Luminescent enzyme-active protein

Abstract

To provide a new luciferase that has a high emission value despite its small molecular weight.SOLUTION: An example of this invention can be a luciferase-active protein that comprises an amino acid sequence within the molecule, in which the oligopeptide mentioned in (I) is connected to the C-terminus of the polypeptide described in (II): (I) an oligopeptide comprising a polar amino acid at the C-terminus and (II) a polypeptide comprising an amino acid sequence derived from copepods, the polypeptide having luciferase activity.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] The present invention belongs to the technical field of luminescent enzymes. The present invention relates to a protein having luminescent enzyme activity.

Background Art

[0002] Conventionally, in the fields of basic biology, medical diagnosis, drug screening, food and hygiene management, etc., luminescent enzymes have been used as reporter proteins for detecting targets. In basic biology, luminescent enzymes are used in reporter assays and imaging to analyze the expression level and location of targets. In medical diagnosis, they are used for detecting disease markers, etc. In drug screening, the effects of drugs are analyzed by fusing them with drug targets or target-related factors. Also, in places where food and hygiene management are required, luminescent enzymes are used for detecting bacteria, etc.

[0003] Examples of other uses of reporter proteins include fluorescent proteins, etc. However, in order to perform observations, etc. using fluorescent proteins, excitation light is required, whereas luminescent enzymes have the characteristic of not requiring excitation light. Therefore, compared with fluorescent proteins, luminescent enzymes have the following advantages : 1) There is no phototoxicity due to excitation light irradiation on living organisms and cells; 2) Since autofluorescence of cells due to excitation light irradiation is not observed, the background signal is low; 3) Excitation light does not become the background signal; 4) There is no need to equip the measuring machine with an excitation light irradiation device.

[0004] If a luminescent enzyme is large in size (molecular weight), the fusion protein formed from the luminescent enzyme and the target protein may not be properly expressed in the cell, or the target protein may not function properly due to steric hindrance. Furthermore, large luminescent enzymes are more susceptible to the influence of small molecule compounds on their luminescence, making them unsuitable for drug screening. In addition, 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, the smaller the size of the luminescent enzyme, the stronger the signal.

[0005] Therefore, the small size of luminescent enzymes offers several advantages: 1) ease of expression, 2) low steric hindrance to targets and target binding factors, 3) reduced susceptibility to small molecule compounds, and 4) increased signal strength in intermolecular interaction detection using BRET. For this reason, even smaller luminescent enzymes are desired. To date, the inventors have developed the world's smallest luminescent enzyme (picALuc®, hereinafter the same) with luminescence characteristics at a practical level of brightness (Non-Patent Literature 1). [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] ACS Chem. Biol., 2022, Vol.17, Issue 4, p.864-872 [Overview of the project] [Problems that the invention aims to solve]

[0007] Generally, luminescent enzymes tend to have lower (dimmer) emission values ​​compared to fluorescent proteins. Therefore, there is a need to develop luminescent enzymes that have a size (molecular weight) comparable to small luminescent enzymes, such as the luminescent enzyme described in Non-Patent Document 1, while possessing brighter emission characteristics.

[0008] The main objective of this invention is to provide a novel luminescent enzyme with a small molecular weight but a higher luminescence value. [Means for solving the problem]

[0009] As a result of diligent research, the present inventors have discovered that by adding an oligopeptide having a polar amino acid (preferably an charged amino acid) at its C-terminus to the C-terminus of a luminescent enzyme (such as picALuc) containing a polypeptide structure derived from copepods, the luminescence value of the luminescent enzyme increases, thus completing the present invention. Examples of the present invention include the following embodiments. [1] A protein having luminescent enzyme activity, characterized in that it contains within its molecule an amino acid sequence in which the oligopeptide described in (I) below is linked to the C-terminus of the polypeptide described in (II) below; (I) Oligopeptides having polar amino acids at the C-terminus, (II) A polypeptide comprising an amino acid sequence derived from copepods, wherein the polypeptide has bioluminescent enzyme activity. [2] The protein according to [1] above, wherein the polar amino acid is an amino acid having an electric charge. [3] The protein described in [1] above, wherein the number of amino acid residues constituting the oligopeptide is in the range of 4 to 15. [4] The protein according to [1] above, wherein the polypeptide is a polypeptide comprising the amino acid sequence described in (A) or (B) below; (A) Amino acid sequences in which amino acid residues at positions 1-69 and 204-221 are missing from the amino acid sequence represented by 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. [5] The protein according to [1], wherein the polypeptide comprises the amino acid sequence described in (A) or (B) above, and further comprises an amino acid sequence in which at least one amino acid residue between positions 70 and 74 of the amino acid sequence represented by Sequence ID No. 1 is missing. [6] The protein according to [1] above, wherein the polypeptide is a polypeptide comprising any of the sequences described in (a) to (c) below; (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 obtained by deleting, substituting, inserting, or adding one or more amino acid residues to the amino acid sequence described in any of Sequence IDs 51-56. [7] A nucleic acid that codes for the amino acid sequence of any one of the proteins described in [1] to [6] above. [8] An expression vector into which a gene sequence for expressing any of the proteins described in [1] to [6] above has been inserted. [9] Transformed cells into which a gene sequence for expressing any one of the proteins described in [1] to [6] above has been introduced.

[10] A fusion protein comprising a protein described in any one of the above items [1] to [6] and a target protein or a protein that binds to a target protein.

[11] A nucleic acid encoding the amino acid sequence of the fusion protein described in

[10] above.

[12] An expression vector into which a gene sequence for expressing the fusion protein described in

[10] above has been inserted.

[13] Transformed cells into which the gene sequence for expressing the fusion protein described in

[10] above has been introduced.

[14] A bioluminescent resonance energy transfer (BRET) probe comprising a protein described in any one of the above items [1] to [6] and a fluorescent substance.

[0010]

[15] A method for improving the luminescence value of a luminescent enzyme, characterized by linking an amino acid sequence of an oligopeptide having a polar amino acid at the end to the end of the amino acid sequence of the luminescent enzyme.

Advantages of the Invention

[0011] According to the present invention, it is possible to provide a luminescent enzyme with an improved luminescence value while having a small molecular weight. Further, according to the present invention, it is possible to improve the luminescence value of a luminescent enzyme such as picALuc while minimizing an increase in size.

Brief Description of the Drawings

[0012] [Figure 1] It is a graph showing the luminescence values of each luminescent enzyme at each substrate concentration shown on the horizontal axis (Experiment 1). The vertical axis indicates the luminescence value (s-1). [Figure 2] It is a graph showing the luminescence values of each luminescent enzyme having a structure in which each oligopeptide shown on the horizontal axis is added to the C-terminus of picALuc30 (Experiment 2). The vertical axis indicates the luminescence value (108·s-1). [Figure 3] It is a graph showing the luminescence values of picALuc30 (WT) and each luminescent enzyme having a structure in which each oligopeptide shown on the horizontal axis is added to the C-terminus of picALuc30 (Experiment 2). The vertical axis indicates the luminescence value (107·s-1). [Figure 4] It is a graph showing the luminescence values of WT and each luminescent enzyme having a structure in which each amino acid shown on the horizontal axis is added to the C-terminus of an oligopeptide (Experiment 3). The vertical axis indicates the luminescence value (107·s-1). [Figure 5] It is a graph showing the luminescence values of each luminescent enzyme having a structure in which each oligopeptide shown on the horizontal axis is added to the C-terminus of picALuc30 (Experiment 4). The vertical axis indicates the luminescence value (108·s-1). [Figure 6] It is a graph showing the luminescence values of WT and each luminescent enzyme having a structure in which each amino acid shown on the horizontal axis is added to the C-terminus of an oligopeptide (Experiment 4). The vertical axis indicates the luminescence value (106·s-1). [Figure 7] It is a graph showing the luminescence values of each luminescent enzyme at each substrate concentration (Experiment 4). The horizontal axis represents the substrate concentration (μM), and the vertical axis represents the luminescence value (108·s-1).

Embodiments for Carrying Out the Invention

[0013] Hereinafter, the present invention will be described in detail. 1 Protein According to the Present Invention The protein according to the present invention (hereinafter referred to as "the protein of the present invention") is a protein having luminescent enzyme activity, and is characterized in that it contains, within the molecule, an amino acid sequence having a structure in which the oligopeptide described in the following (I) is linked to the C-terminus of the polypeptide described in the following (II). (I) An oligopeptide having a polar amino acid at the C-terminus. (II) A polypeptide having luminescent enzyme activity and containing an amino acid sequence derived from Cyathus.

[0014] The protein of 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. The luminescent enzyme activity in this specification represents the activity of the enzyme reaction between the luminescent enzyme and the substrate, and is measured by detecting the light (luminescence spectrum) emitted when the substrate returns to the ground state after being excited by the enzyme reaction with the luminescent enzyme. The light emitted when returning to the ground state can be detected using a known luminometer (for example, "GloMax" series manufactured by Promega) or a spectrophotometer (for example, "Infinite200PRO" manufactured by TECAN). By measuring the intensity at a specific wavelength every minute, the temporal change and stability of the luminescence can be detected. The shift of the luminescence to a longer wavelength can be detected by measuring the entire wavelength.

[0015] The pH and temperature of the luminescent enzyme activity may be the same as those of known luminescent enzymes (e.g., copepod luminescent enzymes or artificial luminescent enzymes). For these indicators, the optimal values ​​for the luminescent enzyme activity of the protein of the present invention may be the same as those for the luminescent enzyme activity of copepods. For example, a suitable pH range for luminescent enzyme activity is 5.0 to 9.5, with an optimal pH of 7.0. A suitable temperature range is 4°C to 30°C, with an optimal temperature of 25°C.

[0016] The luminescent substrate is not particularly limited and can be appropriately selected in accordance with the luminescent enzyme. The luminescent substrate may be a known substrate such as a coelenterazine-based substrate, a firefly luciferin-based substrate, a sea firefly luciferin-based substrate, or a flimazine-based substrate, but a coelenterazine-based substrate is preferred. 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. The luminescent substrate preferably contains coelenterazine or coelenterazine h.

[0017] 1.1 Oligopeptides The protein of the present invention contains an oligopeptide having a polar amino acid at its C-terminus (hereinafter referred to as "the constituent oligopeptide of the present invention") in its molecular structure.

[0018] There are no particular restrictions on the number of amino acid residues in the constituent oligopeptide of the present invention, but for example, it is suitable to have a range of 4 to 15. In particular, it is preferable to have a range of 5 to 12, and more preferably to have a range of 6 to 10.

[0019] Here, "polar amino acids" refers to amino acids that have polar or hydrophilic side chains, and as long as they have such side chains, this includes both charged and uncharged amino acids.

[0020] Among polar amino acids, charged amino acids are those that ionize in a neutral pH aqueous solution to acquire a positive or negative charge, and include both basic and acidic amino acids. Specific examples of basic amino acids include lysine (K), histidine (H), and arginine (R). Specific examples of acidic amino acids include aspartic acid (D) and glutamic acid (E).

[0021] Examples of polar amino acids that do not possess an electric charge (polar uncharged amino acids) include, for example, asparagine (N), glutamine (Q), serine (S), threonine (T), and cysteine ​​(C).

[0022] Among the above, it is preferable that the polar amino acid is an amino acid with an electric charge, i.e., a basic amino acid (an amino acid with a positive charge) or an acidic amino acid (an amino acid with a negative charge). By including an oligopeptide having a highly polar amino acid at its C-terminus, it is thought that the interaction between the highly polar amino acid and the vicinity of the substrate binding site within the enzyme stabilizes the enzyme activity, allowing for more appropriate binding of the substrate, and thus the protein of the present invention can be made into a bright luminescent enzyme exhibiting a higher luminescence value.

[0023] The polar amino acid is located at the C-terminus of the amino acid sequence of the constituent oligopeptide of the present invention. However, within the protein molecule of the present invention, other amino acids or peptides (such as dipeptides, tripeptides, oligopeptides, polypeptides, etc.) may be further linked to the C-terminus of the polar amino acid. Examples of such other amino acids or peptides include functional peptides such as antibody recognition sites as exemplified in Table 4 below, and signal peptides as exemplified in Table 5 below. Furthermore, the number of amino acid residues in the amino acid sequence of the portion protruding from the C-terminus polar amino acid of such constituent oligopeptide of the present invention can range from 1 to 20 to 1 to 5 or 1 to 3. Molecular structures with such configurations may also be included in the protein of the present invention.

[0024] 1.2 Polypeptides The protein of the present invention is a polypeptide containing an amino acid sequence derived from copepods, and its molecular structure includes a polypeptide having luminescent enzyme activity (hereinafter referred to as "the constituent polypeptide of the present invention"). In the protein of the present invention, the constituent oligopeptide of the present invention is linked to the C-terminus of the constituent polypeptide of the present invention.

[0025] Here, "amino acid sequence derived from copepods" refers to at least a portion of the amino acid sequence possessed by organisms belonging to the subclass Copepoda (copepods). The polypeptide constituting the present invention is preferably composed of at least a portion of the amino acid sequence of the luminescent enzyme possessed by copepods. Examples of polypeptides containing an amino acid sequence derived from copepods and possessing luminescent enzyme activity include TurboLuc, GLuc, MLuc, ALuc®, and picALuc® (see Japanese Patent Publication No. 2014-100137, International Publication No. 2017 / 057752, Mol.Biol.Evol.29(6):1669-1681.2012, Non-Patent Literature 1, etc.).

[0026] The polypeptide comprising the present invention preferably contains the amino acid sequence represented by SEQ ID NO: 1, or an amino acid sequence in which at least a portion of said sequence is missing. In particular, the polypeptide comprising the present invention is more preferably a polypeptide in which at least a portion of the amino acid sequence represented by SEQ ID NO: 1 is missing, as described below.

[0027] [ka]

[0028] Here, among the amino acid residues represented by X 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 a PTENKDDI sequence (2 residues deleted, SEQ ID NO: 2), an ATINEDI sequence (2 residues deleted, SEQ ID NO: 3), an ATINENFEDI sequence (SEQ ID NO: 4), an HHHHHHHH sequence (2 residues deleted, SEQ ID NO: 5), an EKLISEE sequence (2 residues deleted, SEQ ID NO: 6), an MMYPYDVP sequence (2 residues deleted, SEQ ID NO: 7), or an 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 an ANS sequence or a DAN sequence, the amino acid residue at position 69 is D or G, and the amino acid residues at positions 76-77 are a 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 may be completely 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.

[0029] 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.

[0030] 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.

[0031] 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 SEQ 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 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 may be G, A, or deleted, S, or F; the amino acid residue at position 40 may be T or 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 may be G or deleted; the amino acid residue at position 137 is G or A; and the amino acid residue at position 203 is T or G.

[0032] 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, and 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 deleted, the amino acid residue at position 97 is K or R, and the amino acid residue at position 110 is H or K.

[0033] 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.

[0034] One embodiment of the constituent polypeptide of the present invention is: (A) The amino acid sequence represented by Sequence ID No. 1 includes an amino acid sequence in which amino acid residues at positions 1-69 and 204-221 are missing.

[0035] One embodiment of the constituent polypeptide of the present invention is: (A1) The polypeptide may contain amino acid residues from positions 75 to 203 of the amino acid sequence represented by Sequence ID No. 1, and the number of amino acid residues may be 140 or less. The polypeptide of the present invention may consist of amino acid residues from positions 75 to 203 of the amino acid sequence represented by Sequence ID No. 1.

[0036] (A2) The polypeptide comprising the present invention may include amino acid residues from positions 75 to 203 of the amino acid sequence represented by Sequence ID No. 1, and the molecular weight of the polypeptide may be 20 kDa or less.

[0037] One embodiment of the constituent polypeptide of the present invention is: (B) The amino acid sequence represented by Sequence ID No. 1 includes an amino acid sequence in which 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.

[0038] One embodiment of the constituent polypeptide of the present invention is: (B1) The amino acid sequence may be one in which at least one amino acid residue between positions 146 and 156 is missing or substituted, and the amino acid sequence represented by SEQ ID NO: 1 includes amino acid residues between positions 70 and 221.

[0039] The polypeptide of the present invention may have at least one amino acid residue between positions 70 and 74 of the amino acid sequence represented by SEQ ID NO: 1 further missing, or all of the amino acid residues between positions 70 and 74 may be missing.

[0040] The polypeptide of 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 represented by SEQ ID NO: 1. A linker sequence of one to several bases may be inserted in place of the missing sites. The polypeptide of 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 represented by SEQ ID NO: 1.

[0041] The molecular weight of the polypeptide constituting 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 constituting the present invention is, for example, 10 kDa or more.

[0042] The number of amino acid residues in the polypeptide constituting 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 constituting the present invention is, for example, 100 or more.

[0043] Specific examples of luminescent enzymes having the amino acid sequence represented by SEQ ID NO: 1 include, for example, the group of luminescent enzymes shown in Table 1 (1A-1H). Luminescent enzymes having the amino acid sequence represented by SEQ 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.

[0044] [Table 1A]

[0045] [Table 1B]

[0046] [Table 1C]

[0047] [Table 1D]

[0048] [Table 1E]

[0049] [Table 1F]

[0050] [Table 1G]

[0051] [Table 1H]

[0052] The amino acid sequence represented by Sequence ID No. 1 may have the amino acid sequence represented by 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.

[0053] [ka]

[0054] The amino acid sequence represented by Sequence ID No. 1 may have the amino acid sequence represented by 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.

[0055] [ka]

[0056] The constituent polypeptide of the present invention preferably contains (a) an amino acid sequence represented by any of SEQ ID NOs. 51 to 56, and may consist of any of the amino acid sequences shown in Table 2 (SEQ ID NOs. 51 to 56). The amino acid sequence represented by SEQ ID NOs. 51 is the sequence obtained by removing the N-terminus and C-terminus from the amino acid sequence of ALuc30, one of the artificial luminescent enzymes. The amino acid sequence represented by SEQ ID NOs. 54 is the sequence obtained by removing the N-terminal sequence and intermediate sequence from the amino acid sequence of ALuc30. Similarly, the amino acid sequences represented by SEQ ID NOs. 52 and 53 are the 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. The amino acid sequences represented by SEQ ID NOs. 55 and 56 are the sequences obtained by removing the N-terminal sequence and intermediate sequence, respectively, from the amino acid sequences of ALuc16 and ALuc48.

[0057] [Table 2]

[0058] The constituent polypeptide of the present invention preferably contains an amino acid sequence having 85% or more identity with the amino acid sequence represented by 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 represented by any of SEQ ID NOs. The constituent polypeptide of the present invention 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 represented by any of SEQ ID NOs. 51 to 56.

[0059] The constituent polypeptide of the present invention preferably includes an amino acid sequence in which one or several amino acid residues are deleted, substituted, inserted, or added to the amino acid sequence represented by 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 represented by any of SEQ ID NOs: 51 to 56. In this specification, several may be, for example, 2 to 20, 2 to 10, 2 to 5, or 2 to 3.

[0060] According to the polypeptide composition of the present invention, which includes any of the sequences (a) to (c) above, a protein of the present invention with high luminescence activity can be obtained.

[0061] The protein or polypeptide comprising the present invention may contain an amino acid corresponding to the start codon (often methionine) before the amino acid residue at position 1. Table 3 (Sequences 57-62) shows sequences in which methionine is added to position 1 of the amino acid sequences of SEQ ID NOs. 51-56.

[0062] [Table 3]

[0063] One embodiment of the constituent polypeptide of the present invention may include (a1) to (c1) below, or consist of (a1) to (c1) below. (a1) Amino acid sequence represented by any of sequence numbers 57-62, (b1) An amino acid sequence that has 85% or more identity with any of the amino acid sequences represented by sequence numbers 57-62, or (c1) An amino acid sequence in which one or more amino acid residues are deleted, substituted, inserted, or added to an amino acid sequence represented by any of sequence numbers 57-62. 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 represented by any of SEQ ID NOs. 57 to 62.

[0064] 1.3 Physical properties of the protein of the present invention According to the present invention, the luminescence value can be improved while maintaining the small size of the luminescent enzyme (polypeptide constituting the present invention) as much as possible. With a small luminescent enzyme, when a fusion protein of the luminescent enzyme and a target protein or antibody is expressed in cells, the fusion protein is more likely to be expressed normally, and the possibility of the target protein not functioning properly can be reduced. Because the luminescence value of a small luminescent enzyme is less affected by small molecule compounds, it can be suitably used as a reporter protein for drug or ligand screening. When analyzing intermolecular interactions using bioluminescence resonance energy transfer (BRET), a small luminescent enzyme can be used to detect a strong signal. A small luminescent enzyme can be used as a secreted luminescent enzyme. With a secreted luminescent enzyme, it is not necessary to lyse cells to measure the luminescence value, and changes in gene expression over time can be measured. Because a small luminescent enzyme is easily expressed in cells, it can be expressed and purified in large quantities. A small luminescent enzyme can be expressed in various expression systems. Furthermore, a small luminescent enzyme has excellent structural stability.

[0065] The protein of the present invention can have a high luminescence value. Preferably, the peak luminescence value of the protein of the present invention is at or above that of known luminescent enzymes, such as NanoLuc, ALuc, etc. Luminescent enzymes with high luminescence values ​​enable highly sensitive detection of luminescence, and the detection limit concentration can be lowered.

[0066] The protein of 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 lose their activity when exposed to temperature increases during transport, etc., and are highly practical in clinical settings such as diagnostics and testing.

[0067] The enzyme activity of the protein of the present invention preferably has a broad tail on the long-wavelength side of its emission spectrum, and the emission spectrum is shifted to the long-wavelength side compared to, for example, conventional copepod luminescent enzymes. Long wavelengths have excellent biological permeability, resulting in excellent detection sensitivity. 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 protein of 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 protein of the present invention is preferably 470 nm to 490 nm, more preferably about 488 nm.

[0068] The protein of the present invention may have an antibody recognition site inside or at its end. Examples of antibody recognition sites are shown in Table 4, but are not limited to these.

[0069] [Table 4]

[0070] The protein of 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.

[0071] 2. Nucleic acids encoding the protein of the present invention A nucleic acid according to one embodiment of the present invention encodes the protein of the present invention described above. The protein of the present invention can be produced from the nucleic acid encoding the protein of the present invention described above. The nucleic acid is preferably DNA or RNA. The nucleic acid encoding the protein of the present invention may contain a start codon at the 5' end and a stop codon at the 3' end of the base sequence corresponding to the protein of the present invention described above. The nucleic acid may also contain an intron sequence. The nucleic acid according to one embodiment of the present invention can be obtained by chemical synthesis or PCR, etc.

[0072] 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.

[0073] 3 Vectors 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 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.

[0074] 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.

[0075] The vector allows for easy amplification and maintenance of the nucleic acids described above. Furthermore, the vector can also be used to produce the protein of the present invention.

[0076] 4 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 a form contained in a vector. Transformed cells can express a luminescent enzyme. The luminescent enzyme 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.

[0077] Cells into which nucleic acids are introduced include eukaryotic or prokaryotic cells, specifically bacteria, fungi, plant cells, animal cells, and insect cells. These cells may also be yeast, Escherichia coli, or mammalian cells. Mammals include humans, cattle, horses, sheep, monkeys, pigs, mice, rats, hamsters, guinea pigs, rabbits, dogs, and the like.

[0078] The transformed cells can express the protein of the present invention (luminescent enzyme). The luminescent enzyme may be secreted into the supernatant.

[0079] 5. Reporter Proteins and Reporter Analysis Methods The protein of the present invention can be used as a reporter protein to perform reporter analysis. The protein of 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.

[0080] 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 this protein 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.

[0081] 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 aforementioned protein 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.

[0082] Below, the reporter analysis methods 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 their application to each of the protein analysis methods of the present invention will be explained.

[0083] 5.1 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 studied. When applying the protein of the present invention as a reporter protein to the basic method, a fusion protein containing the protein of 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.

[0084] 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.

[0085] Fusion proteins include (i) those expressed as a whole from a nucleic acid encoding a fusion protein containing the protein of the present invention and a target protein or a protein (including peptides) that recognizes the target protein, and (ii) those expressed separately from the protein of the present invention and the target protein or a protein that recognizes the 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.

[0086] 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.

[0087] 5.2 Inducible Method 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.

[0088] 5.2.1 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.

[0089] In reporter gene assays, conventionally widely used firefly luciferases have the following 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 protein of the present invention as the reporter protein, these problems are resolved.

[0090] Using the protein of this invention as a reporter protein offers the advantage of extremely high luminescence intensity, allowing for measurements to be taken very quickly after stimulation. Therefore, measurement time can be significantly reduced compared to conventional reporter proteins, and the stability of luminescence over time is also high, enabling luminescence measurements 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 results in higher measurement accuracy.

[0091] For example, to apply the protein of 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 protein of the present invention, and it can be easily loaded using a known method.

[0092] 5.2.2 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, and the DNA-binding domain (DBD) binds to the "UASG" base sequence, 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.

[0093] 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.

[0094] Examples of vectors that can be used in budding yeast as a host cell and contain DNA encoding the DNA-binding region 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 region of GAL4 include plasmid pGAD424 (Clontech). Examples of vectors that can be used in mammalian cells and contain DNA encoding the DNA-binding region 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 region 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 region of LexA include pLexA (Clontech). Examples of vectors that can be used in mammalian cells and contain DNA encoding B42 include pB42AD (Clontech).

[0095] For example, a vector can be constructed by inserting the protein of the present invention as a reporter gene downstream of the region where 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 protein of 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).

[0096] 5.3 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).

[0097] 5.3.1 Manufacturing of luminescent fusion proteins (luminescent capsules) By attaching a membrane localization signal (MLS) to the C-terminus of the present invention's protein, 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 process, it is possible to insert the gene for any polypeptide or protein as cargo between the luminescent enzyme itself and the nucleic acid encoding the signal peptide. This allows for efficient delivery of the cargo protein to the cell membrane surface, and the location where it is delivered 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 and visualize the activity of caspase-3 and caspase-8 during cell death. 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.

[0098] 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 be linked in tandem with the protein of the present invention to enhance the amount of luminescence. 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.

[0099] 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-terminus of the present invention protein 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 protein of the present invention) is inserted between the C-terminus of the protein of the present invention and the membrane localization signal (MLS), or (b) The present invention may be a luminescent fusion protein 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 said polypeptide is inserted between the C-terminus of 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.

[0100] 5.3.2 Applications to Luminescent Probes By incorporating the protein of 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. A high-performance luminescent probe can be constructed by linking [1] a luminescent enzyme (N and C-terminal fragments) that has been split into two parts, [2] a ligand-binding protein that responds to a target ligand, and [3] a recognition protein that recognizes that a ligand has bound to the ligand-binding protein, near these two parts. In this luminescent probe, when the recognition protein recognizes that a 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 achieved.

[0101] 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, it is a fusion protein containing the N and C-terminal fragments obtained by splitting the protein of the present invention into two parts, with 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 protein of the present invention are respectively present within a fusion protein containing a ligand-binding protein and a fusion protein containing a recognition protein.

[0102] When using the protein of 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 correspond to the cleavage positions 125 / 126, 129 / 130, 133 / 134, 137 / 138, 141 / 142, and 146 / 147 of ALuc16.

[0103] The specific method for using the protein of the present invention as a single-molecule luminescent probe follows known methods. Specifically, the protein of 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 with 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.

[0104] Since the bioluminescent probe using the protein of the present invention is introduced into cells as chimeric DNA and then expressed as a fusion protein within the cells, the properties and activity level of the ligand can be evaluated by measuring the change in the amount of light emitted from the transformed cells after ligand stimulation.

[0105] When the protein of the present invention is incorporated into a bioluminescent probe, the "ligand-binding protein" that can be loaded 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 be, for example, 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.

[0106] 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.

[0107] 5.3.3 Bioluminescence Resonance Energy Transfer (BRET) Molecular Probes The protein of 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 protein of 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 protein of 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 protein of 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 protein of 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).

[0108] The absorption spectrum of the fluorescent substance typically overlaps with the emission spectrum of the protein of 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).

[0109] Another example of a BRET molecular probe is a fusion protein in which the protein of the present invention and a fluorescent substance are linked 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 highly sensitive detection of the presence of a specific protease.

[0110] 5.3.4 Protein Complementation Assay (PCA) The protein of 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 protein of the present invention is fragmented into two and each is fused to a molecule whose proximity is to be verified. When the target molecules interact, the two fragmented proteins interact to form a complete luminescent enzyme, and luminescence is detected.

[0111] 5.3.5 Intracellular Imaging The gene encoding the protein of 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.

[0112] By linking the protein of the present invention with a suitable signal peptide, as exemplified in Table 5, 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 protein of the present invention 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 immunohistochemical staining, isolation, and purification in non-cellular systems. In such cases, well-known immunohistochemical staining methods and immunocytochemistry techniques can be applied.

[0113] [Table 5]

[0114] 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 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: https: / / www.ncbi.nlm.nih.gov / ), etc. [Examples]

[0115] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples.

[0116] <Experiment 1> A sequence containing the DNA sequences encoding ENLYFQSK (SEQ ID NO: 73) and ENLYFQSD (SEQ ID NO: 74) was added to the 3' end of the DNA sequence encoding picALuc30 (SEQ ID NO: 51) and inserted into a pET32 vector. E. coli Shuffle T7 express lysY (New England Biolab) was transformed with this plasmid and expressed intracellularly. Protein purification was performed using TALON metal affinity resin (TAKARA) and His TALON buffer set (TAKARA). picALuc (WT) without the added sequences, picALuc-ENLYFQSK, and picALuc-ENLYFQSD were each diluted to 320 nM, and the substrate CTZh was diluted to 0.0125-125 nM in PBS (Phosphate Buffered Salts, TAKARA) and mixed. The luminescence value immediately after substrate addition was measured using a GloMax (Promega) luminometer. As a result, an increase in luminescence value was observed with the added sequences (Figure 1).

[0117] <Experiment 2> The additional sequences listed in Table 6 were investigated. E. coli cells from 5 mL of E. coli SHuffle T7 express lysY culture medium expressing picALuc with each additional sequence were pelleted, suspended in 200 μL of B-PER Reagent (Thermo), and lysed by rotation at room temperature for 15 minutes. The cells were then centrifuged at 20000 g for 5 minutes, and the supernatant was collected as lysate. Each lysate was subjected to electrophoresis to confirm the expression level of each protein. Each lysate was then diluted to ensure the same protein levels, and the luminescence value immediately after reaction with 125 nM CTZh was measured. The results revealed that the luminescence value differed depending on the additional sequence (Figure 2).

[0118] [Table 6]

[0119] Next, comparing the emission values ​​of the four rightmost additional sequences in Figure 2 (sequence numbers 101-104) and WT using the same method, we found that WT exhibited emission values ​​between GSGD and GSGGSGGD (Figure 3), demonstrating that most of the additional sequences shown in Figure 2 can improve emission values.

[0120] <Experiment 3> To confirm that attaching an amino acid with a charge to the C-terminus is necessary for increased luminescence, the luminescence values ​​of each attached sequence, ENLYFQSK (+charge), ENLYFQSR (+charge), ENLYFQSD (-charge), ENLYFQSE (-charge), and ENLYFQSA (no charge), as shown in Table 7, were compared. When each enzyme, purified using the same method as in Experiment 1, was mixed with 1.25 μM of CTZh, it was confirmed that the sequences with a charge had higher luminescence values ​​than those without a charge (Figure 4).

[0121] [Table 7]

[0122] <Experiment 4> In Figure 2, the sequences that showed relatively high luminescence values ​​(excluding K and D), ANLYFQS·EALYFQS·ENAYFQS·ENLAFQS·ENLYAQS·ENLYFAS·ENLYFQA, were examined using lysates prepared in the same manner as in Experiment 2, with E appended to the end of C (Table 8).

[0123] [Table 8]

[0124] As a result, when combined with the results in Figure 2, it was shown that amino acids with a C-terminal charge, regardless of whether they were K, D, or E, showed high luminescence values ​​when ENLYFQA was used (Figure 5). Therefore, ENLYFQAK (Sequence No. 81) ·ENLYFQAR (Sequence ID 115) ENLYFQAD (Sequence No. 88) ·ENLYFQAE (Sequence ID 114) • The luminescence values ​​of the waste products (WT) were compared. Each enzyme, purified using the same method as in Experiment 1, was mixed with CTZh at concentrations from 50 to 250 μM. The results showed that ENLYFQAK exhibited the highest luminescence value, approximately 50 times higher when using CTZh 25 μM (Figure 6), approximately 28 times higher when using CTZh 62.5 μM, and approximately 25 times higher when using CTZh 125 μM (Figure 7). [Industrial applicability]

[0125] According to the present invention, it is possible to provide a luminescent enzyme with improved luminescence despite having a small molecular weight. Furthermore, according to the present invention, it is possible to improve the luminescence of luminescent enzymes such as picALuc while minimizing the increase in size. Therefore, it is useful in industries such as analytical chemistry, pharmaceuticals, food processing, and hygiene management.

Claims

1. A protein having luminescent enzyme activity, characterized in that it contains within its molecule an amino acid sequence in which the oligopeptide described in (I) below is linked to the C-terminus of the polypeptide described in (II) below; (I) Any oligopeptide selected from Sequence IDs 73-99, 101-103, and 105-115, (II) A polypeptide comprising an amino acid sequence derived from copepods, having luminescent enzyme activity.

2. The protein according to claim 1, wherein the C-terminal amino acid of the oligopeptide is a polar amino acid having an electric charge.

3. The protein according to claim 1, wherein the polypeptide is a polypeptide comprising the amino acid sequence described in (A) or (B) below; (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 missing and at least one amino acid residue at positions 146 to 156 is missing or substituted, as shown in SEQ ID NO:

1.

4. The protein according to claim 3, wherein the polypeptide comprises the amino acid sequence described in (A) or (B), and further comprises 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 missing.

5. The protein according to claim 1, wherein the polypeptide is a polypeptide comprising the sequence described in any of (a) to (c) below; (a) The amino acid sequence described in any of Sequence IDs 51 to 56, (b) An amino acid sequence having 85% or more identity with the amino acid sequence described in any of Sequence IDs 51 to 56, or (c) An amino acid sequence obtained by deleting, substituting, inserting, or adding one or more amino acid residues to the amino acid sequence described in any of Sequence IDs 51 to 56.

6. A nucleic acid encoding the amino acid sequence of a protein according to any one of claims 1 to 5.

7. An expression vector into which a gene sequence for expressing the protein described in any one of claims 1 to 5 is inserted.

8. Transformed cells into which a gene sequence for expressing the protein described in any one of claims 1 to 5 has been introduced.

9. A fusion protein comprising a protein according to any one of claims 1 to 5 and a target protein or a protein that binds to a target protein.

10. A nucleic acid encoding the amino acid sequence of the fusion protein described in claim 9.

11. An expression vector into which a gene sequence for expressing the fusion protein described in claim 9 has been inserted.

12. Transformed cells into which a gene sequence for expressing the fusion protein described in claim 9 has been introduced.

13. A bioluminescent resonance energy transfer (BRET) probe comprising a protein according to any one of claims 1 to 5 and a fluorescent substance.

14. A method for producing a luminescent enzyme, characterized by ligating the amino acid sequence of an oligopeptide described in (I) below to the C-terminus of the amino acid sequence of a luminescent enzyme containing an amino acid sequence derived from copepods; (I) Any oligopeptide selected from Sequence IDs 73-99, 101-103, and 105-115.

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