked to an antibody. In these fusion proteins, chimeric DNA is created in which a probe sequence is linked upstream or downstream of the cDNA for the antibody's single-chain variable fragment (scFv). The DNA is inserted into an appropriate expression vector, introduced into cells, and expressed to obtain the fusion protein.

[0072] (2) Inducible method The inducible method differs from the basic method in that reporter expression is controlled by a promoter. The application of luciferases as reporter proteins to the inducible method has traditionally been used to analyze the timing and expression level of genes when recombinant proteins are produced by recombinant DNA technology, and is particularly widely used as an indicator of changes in expression timing and expression level in response to external stimuli. Examples of analytical systems that can be used in the inducible method include reporter gene assays, yeast two-hybrid assays, mammalian two-hybrid assays, bioluminescence resonance energy transfer (BRET), protein splicing assays (PSA), protein complementation assays (PCA), and circular permutation assays. Using the polypeptides of the present invention as reporter genes in these analytical methods can dramatically improve the measurement performance of the assays.

[0073] (i) Reporter gene assay Reporter gene assays are widely used as a means of analyzing the activation of transcription factors and the regulation of gene expression by external stimuli. One example is the detection of endocrine disruptors (environmental hormones) that disrupt signal transduction mediated by nuclear receptors. Expression of target genes (e.g., hormone-responsive genes) associated with nuclear receptor-mediated signal transduction is triggered by binding of a ligand-receptor complex to the cis-region (hormone response element) that regulates the transcription of the gene. Plasmids incorporating reporter genes downstream of the cis-region of various hormone-responsive genes are introduced into cells, and the amount of hormone molecules or endocrine disruptors that can act as ligands is detected as luminescence values.

[0074] In the reporter gene assay method, firefly luciferase has traditionally been widely used, but it has the following drawbacks: (1) its large molecular weight means it takes a long time to express, placing a heavy burden on the host cells; and (2) its low luminescence intensity means that it usually takes 1 to 2 days after stimulation for sufficient luciferase (reporter) levels to accumulate. However, these problems can be resolved by selecting the above polypeptide as a probe.

[0075] The use of the above polypeptide as a probe has the advantage that the reporter's luminescence intensity is extremely high, allowing measurements to be made very quickly after stimulation. Therefore, measurement time can be significantly shortened compared to conventional reporter proteins, and the luminescence is highly stable over time, making it possible to measure luminescence even in cell lines with poor gene transfer efficiency. Furthermore, because the wavelength shifts to the long wavelength side, permeability through cell membranes and skin is increased, resulting in reduced background values and high measurement accuracy.

[0076] Specifically, to apply the above polypeptide to a reporter gene assay, the probe sequence is ligated to a known eukaryotic cell expression vector carrying a special promoter upstream, and the vector is introduced into a eukaryotic cell. After a certain period of time, luminescence intensity is measured under conditions with and without a signal (stimulus). The known pTransLucent vector can be used as an expression vector for reporter gene assays that can carry the polypeptide, and the vector can be easily loaded using known methods.

[0077] (ii) Two-hybrid assay The two-hybrid method is a method for investigating protein-protein interactions. The yeast two-hybrid (Y2H) system was first developed in 1989 using yeast (Saccharomyces cerevisiae). Taking advantage of the fact that the DNA-binding domain (GAL4 DBD) and transcription activation domain of the transcriptional activator GAL4 protein can be separated, the GAL4 DBD can be expressed as a fusion protein with a given protein A (bait), and the interaction with a protein B (prey) fused with a transcription activation domain (TA) simultaneously expressed in cells can be determined. When proteins A and B bind, the DBD and TA come into close proximity, and the DNA-binding domain (DBD) binds to the "UASG" base sequence, promoting the expression of the downstream polypeptide. By combining the first and second polypeptides and monitoring bioluminescence in the presence of their specific substrates, the affinity of both proteins A and B can be measured, allowing for screening of proteins or peptides that interact with protein A (bait). In this case, protein B (prey) can also be provided by an expression library.

[0078] Host cells are not limited to yeast cells, but also include bacteria such as Escherichia coli, mammalian cells, and insect cells. In this case, in addition to the yeast-derived transcriptional activator GAL4 DBD, the Escherichia coli-derived repressor protein "LexA" can also be used. DNA encoding these is ligated to DNA encoding a bait protein (i.e., any protein A) such as the ligand-binding domain of a ligand-responsive transcriptional regulator, and then ligated downstream of a promoter functional in the host cell. On the other hand, examples of "transcriptional activation domains of transcriptional activators" include the GAL4 transcriptional activation domain, the E. coli-derived B42 acidic transcriptional activation domain, and the herpes simplex virus VP16 transcriptional activation domain. DNA encoding these transcriptional activation domains is ligated to DNA encoding a prey protein (i.e., any protein B) and then ligated downstream of a promoter functional in the host cell.

[0079] Specifically, an example of a vector that has DNA encoding the DNA-binding domain of the transcriptional regulatory factor GAL4 and can be used in budding yeast as a host cell is the plasmid pGBT9 (Clontech). An example of a vector that has DNA encoding the transcription activation domain of GAL4 and can be used in budding yeast is the plasmid pGAD424 (Clontech). Furthermore, examples of vectors that have DNA encoding the GAL4 DNA-binding domain and can be used in mammalian cells include pM (Clontech) and pBIND (Promega). Examples of vectors that have DNA encoding the transcription activation domain of herpes simplex virus VP16 and can be used in mammalian cells include pVP16 (Clontech) and pACT (Promega). Furthermore, examples of vectors that have DNA encoding the LexA DNA-binding domain and can be used in mammalian cells include pLexA (Clontech). Examples of vectors that have DNA encoding B42 and can be used in mammalian cells include pB42AD (Clontech).

[0080] For example, a vector can be constructed in which the first polypeptide is inserted downstream of the GAL4-binding region ("UASG"), etc. In the case of a mammalian host, the commercially available pG5Luc vector (Promega) or pFR-Luc vector (Stratagene) can be used, and the first polypeptide can be easily inserted in place of the firefly luciferase contained in the vector using well-known methods. It can also be used in place of chloramphenicol acetyltransferase (CAT) in the commercially available pG5CAT vector (Clontech).

[0081] (3) Activatable Method The activatable method is a reporter assay that utilizes the ability of a first polypeptide, in combination with a second polypeptide, to emit light in response to ligand stimulation. Typical examples include single-molecule bioluminescent probes and luminescent capsules, and can also be applied to protein complementation assays (PCAs) and protein splicing assays (PSAs).

[0082] (i) Production of luminescent fusion proteins (luminescent capsules) By attaching a membrane localization signal (MLS) to the C-terminus of the polypeptide, the polypeptide can be localized to the cell membrane. Molecular design of the luminescent enzyme to localize to the cell membrane facilitates the supply of substrate and oxygen, enabling extremely bright and stable bioluminescence visualization. In this case, a gene for any polypeptide or protein can be inserted as cargo between the polypeptide and the nucleic acid encoding the signal peptide. This allows efficient transport of the cargo protein to the cell membrane surface, and the transported site will glow. For example, if the DEVD or IETD sequence, which responds to cell death, is inserted as cargo at the junction of each protein, it will actively respond to the activity of caspase-3 or caspase-8 during cell death and function as a visualization system. Luminescent fusion proteins with this structure are also called "luminescent capsules." Luminescent capsules can also be used to evaluate the toxicity of chemicals.

[0083] Compared to conventional luminescent probes, luminescent capsules have the advantage of exhibiting extremely bright and stable luminescence and responding to analytes that cannot penetrate cell membranes. These luminescent capsules have a basic structure in which a membrane localization signal (MLS) is attached to the C-terminus of the luminescent enzyme itself. The above polypeptides may be connected in tandem to enhance luminescence intensity. The luminescent capsules facilitate observation by visualizing the effects of compounds that induce morphological changes on the cell surface, such as cell death, as morphological changes on the cell membrane surface. Preferably, a polypeptide that induces morphological changes on the cell membrane surface or its partial recognition sequence, specifically, the full-length or partial recognition sequence of a G-protein coupled receptor (GPCR) or c-Src, can be inserted between the C-terminus of the luminescent enzyme itself and the MLS. Cell death can be visualized by inserting a cell death-inducing polypeptide or its recognition sequence as cargo between the C-terminus of the luminescent enzyme itself and the MLS. More specifically, the insertion of peptide sequences (usually 20 amino acids or less, preferably 10 amino acids or less) recognized by various caspases, proteases (e.g., serine proteases, cysteine proteases), and digestive enzymes (e.g., trypsin and amylase) or amino acid sequences containing the DEVD or IETD sequences as cargos enables visualization of cell death due to caspase-3 activity. Furthermore, by linking a fluorescent protein or other luminescent enzyme as cargo between the polypeptide and the MLS, the amount of light emitted at the cell membrane surface is enhanced, making it easier to observe cell membrane morphology. The luminescent capsules respond to ligands that cannot penetrate the cell membrane, enabling screening for a wide range of stimuli.

[0084] The luminescent capsule is a luminescent fusion protein in which any protein or polypeptide to be expressed on the cell membrane surface is inserted between the C-terminal side of the above-mentioned polypeptide and a membrane localization signal (MLS), and typically includes: (a) a luminescent fusion protein in which a fluorescent protein or a luminescent enzyme (which may be an enzyme other than the above-mentioned polypeptides) is inserted between the C-terminus of the polypeptide and the membrane localization signal (MLS), or (b) It may be a luminescent fusion protein in which a polypeptide that alters the morphology of a cell membrane or a polypeptide of 20 amino acids or less, preferably 10 amino acids or less, recognized by the polypeptide is inserted between the C-terminus of the polypeptide and the membrane localization signal (MLS). As a polypeptide that alters the morphology of a cell membrane, a polypeptide that induces cell death is preferred, and a polypeptide of 20 amino acids or less that includes a caspase and its recognition sequence "DEVD" or "IETD" is particularly preferred.

[0085] (ii) Application to luminescent probes By incorporating the above polypeptide into a single-molecule or two-molecule luminescent probe, the presence or absence of a ligand and the activity of the ligand can be observed with high brightness. The components of the probe are [1] a luminescent enzyme split into two (N- and C-terminal fragments), [2] a ligand-binding protein that responds to the target ligand, and [3] a recognition protein that recognizes the binding of the ligand to the ligand-binding protein. When the recognition protein recognizes the binding of a ligand to the ligand-binding protein in this luminescent probe, the two split enzyme fragments complement each other, thereby changing the enzyme activity. The high brightness and stability of the split enzyme enable an improved detection limit and highly reliable measurements.

[0086] A single-molecule luminescent probe is a type of known bioluminescent probe characterized by the integration of all components used for visualization and imaging into a single fusion molecule. For example, it is a fusion protein containing, as basic components, the N- and C-terminal fragments of a luciferase split into two, a ligand-binding protein, and a recognition protein for the ligand-binding protein. A two-molecule luminescent probe refers to a type of bioluminescent probe in which the N- and C-terminal fragments of a luciferase are present in a fusion protein containing the ligand-binding protein and a fusion protein containing the recognition protein, respectively.

[0087] Specific techniques for using the polypeptide as a single-molecule-format luminescent probe are known. Specifically, a chimeric DNA is designed to encode a luminescent probe (fusion protein) in which a first polypeptide, a second polypeptide, a ligand-binding protein, and a peptide sequence that recognizes the conformational change that occurs when a ligand binds to the protein are linearly linked. Generally, the chimeric DNA is subcloned into a vector suitable for the cell in which it is desired to express the chimeric DNA, and the vector is then introduced into the cell for intracellular expression. However, a regulatory sequence can also be attached upstream of the chimeric DNA and introduced directly into the cell. Here, the target cells are preferably mammalian cells, including humans, and may be cells present in vivo or cultured cells that maintain their original functions. Prokaryotic cells, such as yeast cells, insect cells, and Escherichia coli, may also be used. The specific type of vector is not particularly limited, and a vector that can be expressed in the host used for expression can be appropriately selected. Known transfection methods, such as microinjection and electroporation, can be used to introduce the chimeric DNA into cells. Alternatively, a lipid-based intracellular introduction method (BioPORTER (Gene Therapy Systems), Chariot (Active Motif), etc.) can also be used.

[0088] Bioluminescent probes using the above polypeptides are introduced into cells as chimeric DNA and then expressed as fusion proteins within the cells. Therefore, the properties and activity of the ligand can be evaluated by measuring the change in the amount of luminescence emitted from the transformed cells after ligand stimulation.

[0089] When constructing the above-mentioned polypeptide into a bioluminescent probe, the "ligand-binding protein" that can be loaded together with the polypeptide is intended to be a protein whose ligand binds to its ligand-binding site. Ligand-binding proteins can, for example, undergo conformational changes, phosphorylation, or protein-protein interactions upon ligand binding. Examples of such ligand-binding proteins include nuclear receptors (NRs), cytokine receptors, and various protein kinases, whose ligands are hormones, chemicals, or signaling proteins. The ligand-binding protein is appropriately selected depending on the target ligand. The ligand that binds to the ligand-binding protein is not particularly limited as long as it binds to the ligand-binding protein. It can be an extracellular ligand that is internalized into the cell from the outside, or an intracellular ligand that is produced within the cell in response to an extracellular stimulus. The extracellular ligand can be, for example, an agonist or antagonist of a receptor protein (e.g., a nuclear receptor, a G protein-coupled receptor, etc.). Examples of such extracellular ligands include signaling proteins such as cytokines, chemokines, and insulin that specifically bind to proteins involved in intracellular signal transduction, intracellular second messengers, lipid second messengers, phosphorylated amino acid residues, and G protein-coupled receptor ligands.

[0090] For example, when intracellular second messengers, lipid second messengers, etc. are targeted as ligands, the binding domain of each second messenger can be used as the ligand-binding protein. Second messengers refer to different types of intracellular messengers that are newly generated within cells when extracellular messengers such as hormones and neurotransmitters bind to receptors present on the cell membrane. Examples of such second messengers include cGMP, AMP, PIP, PIP2, PIP3, inositol triphosphate (IP3), IP4, Ca 2+ , diacylglycerol, arachidonic acid, etc. For example, the second messenger Ca2+ For this, calmodulin (CaM) can be used as a ligand binding protein.

[0091] (iii) Bioluminescence Resonance Energy Transfer (BRET) The above polypeptides can be used in any method for detecting ligand-protein interactions or protein-protein interactions. Energy transfer from the luminescent donor to the fluorescent acceptor results in a shift in the spectral distribution of light emission. This energy transfer can enable real-time monitoring of protein-protein interactions or ligand-protein interactions in vitro or in vivo. As an example, a fusion protein is prepared by connecting a first polypeptide to a target molecule (target protein, ligand, etc.), and a fusion protein is prepared by connecting a protein or ligand that binds to the target molecule to a fluorescent protein. When the two polypeptides and the second polypeptide are brought into close proximity, a BRET signal is detected.

[0092] (iv) Protein Complementation Assay (PCA) The above polypeptides may be used in methods for detecting ligand-protein or protein-protein interactions or proximity, such as protein complementation assays (PCA) or enzyme fragmentation assays. PCA provides a means for detecting the interaction of two biomolecules, e.g., polypeptides. For example, a first polypeptide and a second polypeptide are fused to molecules whose proximity is to be examined, respectively. If the target molecules interact, the two split polypeptides will interact to form a complete luminescent enzyme, and luminescence will be detected.

[0093] (v) Intracellular imaging Genes encoding the above polypeptides can be stably introduced into various cell lines. Intracellular imaging using luminescent enzymes can be performed using known methods. As an example, the polypeptides can be stably introduced into undifferentiated cells within embryos, ES cells, and iPS cells. Because these cells themselves do not emit light, it has been extremely difficult to explore the molecular phenomena and tissue specificity occurring within them. To overcome this difficulty, a molecular probe containing the polypeptide is first introduced into somatic cells, followed by the creation of embryos and differentiation into various organ tissues. This allows for highly sensitive measurement of the specific molecular phenomena occurring in each organ.

[0094] By linking an appropriate signal peptide to the above polypeptide, it can be used for high-brightness imaging of each organelle. For example, adding the "MLCCMRRTKQV sequence" (SEQ ID NO: 63) derived from GAP-43 to the N- or C-terminus of the polypeptide allows it to be localized to the cell membrane. Adding the "GRKKRRQRRR sequence" (SEQ ID NO: 64) allows it to be localized to the cytoplasm. Adding the "KDEL" (SEQ ID NO: 65) allows it to be localized to the endoplasmic reticulum (ER), and adding the "DPKKKRKV sequence" (SEQ ID NO: 66) allows it to be localized to the cell nucleus. By adding antigen sites such as a HIS-tag (HHHHHH) (SEQ ID NO: 67), a FLAG-tag (DYKDDDDK) (SEQ ID NO: 68), a Myc-tag (EQKLISEEDL) (SEQ ID NO: 69), an HA-tag (YPYDVPDYA) (SEQ ID NO: 70), a V5-tag (GKPIPNPLLGLDST) (SEQ ID NO: 71), or a T7-tag (MASMTGGQQMG) (SEQ ID NO: 72), these proteins can be used for immunostaining and separation / purification in cell-free systems. Well-known immunostaining and immunocytochemistry techniques can be used for this purpose.

[0095] Other terms and concepts in this specification are defined in detail in the description of the embodiments and examples of the invention. The terms are basically those defined by the IUPAC-IUB Commission on Biochemical Nomenclature or based on the meanings of terms commonly used in the relevant field. Furthermore, the various techniques used to implement the invention, except for those specifically cited, can be easily and reliably implemented by those skilled in the art based on known literature.For example, genetic engineering and molecular biological techniques are covered in J. Sambrook, E.F. Fritsch & T. Maniatis, "Molecular Cloning: A Laboratory Manual (2nd edition)", Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (1989); D.M.Glover et al., ed., "DNA Cloning", 2nd ed., Vol. 1 to 4, (The Practical Approach Series), IRL Press, Oxford University Press (1995); Ausubel, F.M. et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York, NY, 1995; "Continued Biochemistry Experimental Lectures 1, Genetic Research Methods II", edited by the Japanese Biochemical Society, Tokyo Kagaku Dojin (1986); "New Biochemistry Experimental Lectures 2, Nucleic Acids III (Recombinant DNA Technology)", edited by the Japanese Biochemical Society, Tokyo Kagaku Dojin (1992); R.Wu, ed., "Methods in Enzymology", Vol. 68 (Recombinant DNA), Academic Press, New New York (1980); R. Wu et al. ed., "Methods in Enzymology," Vol. 100 (Recombinant DNA, Part B) & 101 (Recombinant DNA, Part C), Academic Press, New York (1983); R. Wu et al. ed., "Methods in Enzymology," Vol. 153 (Recombinant DNA, Part D), 154 (Recombinant DNA, Part E) & 155 (Recombinant DNA, Part F), Academic Press, New York (1987), or methods described in the literature cited therein, or methods substantially similar to or modified thereto.Furthermore, various proteins and peptides used in the present invention, as well as the DNA encoding them, can be obtained from existing databases (URL: http: / / www.ncbi.nlm.nih.gov / , etc.). [Example]

[0096] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples.

[0097] Experiment 1: Construction of miniALuc plasmid ALuc30wt is a luciferase with an amino acid sequence corresponding to positions 20 to 221 of SEQ ID NO: 1. The signal sequence of ALuc30 (SEQ ID NO: 28) was removed to obtain the amino acid sequence from positions 20 to 212, designated ALuc30wt. The sequence of ALuc30wt is shown in Figure 4. ALuc30wt has a molecular weight of approximately 21 kDa. From the N-terminus, ALuc consists of two helices, a loop, a helix, a helix-loop-helix, two small helices, a helix, and a small helix. As shown in Figure 4, miniALuc30 (SEQ ID NO: 51) was constructed, containing amino acids 54 to 175 of ALuc30wt, omitting the N- and C-termini. miniALuc30 was inserted into the pcDNA3.1(+) vector (Thermo Fisher Scientific). Using the same method, expression plasmids for miniALuc16 (SEQ ID NO: 52) and miniALuc48 (SEQ ID NO: 53), which have amino acid sequences corresponding to miniALuc30, were prepared using ALuc16 (SEQ ID NO: 15) and ALuc48 (SEQ ID NO: 40) instead of ALuc30. The size of miniALuc30 was 13 kDa. Each mutant was tagged with a His-tag at the N-terminus and a Flag-tag at the C-terminus.

[0098] The amino acid sequence identity between miniALuc30 and miniALuc16 was 96% (Fig. 5), between miniALuc30 and miniALuc48 was 85% (Fig. 6), and between miniALuc48 and miniALuc16 was 90% (Fig. 7).

[0099] Experiment 2: Measurement of luminescence value of miniALuc (1) African green monkey kidney-derived COS-7 cells were seeded onto a 24-well dish and allowed to reach subconfluence the next day. (2) 25 μL of Opti-MEM (Thermo Fisher Scientific) was mixed with 400 ng (2 μL) of plasmid and 1 μL of P3000 (Invitrogen). (3) 25 μL of Opti-MEM was mixed with 1 μL of lipofectamine 3000 (Invitrogen). (4) (2) and (3) were mixed and incubated at room temperature for 5 minutes. (5) The mixture was added to the medium in (1). (6) 500 μL of Dulbecco's modified Eagle's medium was added, and the cells were cultured at 37°C for 1 day, after which the medium was collected. The medium contained the secreted and expressed luciferase. (7) The substrate coelenterazine was added to 100 μL of the medium to a final concentration of 5 μM, and the luminescence intensity was measured using an Enspire multi-mode plate reader (PerkinElmer).

[0100] miniALuc30 showed luminescence values equal to or greater than those of ALuc30wt (Figure 8). Sufficiently high luminescence values were also measured for miniALuc16 and miniALuc48.

[0101] Experiment 3: Construction of Δloop plasmid The predicted three-dimensional structure of miniALuc30 is shown in Figure 9. miniALuc30 has multiple loop structures. We constructed mutants by deleting the amino acid sequences constituting three of these loops (loop 1, loop 2, and loop 3). The amino acid sequences of loop 1 (positions 96-100 of ALuc30wt), loop 2 (positions 122-128 of ALuc30wt), or loop 3 (positions 156-161 of ALuc30wt) were deleted from ALuc30wt, and Gly-Ser was inserted. Furthermore, the N-terminus (positions 1-49) of ALuc30wt was deleted. This resulted in the construction of expression plasmids for ALuc30Δloop1N1, ALuc30Δloop2N1 (SEQ ID NO: 54), and ALuc30Δloop3N1, each lacking the N-terminus and loops. The size of ALuc30Δloop2N1 was 14 kDa.

[0102] Experiment 4: Measurement of luminescence value of ALucΔloop Luminescence levels were measured using the same method as in Experiment 2. ALuc30Δloop2N1 maintained approximately half the luminescence level of ALuc30ΔN1, which had only the N-terminus removed (Figure 10). ALuc30Δloop1N1 and ALuc30Δloop3N1 had significantly lower luminescence levels.

[0103] Experiment 5: Comparison with known luminescent enzymes The miniALuc prepared in Experiment 1 was compared with the known NanoLuc, TurboLuc, and GLuc. NanoLuc is known to be small, approximately 19 kDa, have very high luminescence, and exhibit high thermal stability. TurboLuc is small, approximately 16 kDa, have relatively high luminescence, and exhibit high thermal stability. GLuc is small, approximately 20 kDa, and when secreted and expressed from cells, exhibits lower luminescence than ALuc and exhibits high thermal stability. For NanoLuc, TurboLuc, and GLuc, plasmids containing the sequences set forth in SEQ ID NOs: 73, 74, and 75, respectively, inserted into the pcDNA3.1 vector were used.

[0104] Luminescence values were measured in the same manner as in Experiment 2, except that the final concentration of coelenterazine was 0.5 μM. Luminescence values were highest in the following order: NanoLuc>>miniALuc30>TurboLuc>ALuc30wt>GLuc (Figure 11). Luminescence values were measured in the same manner as in Experiment 2, except that the final concentration of coelenterazine was 5 μM. Luminescence values were highest in the following order: ALuc30wt = miniALuc30>NanoLuc>TurboLuc>GLuc (Figure 12).

[0105] Luminescence values were measured in the same manner as in Experiment 2, except that a final concentration of 5 μM coelenterazine h was used as the substrate. Luminescence values were highest in the following order: TurboLuc = ALuc30wt >> NanoLuc > miniALuc30 > GLuc (Figure 13). Luminescence values were measured in the same manner as in Experiment 2, except that a final concentration of 25 μM coelenterazine h was used as the substrate. Luminescence values were highest in the following order: miniALuc30 >> NanoLuc = TurboLuc = ALuc30wt >> GLuc (Figure 14).

[0106] Luminescence was measured in the same manner as in Experiment 2, except that furimazine, a substrate for NanoLuc sold by Promega, was used at the manufacturer's recommended concentration. Luminescence was detected in GLuc, ALuc30wt, and miniALuc30, but was lower than that in NanoLuc and TurboLuc (Figures 15 and 16). NanoLuc and TurboLuc showed high luminescence, and the luminescence value of NanoLuc was comparable to that when coelenterazine or coelenterazine h was used (Figure 16).

[0107] These results demonstrate that coelenterazine and coelenterazine h are more suitable substrates for miniALuc30 than furimazine, and that secreted and expressed miniALuc30 exhibits luminescence values greater than those of NanoLuc and TurboLuc when reacted with high concentrations of coelenterazine or coelenterazine h as a substrate.

[0108] Experiment 6: Stability of protein termini COS-7 cells were transfected with the plasmids and the culture supernatants were collected as in Experiment 2. The N-terminal Flag-tag and C-terminal His-tag of each secreted luciferase were detected by Western blot (SDS-PAGE, Mini-Protean TGX Gel Stain-Free 4-15% (Bio-Rad)) (Figure 17). Antibodies used were Anti 6xHistidine, Monoclonal Antibody (9C11), Peroxidase Conjugated (Fujifilm Wako Pure Chemical Industries, Ltd., 1:1000) and Monoclonal ANTI-FLAG® M2-Peroxidase (HRP) antibody produced in mouse, clone M2 (Sigma-Aldrich, 1:1000), respectively. Detection was performed using an Amesham Imager 680 (Cytiva). The Flag-tag and His-tag of GLuc were below the detection limit. Comparing the signal intensities of the Flag-tag and His-tag for GLuc and TurboLuc, the Flag-tag of TurboLuc had a low detection value, indicating that the N-terminus was truncated.On the other hand, both termini of ALuc30wt and miniALuc30 were intact, indicating that they are more stable than GLuc and TurboLuc.

[0109] Experiment 7: Measurement of specific activity The specific activities of NanoLuc, ALuc30wt, and miniALuc30 were measured by adjusting the enzyme concentrations based on the signal intensity of the Western blot in Experiment 6. When coelenterazine or coelenterazine h was used as the substrate, the specific activities of ALuc and miniALuc were shown to be comparable (Figures 18 and 19). The maximum luminescence values of ALuc30wt and miniALuc30 were comparable to that of NanoLuc, demonstrating their high luminescence activity.

[0110] Experiment 8: Measurement of Emission Spectrum The wavelength peak of miniALuc30 was 482 nm when reacted with coelenterazine (Figure 20), and 488 nm when reacted with coelenterazine h (Figure 21). The wavelength peak of miniALuc30 was almost the same as that of ALuc30wt. The emission spectrum had a broader base on the long-wavelength side than on the short-wavelength side.

[0111] Experiment 9: Thermal stability verification The plasmid was transfected into COS-7 cells using the same method as in Experiment 2, and the medium was collected. The culture supernatant containing miniALuc30 was incubated for 10 minutes at room temperature (25°C), 40°C, 50°C, 60°C, 70°C, 80°C, or 90°C, and then luminescence intensity was measured. The results showed that 80% or more of the activity remained after 10 minutes of incubation at 50°C or 60°C, and 50% or more remained after 10 minutes of incubation at 70°C (Figure 22), demonstrating that the product is fully practical.

[0112] Experiment 10: Expression of luciferase in E. coli (1) A DNA sequence encoding miniALuc30 was inserted into the pET32 vector to create a plasmid, which was then transformed into Escherichia coli SHuffle T7 express lysY (New England Biolabs). (2) The E. coli from (1) was inoculated onto an LB plate (containing 100 μg / μL ampicillin). (3) The next day, one colony was picked up and placed in a test tube containing 2 mL of LB medium (containing 100 μg / μL ampicillin) and cultured overnight at 30° C. with shaking. (4) Add 1 mL of (3) to 100 mL of LB medium (containing 100 μg / μL ampicillin), and measure the absorbance (OD) using a 500 mL flask. 600 The mixture was cultured with shaking at 30°C until the pH reached approximately 0.4. (5) Absorbance OD 600 When the pH reached approximately 0.4, 40 μL of 1 M isopropyl-β-thiogalactopyranoside was added, and the mixture was cultured at 16°C overnight. (6) The cells were collected, and the protein was purified using HisTALON Buffer Set and TALON Metal Affinity Resin (both from Takara Bio Inc.). 1.7 mg of miniALuc30 was obtained from 100 mL of medium.

[0113] The specific activity was measured by Western blot after matching the concentrations. The specific activity of miniALuc30 secreted and expressed from COS-7 cells was nearly identical to that of miniALuc30 produced in E. coli (Figure 23). This indicates that miniALuc30 can be expressed not only in mammalian cells but also in E. coli, and that it can be produced in large quantities.

[0114] MiniALuc30 purified from E. coli was incubated at room temperature (25°C), 60°C, 70°C, 80°C, or 90°C for 10 minutes, and then luminescence intensity was measured (Figure 24). MiniALuc30 expressed in E. coli lost almost no activity even after incubation at 60°C for 10 minutes, and retained over 90% of its activity after incubation at 70°C for 10 minutes and over 80% of its activity after incubation at 80°C for 10 minutes, demonstrating its excellent thermostability.

[0115] Experiment 11-1: Luminescence activity of the separated enzyme miniALuc30 was fragmented to prepare plasmids (pET32 vectors) expressing polypeptides at positions 4-22, 23-120, 4-47, 48-120, 4-77, 80-120, 4-90, and 91-120 of the amino acid sequence set forth in SEQ ID NO:51. Plasmids were introduced into E. coli SHuffle T7 Express lysY using the same method as in Experiment 10, and lysates were obtained from the cultured cells. The substrate coelenterazine was added to a final concentration of 5 μM to 100 μL of a single lysate or to a mixture of lysates as shown in Figure 25, and luminescence intensity was measured using an Infinite® 200 PRO plate reader (TECAN). Because luciferase fragments are present at high concentrations in the lysate, it is believed that when lysates containing each fragment are mixed, the two fragments are likely to meet, leading to the reconstitution of the original luciferase structure.

[0116] As shown in Figure 25, a lysate containing a luciferase fragment polypeptide alone exhibited low luminescence values, but a mixture of multiple lysates exhibited higher luminescence values than a single lysate. Combination 1 (a combination of a polypeptide consisting of the amino acid sequence of positions 4 to 22 of the amino acid sequence set forth in SEQ ID NO: 51 with a polypeptide consisting of the amino acid sequence of positions 23 to 120) and combination 2 (a combination of a polypeptide consisting of the amino acid sequence of positions 4 to 77 with a polypeptide consisting of the amino acid sequence of positions 78 to 122) do not overlap in their amino acid sequences, and together they have the same sequence as miniALuc30 minus three amino acid residues at the N-terminus and two amino acid residues at the C-terminus. Combination 3 (a combination of a polypeptide consisting of the amino acid sequence of positions 4 to 77 of the amino acid sequence set forth in SEQ ID NO: 51 with a polypeptide consisting of the amino acid sequence of positions 23 to 120) exhibited high luminescence values, despite partial overlap in their amino acid sequences. It has been shown that a polypeptide (first polypeptide) that contains a partial sequence of a luciferase but does not have luciferase activity produces luciferase activity in the presence of another polypeptide (second polypeptide) that contains a partial sequence of a luciferase but does not have luciferase activity.

[0117] Experiment 11-2: Luminescence activity of the separated enzyme MiniALuc30 was further divided to prepare plasmids (pET32 vectors) expressing polypeptides at positions 4 to 58, 4 to 64, 4 to 72, 65 to 120, 73 to 120, and 104 to 120 of the amino acid sequence set forth in SEQ ID NO: 51. In Experiment 11-2, luminescence values were measured in the same manner as in Experiment 11-1, except that luminescence values were detected using an Enspire multi-mode plate reader (PerkinElmer).

[0118] A combination of a polypeptide consisting of the amino acid sequence of positions 4 to 58 of the amino acid sequence set forth in SEQ ID NO: 51 with a polypeptide consisting of the amino acid sequence of positions 23 to 120 showed a high luminescence value (FIG. 26). A combination of a polypeptide consisting of the amino acid sequence of positions 4 to 64 of the amino acid sequence set forth in SEQ ID NO: 51 with a polypeptide consisting of the amino acid sequence of positions 65 to 120 or 73 to 120 showed a high luminescence value (FIG. 27). A combination of a polypeptide consisting of the amino acid sequence of positions 4 to 72 of the amino acid sequence set forth in SEQ ID NO: 51 with a polypeptide consisting of the amino acid sequence of positions 73 to 120, 23 to 120, or 104 to 120 showed a high luminescence value (FIG. 28).

[0119] Experiment 12: Detection of molecular interactions using split luciferase We investigated whether the split luciferase could detect molecular interactions using FKBP (FK506-binding protein) and FKBP12-rapamycin-associated protein 1 (FRB), which are known to bind in a rapamycin (Rap)-dependent manner. The polypeptide combinations tested were combinations 1 and 3 described above. First, fusion proteins were prepared in which FKBP was linked to the N-terminus or C-terminus of the amino acid sequence from positions 4 to 77 of the amino acid sequence set forth in SEQ ID NO: 51 (FKBP-4-77aa, 4-77aa-FKBP), and a fusion protein in which FRB was linked to the N-terminus (FRB-4-77aa); fusion proteins in which FRB was linked to the N-terminus or C-terminus of the amino acid sequence from positions 4 to 22 of the amino acid sequence set forth in SEQ ID NO: 51 (FRB-4-22aa, 4-22aa-FRB); fusion proteins in which FKBP was linked to the N-terminus or C-terminus of the amino acid sequence from positions 23 to 120 of the amino acid sequence set forth in SEQ ID NO: 51 (FKBP-23-120aa, 23-120aa-FKBP), and a fusion protein in which FRB was linked to the N-terminus or C-terminus (FRB-23-120aa, 23-120aa-FRB). Each polypeptide was connected to FKBP or FRB via a linker sequence (SEQ ID NO: 87). The sequences of FKBP and FRB are shown in SEQ ID NO: 88 and SEQ ID NO: 89, respectively.

[0120] The plasmid (pET32 vector) encoding the fusion protein was introduced into Escherichia coli to express the fusion protein. The Escherichia coli was harvested, and the protein was purified using HisTALON Buffer Set (Clontech) and TALON Metal Affinity Resin (Clontech). Luminescence was measured. Rapamycin was added to the purified fusion protein at a concentration of 50 nM. Ethanol was used as a negative control. The remaining experimental procedures were the same as in Experiment 11-2.

[0121] As shown in Figures 29 to 36, the combination of fusion proteins showed high luminescence values in the presence of rapamycin. It was demonstrated that a polypeptide (first polypeptide) containing a partial sequence of a luciferase but lacking luciferase activity can be suitably used, together with a second polypeptide, to detect the interaction between two molecules or the presence or absence of a molecule that induces the interaction. It was also demonstrated that the interaction could be detected whether the polypeptide was attached to the N-terminus or C-terminus of the target protein.

[0122] Experiment 13: Detection of molecular interactions using split luciferase A fusion protein was prepared in which FKBP, FRB, and the split polypeptides were linked by a linker sequence. This fusion protein is a circular permutation mutant in which FRB is linked to the N-terminus of the amino acid sequence from positions 23 to 120 of the amino acid sequence set forth in SEQ ID NO:51 via a linker sequence (SEQ ID NO:87), FKBP is linked to the C-terminus of the amino acid sequence from positions 4 to 22 of the amino acid sequence set forth in SEQ ID NO:51 via a linker sequence (SEQ ID NO:87), and the amino acid residues at positions 120 and 4 of the amino acid sequence set forth in SEQ ID NO:51 are linked by a linker sequence (SEQ ID NO:86).

[0123] The fusion protein was expressed in Escherichia coli, purified, and then the luminescence intensity was measured in the same manner as in Experiment 12. The concentrations of rapamycin added were 0 nM, 12.5 nM, 25 nM, 50 nM, and 100 nM.

[0124] As shown in Figure 34, an increase in luminescence intensity was detected in a rapamycin concentration-dependent manner. This indicates that a fusion protein in which a first polypeptide, a second polypeptide, and a target molecule are linked can be suitably used for detecting interactions with target molecules or molecules that induce such interactions.

[0125] [Aspect] It will be appreciated by those skilled in the art that the exemplary embodiments and examples described above are examples of the following aspects.

[0126] (Section 1) a first polypeptide containing a part of any one of the amino acid sequences (A) to (C); a second polypeptide that contains a part of the amino acid sequence of any one of (A) to (C), has a sequence different from that of the first polypeptide, and produces luciferase activity upon contact with the first polypeptide; (A) an amino acid sequence in which the amino acid sequences at positions 1 to 69 and 204 to 221 in the amino acid sequence shown in SEQ ID NO: 1 are deleted; (B) an amino acid sequence in which the amino acid sequence at positions 1 to 69 is deleted and at least one amino acid residue at positions 146 to 156 is deleted or substituted in the amino acid sequence shown in SEQ ID NO: 1; (C) An amino acid sequence in which at least one of the amino acid residues at positions 70 to 74 in the amino acid sequence shown in (A) or (B) is further deleted.

[0127] The reagent kit described in item 1 can be used as a probe for detecting intermolecular interactions. The polypeptide contained in the reagent kit described in item 1 is small in size and therefore unlikely to inhibit the expression and function of the target molecule with which it interacts.

[0128] (Section 2) In the reagent kit according to item 1, the amino acid sequence of any one of (A) to (C) is any one of the amino acid sequences of (a) to (c). (a) an amino acid sequence set forth in any one of SEQ ID NOs: 51 to 56; (b) an amino acid sequence having 85% or more identity to the amino acid sequence set forth in any one of SEQ ID NOs: 51 to 56, or (c) An amino acid sequence in which one or several amino acid residues have been deleted, substituted, inserted or added to the amino acid sequence set forth in any one of SEQ ID NOs: 51 to 56.

[0129] According to the reagent kit described in item 2, a probe that produces high luminescence activity can be obtained.

[0130] (Section 3) In the reagent kit according to item 1 or 2, at least one of the first polypeptide and the second polypeptide is selected from the group consisting of positions 4 to 20, 4 to 21, 4 to 22, 4 to 23, 4 to 24, 4 to 33, 4 to 34, 4 to 35, 4 to 36, 4 to 37, 4 to 45, 4 to 46, 4 to 47, 4 to 48, 4 to 49, 4 to 56, 4 to 57, and 4 to 58 of the amino acid sequence set forth in SEQ ID NO: 51. , 4th to 59th, 4th to 60th, 4th to 62nd, 4th to 63rd, 4th to 64th, 4th to 65th, 4th to 66th, 4th to 67th, 4th to 70th, 4th to 71st, 4th to 72nd, 4th to 73rd, 4th to 74th, 4th to 75th, 4th to 76th, 4th to 77th 4th to 78th, 4th to 79th, 4th to 88th, 4th to 89th, 4th to 90th, 4th to 91st, 4th to 92nd, 4th to 101st, 4th to 102nd, 4th to 103rd, 4th to 104th, 4th to 105th, 21st to 120th, 22nd to 120th, 23rd 1st to 120th, 24th to 120th, 25th to 120th, 34th to 120th, 35th to 120th, 36th to 120th, 37th to 120th, 38th to 120th, 46th to 120th, 47th to 120th, 48th to 120th, 49th to 120th, 50th to 1st 20th, 57th to 120th, 58th to 120th, 59th to 120th, 60th to 120th, 61st to 120th, 63rd to 120th, 64th to 120th, 65th to 120th, 66th to 120th, 67th to 120th, 68th to 120th, 69th to 120th , 70 to 120, 71 to 120, 72 to 120, 73 to 120, 74 to 120, 75 to 120, 76 to 120, 77 to 120, 78 to 120, 79 to 120, 80 to 120, 89 to 120, 90 to 120, 91 to 120, 92 to 120, 93 to 120, 102 to 120, 103 to 120, 104 to 120, 105 to 120, and 106 to 120.

[0131] According to the reagent kit described in item 3, a probe that produces high luminescence activity can be obtained.

[0132] (Section 4) In the reagent kit according to any one of items 1 to 3, at least one of the first polypeptide and the second polypeptide has an amino acid sequence selected from any one of (1) to (3). (1) an amino acid sequence set forth in any one of SEQ ID NOs: 76 to 85; (2) an amino acid sequence having 85% or more identity to any of the amino acid sequences set forth in SEQ ID NOs: 76 to 85, or (3) An amino acid sequence in which one or several amino acid residues are deleted, substituted, inserted or added to the amino acid sequence set forth in any one of SEQ ID NOs: 76 to 85.

[0133] According to the reagent kit described in item 4, a probe that produces high luminescence activity can be obtained.

[0134] (Section 5) In the reagent kit according to any one of items 1 to 4, the amino acid sequence constituting the first polypeptide and the amino acid sequence constituting the second polypeptide partially overlap.

[0135] The reagent kit described in item 5 can produce high luminescence activity and obtain probes with smaller sizes.

[0136] (Section 6) In the reagent kit according to any one of items 1 to 4, the amino acid sequence constituting the first polypeptide and the amino acid sequence constituting the second polypeptide do not overlap.

[0137] The polypeptide described in item 6 can produce a probe with a higher luminescence activity and a smaller size.

[0138] (Section 7) In the reagent kit according to any one of items 1 to 6, the first polypeptide and the second polypeptide are connected by a linker sequence.

[0139] The reagent kit described in item 7 allows easy detection of luminescent signals.

[0140] (Section 8) In the reagent kit according to any one of items 1 to 7, the first polypeptide is linked to a first target protein, and the second polypeptide is linked to a second target protein.

[0141] The reagent kit described in item 8 can be used to detect the interaction between a first target protein and a second target protein.

[0142] (Section 9) A first polypeptide comprising a part of any one of the amino acid sequences (A) to (C), a luciferase that produces luciferase activity upon contact with a second polypeptide containing a part of the amino acid sequence of any one of (A) to (C), a first polypeptide having a sequence different from said second polypeptide; (A) an amino acid sequence in which the amino acid sequences at positions 1 to 69 and 204 to 221 in the amino acid sequence shown in SEQ ID NO: 1 are deleted; (B) an amino acid sequence in which the amino acid sequence at positions 1 to 69 is deleted and at least one amino acid residue at positions 146 to 156 is deleted or substituted in the amino acid sequence shown in SEQ ID NO: 1; (C) An amino acid sequence in which at least one of the amino acid residues at positions 70 to 74 in the amino acid sequence shown in (A) or (B) is further deleted.

[0143] The polypeptide described in item 9 can be used as a probe for detecting intermolecular interactions. Because the polypeptide described in item 9 is small in size, it is unlikely to inhibit the expression and function of the target molecule with which it interacts.

[0144] (Section 10) A fusion protein comprising the first polypeptide according to any one of items 1 to 9 and a first target protein.

[0145] The fusion protein described in paragraph 10 can be used to detect the interaction of a first target protein with another molecule.

[0146] (Section 11) A nucleic acid encoding the first polypeptide according to any one of items 1 to 9 or the fusion protein according to item 10.

[0147] The nucleic acid described in item 11 can be used to produce the first polypeptide described in items 1 to 9 or the fusion protein described in item 10.

[0148] (Section 12) A vector comprising the nucleic acid of item 11.

[0149] The vector described in item 12 allows for easy amplification and maintenance of the nucleic acid described in item 11. Furthermore, the vector described in item 12 can also be used to produce the first polypeptide described in items 1 to 9 or the fusion protein described in item 10.

[0150] (Section 13) A transformed cell into which the nucleic acid according to item 11 has been introduced.

[0151] The transformed cell according to item 13 is capable of expressing the first polypeptide according to items 1 to 9 or the fusion protein according to item 10.

[0152] (Section 14) A method for analyzing protein interactions using the reagent kit according to any one of items 1 to 8.

[0153] According to the protein interaction analysis method described in paragraph 14, the interaction between two proteins can be detected by luminescence. The polypeptide used in this method has a small molecular weight, which makes it easy to normally express a fusion protein with the target protein. Furthermore, the polypeptide used in this method is unlikely to inhibit the function of the target protein. [Explanation of symbols]

[0154] 11 first polypeptide, 12 second polypeptide, 21 first target protein, 22 second target protein, 30 small molecule.

Claims

1. a first polypeptide; and a second polypeptide having a sequence different from that of the first polypeptide and producing luminescent enzyme activity upon contact with the first polypeptide, a reagent kit, wherein the combination of the first polypeptide and the second polypeptide is any one of the following combinations (A) to (D): (A) Positions 4 to 22, 4 to 23, 4 to 24, 4 to 33, 4 to 34, 4 to 35, 4 to 36, 4 to 37, 4 to 45, 4 to 46, 4 of the amino acid sequence set forth in SEQ ID NO:

51. 4th to 47th, 4th to 48th, 4th to 49th, 4th to 56th, 4th to 57th, 4th to 58th, 4th to 59th, 4th to 60th, 4th to 62nd, 4th to 63rd, 4th to 64th, 4th to 65th, 4th to 66th, 4th place a combination of a polypeptide consisting of an amino acid sequence corresponding to any one selected from positions 4 to 67, 4 to 70, 4 to 71, 4 to 72, 4 to 73, 4 to 74, 4 to 75, 4 to 76, 4 to 77, 4 to 78, 4 to 79, 4 to 88, 4 to 89, and 4 to 90, and a polypeptide consisting of the amino acid sequence of positions 23 to 120 in the amino acid sequence set forth in SEQ ID NO: 51; (B) a polypeptide consisting of an amino acid sequence corresponding to any one selected from positions 4 to 77, 4 to 78, 4 to 79, 4 to 88, 4 to 89, and 4 to 90 of the amino acid sequence set forth in SEQ ID NO: 51; and a polypeptide consisting of amino acids corresponding to positions 48 to 120, 49 to 120, 50 to 120, 57 to 120, 58 to 120, 59 to 120, 60 to 120, and 61 to 120 of the amino acid sequence set forth in SEQ ID NO:

51. a polypeptide consisting of an amino acid sequence corresponding to any one selected from positions 63 to 120, 64 to 120, 65 to 120, 66 to 120, 67 to 120, 68 to 120, 69 to 120, 70 to 120, 71 to 120, 72 to 120, 73 to 120, 74 to 120, 75 to 120, 76 to 120, 77 to 120, and 78 to 120; (C) a combination of a polypeptide consisting of the amino acid sequence of positions 4 to 64 of the amino acid sequence set forth in SEQ ID NO: 51 with a polypeptide consisting of an amino acid sequence corresponding to any one selected from positions 65 to 120, 66 to 120, 67 to 120, 68 to 120, 69 to 120, 70 to 120, 71 to 120, 72 to 120, and 73 to 120 of the amino acid sequence set forth in SEQ ID NO: 51; (D) a polypeptide consisting of the amino acid sequence of positions 4 to 72 of the amino acid sequence set forth in SEQ ID NO: 51, and a polypeptide consisting of positions 23 to 120, 24 to 120, 25 to 120, 34 to 120, 35 to 120, 36 to 120, 37 to 120, 38 to 120, 46 to 120, 47 to 120, 48 to 120, 49 to 120, 50 to 120, 57 to 120, 58 to 120, 59 to 120, 60 to 120, 61 to 120, 63 to 120, 64 to 120, 65 to 120, 66 to 120, 67 to 120, 68 to 120, 69 to 120, 70 to 120, 71 to 120, 72 to 120, 73 to 120, 74 to 120, 75 to 120, 76 to 120, 77 to 120, 78 to 120, 79 to 120, 80 to 120, 81 to 120, 82 to 120, 83 to 120, 84 to 120, 85 to 120, 86 to 120, 87 to 120, 88 to 120, 89 to 120, 90 to 120, 91 to 120, 92 to 120, 93 to 120, 94 to 120, 95 to 120, 96 to 120, a combination with a polypeptide consisting of an amino acid sequence corresponding to any one selected from positions 6 to 120, 67 to 120, 68 to 120, 69 to 120, 70 to 120, 71 to 120, 72 to 120, 73 to 120, 74 to 120, 75 to 120, 76 to 120, 77 to 120, 78 to 120, 79 to 120, 80 to 120, 89 to 120, 90 to 120, 91 to 120, 92 to 120, 93 to 120, 102 to 120, 103 to 120, and 104 to 120.

2. 2. The reagent kit according to claim 1, wherein the amino acid sequence constituting the first polypeptide and the amino acid sequence constituting the second polypeptide partially overlap.

3. 2. The reagent kit according to claim 1, wherein the amino acid sequence constituting the first polypeptide and the amino acid sequence constituting the second polypeptide do not overlap.

4. the first polypeptide and the second polypeptide are connected by a linker sequence, The reagent kit according to any one of claims 1 to 3, wherein the amino acid sequence of the linker sequence is the amino acid sequence shown in SEQ ID NO: 86 or 87.

5. A method for analyzing protein interactions, using the reagent kit according to any one of claims 1 to 4.

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