Polypeptides possessing luminescent enzyme activity

Abstract

To provide novel small molecular weight luminescent enzymes.SOLUTION: Provided herein is a polypeptide having luminescent enzyme activity comprising an amino acid sequence (A) or (B), where (A) is an amino acid sequence having deletion of amino acid residues from position 1 to position 69 and from position 204 to position 221 in the amino acid sequence of SEQ ID NO:1, and (B) is an amino acid sequence having deletion of amino acid residues position 1 to position 69 and deletion or substitution of at least one of the amino acid residues from position 146 to position 156 in the amino acid sequence of SEQ ID NO:1.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] The present invention relates to a polypeptide having luminescent enzyme activity.

Background Art

[0002] In the fields of basic biology, diagnostic techniques, and inspection techniques, luminescent enzymes are used as reporter proteins for detecting target proteins. In addition to luminescent enzymes, fluorescent proteins, fluorescent dyes, quantum dots, peroxidases, etc. are widely used as reporter proteins. Although fluorescent proteins, fluorescent dyes, and quantum dots have high fluorescence intensity, they require excitation light. Therefore, (1) they have phototoxicity to cells, (2) the excitation light spectrum overlaps with the fluorescence spectrum, making the Signal / Background ratio likely to be low and unsuitable for trace detection, and (3) the detector needs to incorporate an excitation light irradiation device and a spectroscopic filter. Luminescent enzymes do not require excitation light and thus do not have the above disadvantages. Also, detection using luminescent enzymes is more suitable for trace detection than colorimetric methods using peroxidases or the like.

[0003] To date, wild-type firefly-derived luminescent enzyme (FLuc), NanoLuc, TurboLuc, Gaussia princeps-derived luminescent enzyme (GLuc), Renilla reniformis-derived luminescent enzyme (), and Metridia longa-derived luminescent enzyme (MLuc) have been reported as luminescent enzymes. Japanese Patent Application Laid-Open No. 2014-100137 (Patent Document 1) and International Publication No. 2017 / 057752 (Patent Document 2) disclose an artificial luminescent enzyme (Aluc) prepared by selecting frequently occurring amino acids from the amino acid sequence of a luminescent enzyme of Gaussia princeps.

Prior Art Documents

Patent Documents

[0004]

Patent Document 1

[0005] When expressing a fusion protein of a luminescent enzyme and a target protein in cells, if the luminescent enzyme is large, the fusion protein may not be expressed properly, or the target protein may not function properly due to steric hindrance. Furthermore, it has been reported that large luminescent enzymes are more susceptible to the influence of small molecule compounds on their luminescence, making them unsuitable for drug screening. When analyzing intermolecular interactions using bioluminescence resonance energy transfer (BRET), the signal intensity is inversely proportional to the sixth power of the distance between the luminescent enzyme and the fluorescent protein; therefore, a smaller luminescent enzyme results in a stronger signal.

[0006] Therefore, a small size of luminescent enzyme is useful when utilizing them. The present invention aims to provide a novel luminescent enzyme with a small molecular weight. [Means for solving the problem]

[0007] The present invention relates to a polypeptide having luminescent enzyme activity as described in (A) or (B). (A) Amino acid sequences in which amino acid residues at positions 1-69 and 204-221 are missing from the amino acid sequence shown in Sequence ID No. 1, (B) An amino acid sequence in which the amino acid residues at positions 1 to 69 are deleted and at least one amino acid residue at positions 146 to 156 is deleted or substituted. [Effects of the Invention]

[0008] According to the present invention, a luminescent enzyme with a small molecular weight is provided. [Brief explanation of the drawing]

[0009] [Figure 1]This figure shows the structure of the ALuc30 mutant prepared in the example. [Figure 2] This figure shows the amino acid sequence identity between picALuc30 and picALuc16. [Figure 3] This figure shows the amino acid sequence identity between picALuc30 and picALuc48. [Figure 4] This figure shows the amino acid sequence identity between picALuc48 and picALuc16. [Figure 5] This graph shows the luminescence values ​​of picALuc in Experiment 2. Error bars indicate ±1 SD (n=3). [Figure 6] This is a diagram showing the three-dimensional structure of picALuc30. [Figure 7] This graph shows the emission values ​​of ΔloopN1 in Experiment 4. [Figure 8] This graph shows the luminescence values ​​when coelenterazine (0.5 μM) was added as a substrate to the luminescent enzyme in Experiment 5. Error bars indicate ±1 SD (n=3). [Figure 9] This graph shows the luminescence values ​​when coelenterazine (5 μM) was added as a substrate to the luminescent enzyme in Experiment 5. Error bars indicate ±1 SD (n=3). [Figure 10] This graph shows the luminescence values ​​when coelenterazine h (5 μM) was added as a substrate to the luminescent enzyme in Experiment 5. Error bars indicate ±1 SD (n=3). [Figure 11] This graph shows the luminescence values ​​when coelenterazine h (25 μM) was added as a substrate to the luminescent enzyme in Experiment 5. Error bars indicate ±1 SD (n=3). [Figure 12] This graph shows the luminescence values ​​when flimazine was added as a substrate to the luminescent enzyme in Experiment 5. Error bars indicate ±1 SD (n=3). [Figure 13] This graph shows the luminescence values ​​when flimazine was added as a substrate to the luminescent enzyme in Experiment 5. Error bars indicate ±1 SD (n=3). [Figure 14]This is a figure showing the detection of the enzyme protein in the supernatant by Western blot in Experiment 6. The figure on the left detected the flag-tag, and the figure on the right detected the His-tag. [Figure 15] This is a graph showing the specific activity when coelenterazine was added as a substrate to the luminescent enzyme in Experiment 7. [Figure 16] This is a graph showing the specific activity when coelenterazine h was added as a substrate to the luminescent enzyme in Experiment 7. [Figure 17] This is a figure showing the luminescence spectrum when coelenterazine was added as a substrate to the luminescent enzyme in Experiment 8. [Figure 18] This is a figure showing the luminescence spectrum when coelenterazine h was added as a substrate to the luminescent enzyme in Experiment 8. [Figure 19] This is a graph showing the luminescence value of secreted and expressed picALuc after heat treatment in Experiment 9. [Figure 20] This is a graph showing the luminescence values of secreted and expressed picALuc and E. coli-expressed picALuc in Experiment 10. [Figure 21] This is a graph showing the luminescence value of E. coli-expressed picALuc after heat treatment in Experiment 10. Error bars indicate ±1 SD (n = 3). [Figure 22] This is a figure showing the plasmid map used in Experiment 11. [Figure 23] This is a figure showing the spectrum when coelenterazine h was added as a substrate to the fusion protein of NanoLuc and YFP in Experiment 11. [Figure 24] This is a figure showing the spectrum when coelenterazine h was added as a substrate to the fusion protein of picALuc30 and YFP in Experiment 11. [Figure 25] This is a figure showing the BRET signal of the fusion protein of NanoLuc and YFP in Experiment 11. [Figure 26] This is a figure showing the BRET signal of the fusion protein of picALuc30 and YFP in Experiment 11. [Modes for carrying out the invention]

[0010] <prototype> The polypeptide according to the present invention has luminescent enzyme activity. A luminescent enzyme (luciferase) is an enzyme that oxidizes a luminescent substrate (luciferin), and emits light during the oxidation process. In this specification, luminescent enzyme activity refers to the activity of the enzymatic reaction between the luminescent enzyme and the substrate, and is measured by detecting the light (emission spectrum) emitted when the substrate returns to the ground state after being 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., Promega's "GloMax" series) or a spectrophotometer (e.g., TECAN's "Infinite200PRO"). By measuring the intensity at a specific wavelength every minute, the temporal change in emission and its stability can be detected. The shift of emission to longer wavelengths can be detected by measuring all wavelengths.

[0011] The optimal pH and temperature for luminescent enzyme activity may be the same as those of known luminescent enzymes (e.g., copepod luminescent enzymes or artificial luminescent enzymes). The luminescent enzyme activity is preferably the same as that of copepod luminescent enzymes. The optimal pH for luminescent enzyme activity is 5.0 to 8.0, preferably 7.0, and the optimal temperature is 4°C to 30°C, preferably 25°C.

[0012] The luminescent substrate is not particularly limited and can be appropriately selected in accordance with the luminescent enzyme. The luminescent substrate may be any known substrate such as coelenterazine, firefly luciferin, sea firefly luciferin, or flimazine, but a coelenterazine substrate is preferred. Examples of coelenterazine substrates include natural coelenterazine, coelenterazine ip, coelenterazine i, coelenterazine hcp, coelenterazine 400A, coelenterazine, coelenterazine cp, coelenterazine f, coelenterazine h, and coelenterazine n. The luminescent substrate preferably contains coelenterazine or coelenterazine h.

[0013] One aspect of the polypeptide according to the present invention is (A) The amino acid sequence shown in Sequence ID No. 1 includes an amino acid sequence in which amino acid residues at positions 1-69 and 204-221 are missing.

[0014] One aspect of the polypeptide according to the present invention is (A1) The polypeptide may consist of amino acid residues from positions 75 to 203 of the amino acid sequence shown in Sequence ID No. 1, and the total number of amino acid residues may be 140 or less.

[0015] (A2) The polypeptide according to the present invention may contain amino acid residues from positions 75 to 203 of the amino acid sequence shown in Sequence ID No. 1, and the molecular weight of the polypeptide may be 20 kDa or less.

[0016] One aspect of the polypeptide according to the present invention is (B) The amino acid sequence shown in Sequence ID No. 1 includes an amino acid sequence in which the amino acid residues at positions 1 to 69 are missing, and at least one amino acid residue at positions 146 to 156 is missing or substituted.

[0017] One aspect of the polypeptide according to the present invention is (B1) The amino acid sequence may include amino acid residues from positions 70 to 221 of the amino acid sequence shown in Sequence ID No. 1, with at least one amino acid residue from positions 146 to 156 being deleted or substituted. The polypeptide according to the present invention may consist of amino acid residues from positions 70 to 221 of the amino acid sequence shown in Sequence ID No. 1, with at least one amino acid residue from positions 146 to 156 being deleted or substituted.

[0018] The polypeptide according to the present invention may have at least one amino acid residue between positions 70 and 74 of the amino acid sequence shown in SEQ ID NO: 1 further missing, or all of the amino acid residues between positions 70 and 74 may be missing.

[0019] The polypeptide according to the present invention has, preferably, two or more, three or more, four or more, five or more, six or more, or seven or more amino acid residues missing from 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 in place of the missing sites. The polypeptide according to the present invention may have six or fewer, five or fewer, four or fewer, three or fewer, two or fewer, one or fewer, or zero amino acid residues between positions 146 to 156 of the amino acid sequence shown in SEQ ID NO: 1.

[0020] The molecular weight of the polypeptide according to the present invention is preferably 20 kDa or less, more preferably 18 kDa or less, even more preferably 15 kDa or less, even more preferably 14 kDa or less, and particularly preferably 13 kDa or less. The molecular weight of the polypeptide according to the present invention is, for example, 10 kDa or more.

[0021] The number of amino acid residues in the polypeptide according to the present invention is, for example, 160 or less, 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 the polypeptide according to the present invention is, for example, 100 or more.

[0022] Among the amino acids represented by Xaa in Sequence ID No. 1, the amino acid residues 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 may be any amino acid. Of these, the amino acid residues at positions 22-23, 39-40, 76-77, 140, and 148-151 may be deleted. Preferably, the amino acid residue at position 3 is E or G, the amino acid residues at positions 20-29 are PTENKDDI sequence (2 residues deleted, SEQ ID NO: 2), ATINEDI 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), the amino acid residue at position 31 is I, L, Y, or K, the amino acid residue at position 32 is V or A, the amino acid residue at position 35 is E or G, the amino acid residue at position 37 is K or S, the amino acid residues at positions 64-66 are ANS sequence or DAN sequence, the amino acid residue at position 69 is D or G, and the amino acid residues at positions 76-77 are GG sequence or K (1 residue deleted) or may be deleted. The amino acid residues at positions 85-86 are LE, KA, or KE sequences, the amino acid residues at positions 89-90 are KE, IE, LE, or KI sequences, the amino acid residue at position 129 is E, G, or A, the amino acid residues at positions 140-144 are TEEET sequences (SEQ ID NO: 9), GEAI sequences (1 residue deletion, SEQ ID NO: 10), or VGAI sequences (1 residue deletion, SEQ ID NO: 11), and the amino acid residues at positions 148-151 are GVLG sequences. The sequence is (SEQ ID NO: 12) or I (with 3 residues deleted), or all residues may be deleted, wherein the amino acid residue at position 159 is D, E, N, F, Y, or W, the amino acid residue at position 161 is E, A, or L, the amino acid residue at position 188 is K, F, Y, or W, the amino acid residue at position 191 is D, A, N, F, Y, or W, the amino acid residue at position 202 is A or K, and the amino acid residue at position 206 is S, D, N, F, Y, or W.

[0023] The amino acid residues at positions 13, 16, 174, and 218 of Sequence ID No. 1 are hydrophobic amino acids (e.g., V, F, A, L, I, G), preferably with the amino acid residue at position 13 being V or F, the amino acid residue at position 16 being V or A, the amino acid residue at position 174 being V or A, and the amino acid residue at position 218 being A or L.

[0024] The amino acid residues at positions 5, 67, 75, 101, 119, and 214 of Sequence ID No. 1 are hydrophilic amino acids (e.g., Q, K, D, R, H, E, T), preferably the amino acid residue at position 5 is Q or K, the amino acid residue at position 67 is D or R, the amino acid residue at position 75 is K, H, R, or E, the amino acid residue at position 101 is T or H, the amino acid residue at position 119 is K, E, or Q, and the amino acid residue at position 211 is K or T.

[0025] The amino acid residues at positions 4, 6, 7, 10, 11, 15, 33, 34, 39-41, 63, 68, 74, 78, 83, 137, 160, and 203 of Sequence ID No. 1 are aliphatic amino acids. However, the amino acid residues at positions 39, 40, and 70 may be deleted. The amino acid residues at positions 4, 6, 7, 10, 11, 15, 34, 63, 78, 83, and 160 are preferably high molecular weight aliphatic amino acids (e.g., I, V, L, M), but low molecular weight aliphatic amino acids may also be present at a low frequency. More preferably, the amino acid residue at position 4 is I or V, the amino acid residue at position 6 is V or L, the amino acid residue at position 7 is L or I, the amino acid residue at position 10 is L or V, the amino acid residue at position 11 is I or L, the amino acid residue at position 15 is L or V, the amino acid residue at position 34 is I or V, the amino acid residue at position 63 is L or V, the amino acid residue at position 78 is L or M, the amino acid residue at position 83 is L or M, and the amino acid residue at position 160 is L or M. The amino acid residues at positions 33, 39-41, 68, 74, 137, and 203 are preferably low molecular weight aliphatic amino acids (e.g., A, G, T), but high molecular weight aliphatic amino acids may be present at a low frequency. More preferably, the amino acid residue at position 33 is G, L, or A; the amino acid residue at position 39 is G, A, or may be deleted, S, or F; the amino acid residue at position 40 is T or may be deleted; the amino acid residue at position 41 is T or A; the amino acid residue at position 68 is A or G; the amino acid residue at position 74 is G or may be deleted; the amino acid residue at position 137 is G or A; and the amino acid residue at position 203 is T or G.

[0026] The amino acid residues at positions 72, 73, 97, and 110 of Sequence ID No. 1 are positively charged amino acids (basic amino acids; for example, K, R, H). However, the amino acid residues at positions 72 and 73 may be deleted. Preferably, the amino acid residues at positions 72 and 73 are R or may be deleted, the amino acid residue at position 97 is K or R, and the amino acid residue at position 110 is H or K.

[0027] The amino acid residues at positions 62 and 211 of Sequence ID No. 1 are negatively charged amino acids (acidic amino acids; for example, N, D, Q, E), preferably, the amino acid residue at position 62 is N or D, and the amino acid residue at position 211 is Q or E.

[0028] Specific examples of luminescent enzymes 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), ALuc Examples include uc32 (sequence number 30), ALuc33 (sequence number 31), ALuc34 (sequence number 32), ALuc41 (sequence number 33), Aluc42 (sequence number 34), ALuc43 (sequence number 35), Aluc44 (sequence number 36), ALuc45 (sequence number 37), Aluc46 (sequence number 38), ALuc47 (sequence number 39), ALuc48 (sequence number 40), ALuc49 (sequence number 41), Aluc50 (sequence number 42), ALuc51 (sequence number 43), Aluc52 (sequence number 44), ALuc53 (sequence number 45), ALuc55 (sequence number 46), Aluc56 (sequence number 47), ALuc57 (sequence number 48), and so on. The luminescent enzyme having the amino acid sequence shown in Sequence ID No. 1 may have some or all of the amino acid residues at positions 1-19 (secretion signal), positions 20-31 (antigen recognition site, etc.), and positions 217-221 (GS linker sequence) missing.

[0029] The amino acid sequence shown in Sequence ID No. 1 may have the amino acid sequence shown in Sequence ID No. 49 in the region from positions 1 to 71. Typical examples of luminescent enzymes having this sequence include ALuc15, ALuc16, ALuc17, ALuc18, and ALuc24.

[0030] The amino acid sequence shown in Sequence ID No. 1 may have the amino acid sequence shown in Sequence ID No. 50 in the region from positions 1 to 157. Typical examples of luminescent enzymes having this sequence include ALuc22, ALuc25, ALuc26, ALuc27, ALuc28, and ALuc29.

[0031] The polypeptide according to the present invention allows for a reduction in the size of luminescent enzymes. A smaller luminescent enzyme makes it easier for the fusion protein, such as a target protein or antibody, to be expressed normally in cells, and reduces the likelihood of the target protein malfunctioning. Because the luminescence of a small luminescent enzyme is less affected by small molecule compounds, it is suitable for use as a reporter protein in drug or ligand screening. Even when analyzing intermolecular interactions using bioluminescence resonance energy transfer (BRET), a small luminescent enzyme allows for the detection of a strong signal. Small luminescent enzymes can be used as secreted luminescent enzymes. With secreted luminescent enzymes, there is no need to lysate cells to measure luminescence values, and changes in gene expression over time can be measured. Because small luminescent enzymes are easily expressed in cells, large-scale expression and purification are possible. Small luminescent enzymes can be expressed in various expression systems. Furthermore, small luminescent enzymes exhibit excellent structural stability.

[0032] The polypeptide according to the present invention can have a high luminescence value. Preferably, the peak luminescence value of the polypeptide according to the present invention is the same as or higher than that of known luminescent enzymes, such as NanoLuc, ALuc, etc. Luminescent enzymes with high luminescence values ​​enable highly sensitive detection of luminescence and allow for a lower detection limit concentration.

[0033] The polypeptide according to the present invention preferably has high thermal stability, for example, retaining 80% or more of its activity after heat treatment at 50°C for 10 minutes, and preferably retaining 80% or more of its activity after heat treatment at 60°C for 10 minutes. Luminescent enzymes with high thermal stability are less likely to be deactivated by temperature increases during transport, etc., and are highly practical in field applications such as diagnosis and testing.

[0034] The enzyme activity of the polypeptide according to the present invention preferably has a broad tail 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 luminescent enzymes. Since long wavelengths have excellent biological permeability, luminescent enzymes having an emission spectrum with a broad tail on the long-wavelength side are suitable for live imaging. When coelenterazine is used as a substrate, the emission wavelength peak of the polypeptide according to the present invention is preferably 470 nm to 490 nm, more preferably about 482 nm. When coelenterazine h is used as a substrate, the emission wavelength peak of the polypeptide according to the present invention is preferably 470 nm to 490 nm, more preferably about 488 nm.

[0035] The C-terminus of ALuc was thought to be essential for binding to the substrate. A polypeptide according to one aspect of the present invention lacks the C-terminus of ALuc and possesses luminescent enzyme activity. Therefore, the substrate binding site of a polypeptide lacking the C-terminus of ALuc is thought to have a different structure from that of ALuc.

[0036] The polypeptide according to the present invention 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).

[0037] The polypeptide according to the present invention may have a functional peptide attached to its N-terminus or C-terminus. For example, by attaching a membrane localization signal (MLS) to the N-terminus or C-terminus, the luminescent enzyme can be localized to the cell membrane. In this specification, unless otherwise specified, when two or more peptides, including a signal peptide, are attached, the length, reading frame, etc., may be adjusted using a well-known linker as appropriate. Localization of the luminescent enzyme to the cell membrane facilitates the supply of substrates and oxygen from the outside, and in the case of a luminescent probe (e.g., a luminescent capsule) based on the luminescent enzyme, there is the advantage of being able to react quickly to external signals.

[0038] The polypeptide according to the present invention preferably contains (a) an amino acid sequence described in any of SEQ ID NOs. 51 to 56, and may consist of any of the amino acid sequences described in SEQ ID NOs. 51 is the amino acid sequence obtained by removing the N-terminus and C-terminus from the amino acid sequence of ALuc30, one of the artificial luminescent enzymes (picALuc30 in Figure 1). 54 is the amino acid sequence obtained by removing the N-terminal sequence and intermediate sequence from the amino acid sequence of ALuc30 (ALuc30Δloop2N1 in Figure 1). Similarly, the amino acid sequences described in SEQ ID NOs. 52 and 53 are the amino acid sequences obtained by removing the N-terminus and C-terminus, respectively, from the amino acid sequences of ALuc16 and ALuc48, two of the artificial luminescent enzymes. 55 and 56 are the amino acid sequences obtained by removing the N-terminal sequence and intermediate sequence, respectively, from the amino acid sequences of ALuc16 and ALuc48.

[0039] The polypeptide according to the present invention preferably contains an amino acid sequence having 85% or more identity with the amino acid sequence described in any of SEQ ID NOs. 51 to 56, and may consist of an amino acid sequence having 85% or more identity with the amino acid sequence described in any of SEQ ID NOs. The polypeptide 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 with the amino acid sequence described in any of SEQ ID NOs. 51 to 56.

[0040] The polypeptide according to the present invention preferably comprises an amino acid sequence in which one or several amino acid residues are deleted, substituted, inserted, or added to the amino acid sequence described in any of SEQ ID NOs. 51 to 56, and may consist of an amino acid sequence in which one or several amino acid residues are deleted, substituted, inserted, or added to the amino acid sequence described in any of SEQ ID NOs. In this specification, "several" may be, for example, 2 to 20, 2 to 10, 2 to 5, or 2 to 3.

[0041] The polypeptide according to the present invention may contain an amino acid corresponding to the start codon (often methionine) before the amino acid residue at position 1. Sequences 57-62 show the amino acid sequences of SEQ ID NOs. 51-56 with methionine added to position 1. One embodiment of the polypeptide according to the present invention may include (a1) to (c1) below, or may consist of (a1) to (c1) below. (a1) Amino acid sequence described in any of Sequence IDs 57-62, (b1) An amino acid sequence having 85% or more identity with any of the amino acid sequences described in Sequence ID No. 57 to 62, or (c1) An amino acid sequence in which one or more amino acid residues are deleted, substituted, inserted, or added to any of the amino acid sequences described in SEQ ID NOs. 57 to 62. The polypeptide 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 with the amino acid sequence described in any of SEQ ID NOs. 57 to 62.

[0042] <Nucleic acid that codes for a polypeptide> A nucleic acid according to one embodiment of the present invention encodes the polypeptide described above. The polypeptide described above can be produced from the nucleic acid encoding the polypeptide. The nucleic acid is preferably DNA or RNA. The nucleic acid encoding the polypeptide may contain a start codon at the 5' end and a stop codon at the 3' end of the base sequence corresponding to the polypeptide described above. The nucleic acid may contain an intron sequence. The nucleic acid according to one embodiment of the present invention can be obtained by chemical synthesis or PCR, etc.

[0043] One embodiment of nucleic acids includes nucleic acids containing a base sequence in which codons encoding each amino acid in the coding region are replaced with other codons encoding the same amino acid. From the viewpoint of improving polypeptide expression, the nucleic acids according to the present invention may include nucleic acids containing a base sequence in which the codon usage has been modified to suit the host organism or transformed cell species.

[0044] <Vector> A vector according to one embodiment of the present invention includes the nucleic acid described above. The vector is a nucleic acid molecule capable of amplifying or maintaining DNA, and examples include expression vectors and cloning vectors. In one example, the nucleic acid described above is introduced into a host cell or the like in the form of an insertion into an expression vector, and expresses a polypeptide having luminescent enzyme activity. The expression vector may have a promoter sequence and a terminator sequence for expressing the incorporated gene. A vector according to one embodiment of the present invention can be obtained by inserting the nucleic acid described above into a suitable vector.

[0045] 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, or an artificial chromosome vector. Examples of vectors include pBR322, pUC plasmid vectors, and pET plasmid vectors. Specifically, when using E. coli as the host cell, examples include pUC19, pUC18, pUC119, pBluescriptII, and pET32. When using mammalian cells as the host cell, examples include pRc / RSV, pRc / CMV, retroviral vectors, adenovirus vectors, and adeno-associated virus vectors.

[0046] <Transformed cells> Transformed cells according to one embodiment of the present invention are cells into which the above-mentioned nucleic acid has been introduced. The nucleic acid may be introduced into the cells in the form contained in the vector. Transformed cells can express a luminescent enzyme. The luminescent enzyme may be secreted into the supernatant. Examples of methods for introducing nucleic acid 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.

[0047] Cells into which nucleic acids are introduced can be eukaryotic or prokaryotic cells, including bacterial, fungal, plant, animal, and insect cells. The cells may be yeast, E. coli, or mammalian cells. Mammals include humans, cattle, horses, sheep, monkeys, pigs, mice, rats, hamsters, guinea pigs, rabbits, dogs, etc.

[0048] <Reporter proteins and reporter analysis methods> Reporter analysis can be performed using the polypeptide according to the present invention as a reporter protein. The polypeptide according to the present invention can be used as a substitute for luminescent or fluorescent substances in conventional reporter analysis methods using luminescent enzymes or various fluorescent proteins.

[0049] In this specification, a reporter protein is a luminescent label used to investigate the behavior of a target protein, target nucleic acid, or target gene within a cell. In this specification, a reporter analysis method is an analytical method that uses the polypeptide according to the present invention as a reporter protein to observe the behavior of a target protein or target gene within a cell in response to an external stimulus, by replacing it with the presence or absence of luminescence, the amount of luminescence, the timing of luminescence, or the location of luminescence. Specifically, a reporter analysis method can be described as a method for qualitatively or quantitatively measuring the expression location, timing of expression, or expression level of a target gene as the luminescence location, timing of luminescence, or amount of luminescence of the reporter protein. The reporter assay may be used in multiplexing with multiple enzymes or proteins that emit light of different wavelengths.

[0050] Reporter analysis can be performed in vivo, in cultured cells, or in test tubes in mammals and other organisms. When used under in vivo conditions, for example, a reporter gene consisting of nucleic acids encoding the amino acid sequence of the polypeptide described above is linked to a target gene and incorporated into a vector, which is then introduced into the target cell. Examples of cultured cells include mammalian cells commonly used in genetic recombination, such as COS cells, CHO-K1 cells, HeLa cells, HEK293 cells, and NIH3T3 cells, as well as yeast, bacteria such as Escherichia coli, and insect cells.

[0051] Below, the reporter analysis method according to the present invention will be divided into three categories: "basic," "inducible," and "activatable," as presented in Niu et al., Theranostics, 2, 2012, 413., and its application to each of the polypeptide analysis methods according to the present invention will be explained.

[0052] (1)Basic method The basic method is the simplest reporter analysis system, in which a luminescent enzyme is linked to and labeled the target protein whose behavior is to be investigated. When applying the polypeptide according to the present invention as a reporter protein to the basic method, a fusion protein containing the polypeptide according to the present invention and the target protein or a protein that binds to the target protein should be prepared. This differs from other reporter analysis methods in that the fusion protein is expressed with an unregulated promoter. The fusion protein can also be used for in vivo imaging of the target protein.

[0053] Fusion proteins are typically created by fusing a reporter protein to the N-terminus or C-terminus of a target protein, but they can also be fused directly to the target protein with a reporter protein that has been split into two parts, one at the N-terminus and the other at the C-terminus, via other peptide sequences.

[0054] Fusion proteins include (i) those expressed as a whole from a nucleic acid encoding a fusion protein comprising the polypeptide according to the present invention and a target protein or a protein (including peptides) that recognizes a target protein, and (ii) those expressed separately from the polypeptide according to the present invention and a target protein or a protein that recognizes a target protein, and linked by a chemical reaction. Examples of means for linking separately expressed proteins by a chemical reaction include linking by crosslinkers, linking utilizing the binding ability of avidin-biotin, and linking utilizing the chemical reactivity of amino acid residues.

[0055] One example of a fusion protein is a luminescent enzyme-labeled antibody, in which a luminescent enzyme is linked to an antibody. In this fusion protein, for example, a chimeric DNA is created by linking a reporter gene upstream or downstream of the cDNA of a single-chain variable region fragment (scFv) of the antibody. The DNA can then be inserted into a suitable expression vector, introduced into cells, and expressed to obtain the fusion protein.

[0056] (2) Inducible Law The inducible method differs from the basic method in that reporter expression is controlled by a promoter. Applying luminescent enzymes as reporter proteins to the inducible method has been conventionally used to analyze the timing and expression levels 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 levels in response to external stimuli. Analytical systems included 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. By using the polypeptide of the present invention as the reporter gene used in these analytical methods, the measurement performance of the assays can be dramatically improved.

[0057] (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 in response to external stimuli. One example is their use in detecting endocrine disruptors (environmental hormones) that disrupt signal transduction via nuclear receptors. The expression of target genes (e.g., hormone-responsive genes) associated with signal transduction via nuclear receptors is triggered by the binding of ligand-receptor complexes to the cis region (hormone response element) of the gene that regulates transcription. Plasmids containing reporter genes downstream of the cis region of these hormone-responsive genes are introduced into cells, and the amount of potential ligand hormone molecules or endocrine disruptors is detected as a luminescence value.

[0058] In reporter gene assays, conventionally widely used firefly luciferases have several drawbacks: [1] their large molecular weight and the time it takes for them to be expressed place a heavy burden on host cells; and [2] their low luminescence intensity means that it is usually necessary to wait 1 to 2 days after stimulation for a sufficient amount of luciferase (reporter) to accumulate. However, by selecting the polypeptide according to the present invention as the reporter protein, these problems are resolved.

[0059] Using the polypeptide according to the present invention as a reporter protein offers the advantage of extremely high luminescence intensity, allowing for measurement in a very short time after stimulation. Therefore, it significantly reduces measurement time compared to conventional reporter proteins, and its high stability of luminescence over time enables luminescence measurement even in cell lines with poor gene transfer efficiency. Furthermore, because the luminescence is shifted to longer wavelengths, its permeability through cell membranes and skin is increased. The reduced background level also leads to higher measurement accuracy.

[0060] For example, to apply the polypeptide according to the present invention to a reporter gene assay, the luminescent enzyme can be linked to a known eukaryotic cell expression vector that has a special promoter upstream, introduced into eukaryotic cells, and after a certain period of time, the luminescence value can be measured under conditions with and without a signal (stimulus). A known pTransLucent vector can be used as the expression vector for the reporter gene assay that can carry the polypeptide according to the present invention, and it can be easily loaded using a known method.

[0061] (ii) Two-hybrid method The two-hybrid method is a technique for investigating protein-protein interactions, and the yeast two-hybrid (Y2H) system using yeast (Saccharomyces cerevisiae) was first established in 1989. By utilizing the fact that the DNA-binding domain (GAL4 DBD) and transcriptional activation domain of the transcriptional activator GAL4 protein are separable, it is possible to express a fusion protein of GAL4 DBD and an arbitrary protein A (bait), and simultaneously determine whether it interacts with protein B (prey), which is a fusion protein of the transcriptional activation domain (TA) expressed in the cell. If protein A and protein B bind, the DBD and TA are in close proximity, causing the DNA-binding domain (DBD) to bind to the "UASG" base sequence, thereby promoting the expression of a reporter gene linked downstream. If the reporter gene is a luminescent enzyme, monitoring bioluminescence in the presence of its specific substrate allows for the measurement of affinity between both protein A and protein B, enabling screening of proteins or peptides that interact with protein A (bait). The protein B (prey) in this case can also be provided through an expression library.

[0062] The host cells used are not limited to yeast cells; bacteria such as E. coli, mammalian cells, and insect cells can also be used. In this case, in addition to the yeast-derived transcription activator GAL4 DBD, the E. coli-derived repressor protein "LexA" can also be used. The DNA encoding these proteins is linked to the DNA encoding a bait protein (i.e., any protein A mentioned above), such as the ligand-binding domain of a ligand-responsive transcription regulator, and this is linked downstream of a promoter that can function in the host cell. On the other hand, as the "transcriptional activation domain of the transcription activator," for example, the transcriptional activation domain of GAL4, the E. coli-derived B42 acidic transcriptional activation domain, or the transcriptional activation domain of herpes simplex virus VP16 can be used. The DNA encoding these transcriptional activation domains is linked to the DNA encoding a prey protein (i.e., any protein B mentioned above), and this is linked downstream of a promoter that can function in the host cell.

[0063] Examples of vectors that can be used in budding yeast as a host cell and contain DNA encoding the DNA-binding domain of the transcription regulator GAL4 include plasmid pGBT9 (Clontech). Examples of vectors that can be used in budding yeast and contain DNA encoding the transcriptional activation domain of GAL4 include plasmid pGAD424 (Clontech). Examples of vectors that can be used in mammalian cells and contain DNA encoding the DNA-binding domain of GAL4 include pM (Clontech) and pBIND (Promega). Examples of vectors that can be used in mammalian cells and contain DNA encoding the transcriptional activation domain of herpes simplex virus VP16 include pVP16 (Clontech) and pACT (Promega). Examples of vectors that can be used in mammalian cells and contain DNA encoding the DNA-binding domain of LexA include pLexA (Clontech). Examples of vectors that can be used in mammalian cells and contain DNA encoding B42 include pB42AD (Clontech).

[0064] For example, a vector can be constructed by inserting the polypeptide according to the present invention as a reporter gene downstream of the region to which GAL4 binds ("UASG"). In the case of a mammalian host, commercially available pG5Luc vectors (Promega) or pFR-Luc vectors (Stratagene) can be used, and the polypeptide according to the present invention can be easily loaded in place of the firefly luciferase contained in those vectors using well-known methods. It can also be used in place of chloramphenicol acetyltransferase (CAT) in commercially available pG5CAT vectors (Clontech).

[0065] (3) Activatable method The activatable method is a reporter analysis method that utilizes the fact that the reporter itself actively responds to ligand stimulation and emits light. Typical examples include single-molecule bioluminescent probes and luminescent capsules, and it can also be applied to protein complenition assays (PCA) and protein splicing assays (PSA).

[0066] (i) Production of luminescent fusion proteins (luminescent capsules) By attaching a membrane localization signal (MLS) to the C-terminus of the polypeptide according to the present invention, the luminescent enzyme itself can be localized to the cell membrane. By designing the molecule to localize the luminescent enzyme to the cell membrane, the supply of substrate and oxygen becomes smoother, enabling extremely high-brightness and stable bioluminescence visualization. In this case, it is possible to insert the gene of any polypeptide or protein as cargo between the luminescent enzyme itself and the nucleic acid encoding the signal peptide. This allows the cargo protein to be efficiently transported to the cell membrane surface, and the location to which it is transported becomes luminescent. For example, if DEVD sequences or IETD sequences that respond to cell death are inserted as cargo at the junctions of each protein, the system will actively respond to the activity of caspase-3 and caspase-8 during cell death as a signal and function as a visualization system. A luminescent fusion protein with this structure is also called a "luminescent capsule." Luminescent capsules can also be used for toxicity assessment of chemical substances.

[0067] Compared to conventional luminescent probes, the luminescent capsule exhibits extremely high brightness and stable luminescence characteristics, and has the advantage of responding even to samples that cannot penetrate the cell membrane. The basic structure of this luminescent capsule is a membrane localization signal (MLS) attached to the C-terminus of the luminescent enzyme body. The enzyme may have its luminescence enhanced by tandem linking of polypeptides according to the present invention. The luminescent capsule makes it easy to observe the effects of compounds that cause morphological changes on the cell surface, such as cell death, as morphological changes on the cell membrane surface. Preferably, a polypeptide that causes morphological changes on the cell membrane surface, or a partial recognition sequence thereof, specifically a 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 body and the MLS. By inserting a polypeptide that induces cell death, or its recognition sequence thereof, as cargo between the C-terminus of the luminescent enzyme body and the MLS, it is possible to visualize cell death. More specifically, when a peptide sequence (usually 20 amino acid residues or less, preferably 10 amino acid residues or less) or an amino acid sequence containing a DEVD sequence or IETD sequence, which is recognized by various caspases and proteases (serine protease, cysteine ​​protease, etc.) and digestive enzymes (trypsin, amylase, etc.) is inserted as cargo, it is possible to visualize cell death induced by caspase-3 activity. Furthermore, by linking a fluorescent protein or another luminescent enzyme as cargo between the luminescent enzyme body and the MLS, the amount of light generated on the cell membrane surface becomes stronger, making it easier to observe the morphology of the cell membrane. Since the luminescent capsule responds even to ligands that cannot penetrate the cell membrane, it is possible to screen for a wide range of stimuli.

[0068] A 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 polypeptide according to the present invention and the membrane localization signal (MLS), and is typically, (a) A luminescent fusion protein in which a fluorescent protein or luminescent enzyme (which may be an enzyme other than the polypeptide according to the present invention) is inserted between the C-terminus of the polypeptide according to the present invention and the membrane localization signal (MLS), or (b) A luminescent fusion protein may be formed in which a polypeptide that alters the morphology of the cell membrane or a polypeptide of 20 amino acid residues or less, preferably 10 amino acid residues or less, recognized by the polypeptide, is inserted between the C-terminal side of the polypeptide according to the present invention and the membrane localization signal (MLS). As the polypeptide that alters the morphology of the cell membrane, a polypeptide that induces cell death is preferred, and a polypeptide of 20 amino acid residues or less containing caspase and its recognition sequence "DEVD" or "IETD" is particularly preferred.

[0069] (ii) Application to luminescent probes By incorporating the polypeptide according to the present invention into a single-molecule or bi-molecule luminescent probe, the presence or absence of a ligand and the activity intensity of the ligand can be observed with high brightness. As a component of the probe, a high-performance luminescent probe can be constructed in which [1] a luminescent enzyme (N and C-terminal fragments) that has been split into two parts is connected to [2] a ligand-binding protein that responds to a target ligand and [3] a recognition protein that recognizes that the ligand has bound to the ligand-binding protein. In this luminescent probe, when the recognition protein recognizes that the ligand has bound to the ligand-binding protein, the two split enzyme fragments complement each other to change the enzyme activity. At this time, due to the high brightness and stability of the split enzyme, the detection limit can be improved and reliable measurement can be made possible.

[0070] A monomolecule bioluminescent probe is a known bioluminescent probe characterized by the integration of all components used for visualization imaging within a single fusion molecule. For example, the N and C-terminal fragments obtained by dividing the polypeptide according to the present invention into two parts constitute a fusion protein containing a ligand-binding protein and a recognition protein for the ligand-binding protein as basic components. A bimolecule bioluminescent probe refers to a type of bioluminescent probe in which the N-terminal and C-terminal fragments of the polypeptide according to the present invention are respectively present within a fusion protein containing a ligand-binding protein and a fusion protein containing a recognition protein.

[0071] When using the polypeptide according to the present invention in a bioluminescent probe, it is necessary to divide it into an N-terminal fragment and a C-terminal fragment. The division positions may be those corresponding to cleavage positions 125 / 126, 129 / 130, 133 / 134, 137 / 138, 141 / 142, and 146 / 147 of ALuc16.

[0072] The specific method for using the polypeptide according to the present invention as a single-molecule luminescent probe follows known methods. Specifically, the polypeptide according to the present invention is divided into two parts, and a chimeric DNA encoding a luminescent probe is designed by linearly linking a ligand-binding protein and a peptide sequence that recognizes the conformational change that occurs when a ligand binds to the protein. Generally, the chimeric DNA is subcloned in a vector suitable for the cells in which expression is to be desired, and the vector is introduced into the cells for expression. However, it is also possible to directly introduce the chimeric DNA into the cells by linking a control sequence upstream of the chimeric DNA. Here, the target cells are preferably mammalian cells, including human cells, and may be cells in a state that exists in vivo or cultured cells that maintain their original functions. Yeast cells, insect cells, and prokaryotic cells such as E. 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. As for the method of introduction into cells, known transfection methods such as microinjection and electroporation can be used. Alternatively, intracellular delivery methods using lipids (such as BioPORTER (Gene Therapy Systems) or Chariot (Active Motif)) can be employed.

[0073] The bioluminescent probe using polypeptides according to the present invention is introduced into cells as chimeric DNA and then expressed as a fusion protein within the cells. Therefore, after ligand stimulation of the transformed cells, the properties and activity level of the ligand can be evaluated by measuring the change in the amount of light emitted from the cells.

[0074] When a polypeptide according to the present invention is incorporated into a bioluminescent probe, the "ligand-binding protein" that can be carried together with the polypeptide is intended to be a protein to which a ligand binds at its ligand-binding site. The ligand-binding protein may, for example, undergo a change in three-dimensional structure, undergo phosphorylation, or promote protein-protein interactions upon ligand binding. Examples of such ligand-binding proteins include nuclear receptors (NRs), cytokine receptors, or various protein kinases that use hormones, chemical substances, or signaling proteins as ligands. 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, and may be an extracellular ligand taken into the cell from outside the cell, or an intracellular ligand produced inside the cell in response to extracellular stimuli. The extracellular ligand may, for example, be an agonist or antagonist to a receptor protein (e.g., a nuclear receptor, a G protein-bound receptor, etc.). These may include cytokines, chemokines, signaling proteins such as insulin, intracellular second messengers, lipid second messengers, phosphorylated amino acid residues, and G protein-bound receptor ligands that specifically bind to proteins involved in intracellular signal transduction.

[0075] For example, when targeting intracellular second messengers, lipid second messengers, etc., as ligands, the binding domain of each second messenger can be used as the ligand-binding protein. A second messenger is a different type of intracellular signaling molecule newly generated within a cell when extracellular signaling molecules 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, and Ca 2+ Examples include diacylglycerol and arachidonic acid. For example, Ca as a second messenger.2+ For this purpose, calmodulin (CaM) can be used as a ligand-binding protein.

[0076] (iii) Bioluminescence resonance energy transition (BRET) molecular probes The polypeptide according to the present invention can be used in any method for detecting molecular interactions, such as ligand-protein interactions or protein-protein interactions. A BRET molecular probe according to one embodiment comprises the polypeptide according to the present invention and a fluorescent substance. Energy transfer from the luminescent donor to the fluorescent receptor results in a shift in the spectral distribution of light emission. This energy transfer can enable real-time monitoring of intermolecular interactions in vitro or in vivo. As an example, fusion proteins are created by linking the polypeptide according to the present invention with a target molecule (target protein, ligand, nucleic acid, lipid, etc.) and fusion proteins are created by linking a molecule that binds to the target molecule (protein, ligand, nucleic acid, lipid, etc.) with a fluorescent substance. The polypeptide according to the present invention and the target molecule may be linked by a suitable linker (e.g., peptide, nucleic acid, polymer, ester bond, PEG linker, carbon chain, etc.). When the target molecule and the molecule that binds to the target molecule are in close proximity, the polypeptide according to the present invention and the fluorescent substance are in close proximity, and a BRET signal is detected. The assay system including the BRET molecular probe may also include a luminescent substrate. By adding molecules that compete for binding to the target molecule (for example, molecules that bind to the target molecule but are not linked to a fluorescent substance), the BRET signal decreases. This decrease in the BRET signal can also be used to detect intermolecular interactions (competitive binding assay).

[0077] The absorption spectrum of the fluorescent substance typically overlaps with the emission spectrum of the polypeptide according to the present invention. The wavelength peaks of the luminescent enzyme are typically separated from the wavelength peaks of the fluorescent substance, for example, by about 80 nm, 100 nm, 120 nm, and 140 nm. The fluorescent substance may be a fluorescent protein, a fluorescent dye, or a chromophore. Fluorescent substances include xanthene derivatives (e.g., fluorescein, rhodamine, Oregon Green, eosin, Texas Red, etc.), cyanine derivatives (e.g., cyanine, indocarbocyanine, oxacarbocyanine, thiacarbocyanine, merocyanine, etc.), naphthalene derivatives (e.g., dansyl and prodan derivatives), oxadiazole derivatives (e.g., pyridyloxazole, nitrobenzoxadiazole, benzooxadiazole, etc.), pyrene derivatives (e.g., Cascade Blue), oxazine derivatives (e.g., Nile Red, Nile Blue, Cresyl Violet, Oxazine 170, etc.), acridine derivatives (e.g., proflavin, acridine orange, acridine yellow, etc.), arylmethine derivatives (e.g., Auramine, Crystal Violet, Malachite Green, etc.), tetrapyrrole derivatives (e.g., porfin, phthalocyanine, bilirubin, etc.), CF dye (Biotium), BODIPY (Invitrogen), ALEXA FLuoR (Invitrogen), and DYLIGHT. Examples include FLUOR (Thermo Scientific, Pierce), ATTO and TRACY (Sigma-Aldrich), FluoProbes (Interchim), DY and MEGASTOKES (Dyomics), SULFO CY dye (CYANDYE, LLC), SETAU and SQUARE DYES (SETA BioMedicals), QUASAR and CAL FLUOR dyes (Biosearch Technologies), SURELIGHT DYES (APC, RPE, PerCP, phycobilisome) (Columbia Biosciences), APC, APCXL, RPE, BPE (Phyco-Biotech), autofluorescent proteins (e.g., YFP, RFP, mCherry, mKate), quantum dot nanocrystals, etc. The fluorescent substance may also be a rhodamine analog (e.g., a carboxyrhodamine analog).

[0078] Another example of a BRET molecular probe is a fusion protein in which a polypeptide according to the present invention and a fluorescent substance are linked together using a protein digested by a specific protease as a linker. In the absence of the specific protease, the luminescent enzyme and the fluorescent substance are in close proximity, resulting in a BRET fluorescence signal. In the presence of the specific protease, the protein is digested, separating the luminescent enzyme and the fluorescent substance, causing quenching. This method allows for the detection of the presence of a specific protease.

[0079] (iv) Protein complementation assay (PCA) The polypeptide according to the present invention may be used in methods for detecting ligand-protein interactions 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, for example, polypeptides. For example, the polypeptide according to the present invention is fragmented into two parts and each part is fused to a molecule whose proximity is to be verified. When the target molecules interact, the two fragmented polypeptides interact to form a complete luminescent enzyme, and luminescence is detected.

[0080] (v) Intracellular imaging The gene encoding the polypeptide according to the present invention can be stably introduced into various cell lines. Intracellular imaging using luminescent enzymes can be performed by known methods. For example, the polypeptide can be stably introduced into undifferentiated embryonic cells, ES cells, and induced pluripotent stem (iPS) cells.

[0081] By linking a suitable signal peptide to the polypeptide according to the present invention, it can be used for high-intensity imaging of various organelles. For example, adding the "MLCCMRRTKQV sequence" (SEQ ID NO: 63) derived from GAP-43 to the N-terminus or C-terminus of the polypeptide allows for localization to the cell membrane. Adding the "GRKKRRQRRR sequence" (SEQ ID NO: 64) allows for localization to the cytoplasm. Adding "KDEL" (SEQ ID NO: 65) allows for localization to the endoplasmic reticulum (ER), and adding the "DPKKKRKV sequence" (SEQ ID NO: 66) allows for localization to the cell nucleus. By attaching antigen sites such as HIS-tag (HHHHHH) (SEQ ID NO: 67), FLAG-tag (DYKDDDDK) (SEQ ID NO: 68), Myc-tag (EQKLISEEDL) (SEQ ID NO: 69), HA-tag (YPYDVPDYA) (SEQ ID NO: 70), V5-tag (GKPIPNPLLGLDST) (SEQ ID NO: 71), and T7-tag (MASMTGGQQMG) (SEQ ID NO: 72), these can be used for immunohistochemistry, isolation, and purification in non-cellular systems. In such cases, well-known immunohistochemistry methods and immunostaining techniques can be applied.

[0082] Other terms and concepts used herein are defined in detail in the description of embodiments and examples of the invention. The terminology is generally based on the IUPAC-IUB Commission on Biochemical Nomenclature, or on the meanings of terms commonly used in the art. Furthermore, the various techniques used to carry out the invention, except for those techniques whose sources are specifically indicated, can be easily and reliably implemented by those skilled in the art based on known literature, etc.For example, gene engineering and molecular biological techniques are described 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); DMGlover et al. ed., "DNA Cloning", 2nd ed., Vol.1 to 4, (The Practical Approach Series), IRL Press, Oxford University Press (1995); Ausubel, FM et al., "Current Protocols in Molecular Biology", John Wiley & Sons, New York, NY, 1995; Japanese Biochemical Society (ed.), "Continued Biochemistry Experiment Course 1, Gene Research Methods II", Tokyo Kagaku Dojin (1986); Japanese Biochemical Society (ed.), "New Biochemistry Experiment Course 2, Nucleic Acids III (Recombinant DNA Technology)", Tokyo Kagaku Dojin (1992); R. Wu ed., "Methods in Enzymology", Vol.68 (Recombinant DNA), Academic Press, New This can be done by the methods described in 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), etc., or by the methods described in the cited literature, or by methods or modifications substantially similar thereto.Furthermore, the various proteins, peptides, and encoding DNA used in this invention can be obtained from existing databases (URL: http: / / www.ncbi.nlm.nih.gov / ), etc. [Examples]

[0083] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples.

[0084] <Experiment 1: Preparation of picALuc plasmid> ALuc30wt is a luminescent enzyme that has the amino acid sequence corresponding to positions 20-221 of SEQ ID NO: 1, and is derived from ALuc30 (SEQ ID NO: 28) with the amino acid sequence from positions 20-212, excluding the signal sequence. The sequence of ALuc30wt is shown in Figure 1. The molecular weight of ALuc30wt is approximately 21 kDa. ALuc consists of two helical structures, a loop structure, a helical structure, a helical structure, a loop structure, a helical structure, two small helical structures, a helical structure, and a small helical structure, in order from the N-terminus. As shown in Figure 1, picALuc30 (SEQ ID NO: 51) was created by removing the N-terminus and C-terminus of ALuc30wt, and containing amino acids from positions 54-175 of ALuc30wt. picALuc30 was inserted into a pcDNA3.1(+) vector (Thermo Fisher Scientific). Using the same method, expression plasmids for picALuc16 (SEQ ID NO: 52) and picALuc48 (SEQ ID NO: 53), which have amino acid sequences equivalent to picALuc30, were constructed using ALuc16 (SEQ ID NO: 15) and ALuc48 (SEQ ID NO: 40) instead of ALuc30. The size of picALuc30 was 13 kDa. A His-tag was added to the N-terminus and a Flag-tag to the C-terminus of each mutant.

[0085] The amino acid sequence identity between picALuc30 and picALuc16 was 96% (Figure 2), between picALuc30 and picALuc48 was 85% (Figure 3), and between picALuc48 and picALuc16 was 90% (Figure 4).

[0086] <Experiment 2: Measurement of picALuc's luminescence value> (1) African green monkey kidney-derived COS-7 cells were seeded in 24 welldishes and allowed to subconfluence the following 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 and 1 μL of lipofectoamine 3000 (Invitrogen) were mixed. (4) Mix (2) and (3) and incubate at room temperature for 5 minutes. (5) The mixture was added to the culture medium of (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 secreted luminescent enzymes. (7) Coelenterazine, the substrate, was added to 100 μL of culture medium containing the luminescent enzyme to a final concentration of 5 μM, and the luminescence value was measured using an Enspire multi-mode plate reader (PerkinElmer).

[0087] picALuc30 showed an emission value equivalent to or higher than ALuc30wt (Figure 5). Sufficiently high emission values ​​were also measured for picALuc16 and picALuc48.

[0088] <Experiment 3: Creation of Δloop plasmid> The expected three-dimensional structure of picALuc30 is shown in Figure 6. picALuc30 had multiple loop structures. Mutants were created by deleting the amino acid sequences constituting three of these loops (loop 1, loop 2, and loop 3). Amino acid residues from 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 removed. This resulted in the creation of expression plasmids for ALuc30Δloop1N1, ALuc30Δloop2N1 (SEQ ID NO: 54), and ALuc30Δloop3N1, which lacked the N-terminus and loops. The size of ALuc30Δloop2N1 was 14 kDa.

[0089] <Experiment 4: Measurement of luminescence value of ALucΔloop> The emission values ​​were measured using the same method as in Experiment 2. ALuc30Δloop2N1 maintained approximately half the emission value compared to ALuc30ΔN1 with only the N-terminus removed (Figure 7). The emission values ​​of ALuc30Δloop1N1 and ALuc30Δloop3N1 were significantly lower.

[0090] <Experiment 5: Comparison with known luminescent enzymes> We compared the known NanoLuc, TurboLuc, and GLuc with picALuc, which was prepared in Experiment 1. NanoLuc is known to be small in size (approximately 19 kDa), have very high luminescence, and exhibit high thermal stability. TurboLuc is known to be small in size (approximately 16 kDa), have relatively high luminescence, and exhibit high thermal stability. GLuc is small in size (20 kDa), and when secreted and expressed from cells, it is known to have lower luminescence and higher thermal stability compared to ALuc. For the preparation of NanoLuc, TurboLuc, and GLuc, plasmids were used in which the sequences described in SEQ ID NOs. 73, 74, and 75 were inserted into pcDNA3.1 vectors, respectively.

[0091] Except for using a final coelenterazine concentration of 0.5 μM, the luminescence values ​​were measured using the same method as in Experiment 2. The luminescence values ​​were highest in the order of NanoLuc >> picALuc30 > TurboLuc > ALuc30wt > GLuc (Figure 8). Using a final coelenterazine concentration of 5 μM, the luminescence values ​​were measured using the same method as in Experiment 2. The luminescence values ​​were highest in the order of ALuc30wt = picALuc30 > NanoLuc > TurboLuc > GLuc (Figure 9).

[0092] Except for using coelenterazine h with a final concentration of 5 μM as the substrate, the luminescence values ​​were measured using the same method as in Experiment 2. The luminescence values ​​were highest in the order of TurboLuc = ALuc30wt >> NanoLuc > picALuc30 > GLuc (Figure 10). Except for using coelenterazine h with a final concentration of 25 μM as the substrate, the luminescence values ​​were measured using the same method as in Experiment 2. The luminescence values ​​were highest in the order of picALuc30 >> NanoLuc = TurboLuc = ALuc30wt >> GLuc (Figure 11).

[0093] Except for using furimazine, sold by Promega as a substrate for NanoLuc, at the manufacturer's recommended concentration, the luminescence values ​​were measured using the same method as in Experiment 2. Luminescence was detected in GLuc, ALuc30wt, and picALuc30, but it was lower compared to NanoLuc and TurboLuc (Figures 12 and 13). NanoLuc and TurboLuc showed high luminescence values, and the luminescence value of NanoLuc was comparable to that when using coelenterazine or coelenterazine h (Figure 13).

[0094] From these results, it was revealed that coelenterazine and coelenterazine h are more suitable as substrates for picALuc30 than flimazine, and that secreted picALuc30 exhibits luminescence values ​​of NanoLuc and TurboLuc or higher when reacted with high concentrations of coelenterazine or coelenterazine h as substrates.

[0095] <Experiment 6: Stability of protein terminology> Using the same method as in Experiment 2, COS-7 cells were transfected with plasmids, and the culture supernatant was collected. The Flag-tag attached to the N-terminus and His-tag attached to the C-terminus of each secreted luminescent enzyme were detected by Western blotting (SDS-PAGE, Mini-Protean TGX gel, StainFree 4-15% (Bio-Rad)) (Figure 14). Antibodies used were Anti 6×Histidine, Monoclonal Antibody (9C11), Peroxidase Conjugated (Fujifilm Wako Pure Chemical Industries, 1:1000) and Monoclonal ANTI-FLAG(R) M2-Peroxidase (HRP) antibody produced in mouse, clone M2 (Sigma-Aldrich, 1 / 1000), respectively, and 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 detection value of the TurboLuc Flag-tag was low, indicating that the N-terminus was truncated. On the other hand, both ends of ALuc30wt and picALuc30 were not truncated, indicating higher stability compared to GLuc and TurboLuc.

[0096] <Experiment 7: Measurement of specific activity> The specific activities of NanoLuc, ALuc30wt, and picALuc30 were measured by adjusting the enzyme concentrations based on the signal intensities of the Western blots from Experiment 6. When reacted with coelenterazine or coelenterazine h as substrates, the specific activities of ALuc and picALuc were similar (Figures 15 and 16). The maximum luminescence values ​​of ALuc30wt and picALuc30 were similar to those of NanoLuc, indicating that they possess high luminescence activity.

[0097] <Experiment 8: Measurement of emission spectrum> The wavelength peak of picALuc30 was 482 nm when reacted with coelenterazine (Figure 17) and 488 nm when reacted with coelenterazine h (Figure 18). The wavelength peak of picALuc30 was almost the same as that of ALuc30wt. The emission spectrum had a characteristic where the tail on the long wavelength side was broader than the tail on the short wavelength side.

[0098] <Experiment 9: Verification of Thermal Stability> Following the same procedure as in Experiment 2, COS-7 cells were transfected with the plasmid, and the culture medium was collected. The culture supernatant containing picALuc30 was incubated at room temperature (25°C), 40°C, 50°C, 60°C, 70°C, 80°C, or 90°C for 10 minutes, and the luminescence value was measured. As a result, more than 80% of the activity remained after incubation at 50°C for 10 minutes or 60°C for 10 minutes, and more than 50% remained after incubation at 70°C for 10 minutes (Figure 19), confirming that it has sufficient practical use.

[0099] <Experiment 10: Expression of luminescent enzymes in E. coli> (1) A plasmid was prepared by inserting the DNA sequence encoding picALuc30 into the pET32 vector. This plasmid was used to transform E. coli SHuffle T7 express lysY (New England Biolab). (2) E. coli from (1) was seeded onto LB plates (containing 100 μg / μL ampicillin). (3) The following day, one colony was picked and incubated overnight at 30°C in a test tube containing 2 mL of LB medium (containing 100 μg / μL ampicillin). (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 culture was performed with shaking at 30°C until the concentration reached approximately 0.4. (5) Absorbance OD 600 When the ratio reached approximately 0.4, 40 μL of 1 M isopropyl-β-thiogalactopyranoside was added, and the mixture was incubated overnight at 16°C. (6) The bacterial cells were collected and the proteins were purified using HisTALON Buffer Set and TALON Metal Affinity Resin (both from Takara Bio Inc.). 1.7 mg of picALuc30 was obtained from 100 mL of culture medium.

[0100] When the specific activity was measured by matching the concentration using western blot, the specific activity of picALuc30 secreted and expressed from COS-7 cells was almost the same as that of picALuc30 produced in E. coli (Figure 20). This indicates that picALuc30 can be expressed not only from mammalian cells but also from E. coli, and that it can be produced in large quantities.

[0101] picALuc30 purified from E. coli was incubated at room temperature (25°C), 60°C, 70°C, 80°C, or 90°C for 10 minutes, and its luminescence was measured (Figure 21). picALuc30 expressed in E. coli lost almost no activity even after incubation at 60°C for 10 minutes, and retained more than 90% of its activity after incubation at 70°C for 10 minutes, and more than 80% after incubation at 80°C for 10 minutes, indicating excellent thermal stability.

[0102] <Experiment 11: BRET Molecular Probe> The luminescent enzyme (NanoLuc or picALuc30)-Gly-Ser-TEV protease recognition sequence (Glu-Asn-Leu-Tyr-Phe-Gln-Ser)-Ser-YFP was inserted into the pET32 vector (Figure 22). Using this plasmid, the protein was expressed and purified in the same manner as in Experiment 10. The fusion protein was reacted overnight at 30°C with TEV protease (Cosmo Bio Co., Ltd.). A solution without protease was used as a control. The protein after protease treatment was diluted to 40 nM in PBS and reacted with coelenterazine h (500 nM) as a substrate. The wavelength peak of the luminescent enzyme was detected at approximately 488 nm, and the wavelength peak of YFP was detected at approximately 527 nm. The spectrum when NanoLuc was used as the luminescent enzyme is shown in Figure 23, and the spectrum when picALuc30 was used as the luminescent enzyme is shown in Figure 24.

[0103] The BRET ratio was calculated by setting the maximum luminescence of NanoLuc to 100%. As a result, the BRET ratio of the probe containing NanoLuc was 26% (Figure 25), and the BRET ratio of the probe containing picALuc30 was 44% (Figure 26). BRET efficiency is inversely proportional to the sixth power of the distance between the luminescent enzyme and the fluorescent protein. It is thought that ALuc30 obtained a higher BRET ratio compared to NanoLuc because the size of picALuc is smaller than that of NanoLuc. This suggests that the polypeptide according to the present invention is useful as a BRET molecular probe.

[0104] [Pattern] Those skilled in the art will understand that the above-described exemplary embodiments and examples are specific examples of the following embodiments.

[0105] (Section 1) Polypeptides having luminescent enzyme activity, comprising the amino acid sequence described in (A) or (B); (A) Amino acid sequences in which amino acid residues at positions 1-69 and 204-221 are missing from the amino acid sequence shown in Sequence ID No. 1, (B) An amino acid sequence in which the amino acid residues at positions 1 to 69 are deleted and at least one amino acid residue at positions 146 to 156 is deleted or substituted.

[0106] According to the polypeptide in paragraph 1, it is possible to obtain a small-sized luminescent enzyme while maintaining luminescence activity.

[0107] (Section 2) The polypeptide described in paragraph 1 further includes an amino acid sequence in which at least one amino acid residue between positions 70 and 74 of the amino acid sequence shown in SEQ ID NO: 1 is further missing.

[0108] According to the polypeptide in Section 2, it is possible to obtain a smaller luminescent enzyme while maintaining luminescence activity.

[0109] (Section 3) The polypeptide of paragraph 1 or 2 contains any of the sequences (a) to (c); (a) Amino acid sequence described in any of Sequence IDs 51-56, (b) An amino acid sequence having 85% or more identity with any of the amino acid sequences described in Sequence ID No. 51 to 56, or (c) An amino acid sequence in which one or more amino acid residues are deleted, substituted, inserted, or added to any of the amino acid sequences described in SEQ ID NOs. 51 to 56.

[0110] The polypeptides described in paragraph 3 can be used to obtain luminescent enzymes with high luminescence activity.

[0111] (Section 4) The polypeptides described in paragraphs 1 to 3 retain 80% or more of their activity after heat treatment at a temperature of 50°C for 10 minutes.

[0112] The polypeptide described in Section 4 can be used to obtain a luminescent enzyme with high thermal stability.

[0113] (Section 5) In the polypeptides described in paragraphs 1 to 4, the luminescent enzyme activity is such that when coelenterazine is used as a substrate, the emission wavelength peak is between 470 nm and 490 nm, or When coelenterazine h is used as the substrate, the emission wavelength peak is between 470 nm and 490 nm.

[0114] The polypeptides described in Section 5 exhibit a shift in their emission spectrum toward longer wavelengths, resulting in good biopermeability and superior detection sensitivity. Furthermore, the polypeptides described in Section 5 are suitable for live imaging.

[0115] (Section 6) A nucleic acid encoding a polypeptide as described in any of paragraphs 1 to 5.

[0116] According to the nucleic acid described in paragraph 6, polypeptides described in paragraphs 1 to 5 can be produced.

[0117] (Section 7) A vector containing the nucleic acid described in paragraph 6.

[0118] The vector described in Section 7 allows for the easy amplification and maintenance of the nucleic acids described in Section 6. Furthermore, the vector described in Section 7 can also be used to produce the polypeptides described in Sections 1 through 5.

[0119] (Section 8) Transformed cells into which the nucleic acid described in paragraph 6 has been introduced.

[0120] The transformed cells described in paragraph 8 may express a luminescent enzyme. The luminescent enzyme may be secreted into the supernatant.

[0121] (Section 9) A reporter protein comprising a polypeptide as described in any of paragraphs 1 to 5.

[0122] A polypeptide described in any of paragraphs 1 to 5 may be used as a reporter protein.

[0123] (Section 10) A reporter analysis method using the reporter protein described in Section 9.

[0124] The reporter analysis method described in Section 10 allows for the efficient detection of the behavior of target proteins and target genes. In the reporter analysis method described in Section 10, a polypeptide described in any of Sections 1 to 5 can be used as the luminescent enzyme instead of the luminescent enzyme used in known reporter analysis methods.

[0125] (Section 11) A fusion protein comprising a polypeptide described in any of paragraphs 1 to 5 and a target protein or a protein that binds to a target protein.

[0126] According to the fusion protein described in Section 11, the behavior of the target protein can be detected by luminescence.

[0127] (Section 12) A vector containing nucleic acid encoding the fusion protein described in Section 11.

[0128] The vector described in Section 12 allows for the easy amplification and maintenance of nucleic acids encoding the fusion protein described in Section 11. The fusion protein described in Section 11 can be easily produced using the vector described in Section 12.

[0129] (Section 13) A bioluminescent resonance energy transfer (BRET) molecular probe comprising a polypeptide described in any of paragraphs 1 to 5 and a fluorescent substance.

[0130] The molecular probes described in Section 13 can detect proteases and other substances with high sensitivity.

Claims

1. A polypeptide comprising any of the sequences (a) to (b) having luminescent enzyme activity, The polypeptide having 122 or more amino acid residues and 126 or less; (a) The amino acid sequence described in Sequence ID No. 51, or (b) An amino acid sequence having 97% or more identity with the amino acid sequence described in Sequence ID No.

51.

2. The polypeptide according to claim 1, wherein the luminescent enzyme activity retains 80% or more of its activity after heat treatment at a temperature of 50°C for 10 minutes.

3. The aforementioned luminescent enzyme activity is such that when coelenterazine is used as a substrate, the emission wavelength peak is between 470 nm and 490 nm, or The polypeptide according to claim 1 or claim 2, wherein the emission wavelength peak is 470 nm or more and 490 nm or less when coelenterazine h is used as a substrate.

4. A nucleic acid encoding a polypeptide according to any one of claims 1 to 3.

5. A vector comprising the nucleic acid described in claim 4.

6. A transformed cell into which the nucleic acid described in claim 4 has been introduced.

7. A reporter protein comprising the polypeptide described in any one of claims 1 to 3.

8. A reporter analysis method using the reporter protein described in claim 7.

9. A fusion protein comprising a polypeptide according to any one of claims 1 to 3 and a target protein or a protein that binds to a target protein.

10. A vector comprising a nucleic acid encoding the fusion protein described in claim 9.

11. A bioluminescent resonance energy transition (BRET) molecular probe comprising a polypeptide according to any one of claims 1 to 3 and a fluorescent substance.

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