Peptides that catalyze luminescent reactions

Abstract

The present invention provides: a short peptide catalyzing the luminescence reaction of luciferin; a production method of the peptide; a bioluminescence analysis kit containing the peptide; use of the peptide as a bioluminescence probe; a polynucleotide encoding the peptide; an expression vector containing the polynucleotide; a cell having the expression vector transferred thereinto, etc.

Description

Peptides that catalyze luminescent reactions

[0001] The present invention relates to peptides that catalyze bioluminescence reactions. Specifically, the present invention relates to short peptides that catalyze the luminescence reaction of luciferin, methods for producing the peptides, kits for bioluminescence analysis containing the peptides, and uses of the peptides as bioluminescent probes. The present invention also relates to polynucleotides encoding the peptides, expression vectors containing the polynucleotides, cells into which the expression vectors have been introduced, and the like.

[0002] Bioluminescence reactions typically require the enzyme luciferase, which catalyzes the oxidation of the substrate luciferin. Examples of luciferase include the naturally occurring North American firefly luciferase (550 amino acid residues, molecular weight 61 kDa) and deep-sea shrimp luciferase (169 amino acid residues, molecular weight 19 kDa) (see Patent Documents 1 and 2, etc.). Luciferin-luciferase combination probes are becoming indispensable molecular probes in the field of life science.

[0003] JP 2009-65925 A JP 2015-202096 A

[0004] When analyzing biological phenomena using luciferin-luciferase probes, the large molecular weight of luciferase cannot be ignored, and therefore it is necessary to find a substance with a smaller molecular weight that catalyzes the luciferin oxidation luminescence reaction.

[0005] To solve the above problems, the present inventors conducted extensive research and designed and synthesized short peptides incorporating important amino acids involved in the enzymatic reaction of human serum albumin. They then discovered that these peptides catalyze the luminescence produced by the oxidation of luciferin, leading to the completion of the present invention. The peptides of the present invention that catalyze the luminescence produced by the oxidation of luciferin can be said to be the world's smallest luciferase.

[0006] That is, the present invention provides the following: (1) A peptide that catalyzes a luminescent reaction by oxidizing luciferin, comprising the following amino acid sequence: (a) RLRFLLHRLAIIAE (SEQ ID NO: 9), or (b) an amino acid sequence in which one to several amino acids have been substituted, deleted, inserted, or added in the amino acid sequence shown in (a). (2) The peptide according to (1), comprising an amino acid sequence in which one or more amino acids have been substituted at positions 1, 4, 5, 6, 7, 8, 9, 10, 13, and 14 in the amino acid sequence shown in SEQ ID NO: 9. (3) The peptide according to (1) or (2), comprising an amino acid sequence in which one or more amino acids have been substituted at positions 4, 7, and 9 in the amino acid sequence shown in SEQ ID NO: 9. (4) The peptide according to (3), comprising the amino acid sequence shown in SEQ ID NO: 9, in which the amino acid at position 4 is not substituted or is substituted with isoleucine, and / or the amino acid at position 7 is substituted with glycine, asparagine, glutamine, serine, or threonine, and / or the amino acid at position 9 is substituted with tyrosine, phenylalanine, or isoleucine. (5) Any of the following amino acid sequences (i) to (x): (i) RLRFLLGRLAIIAE (SEQ ID NO: 18) (ii) RLRFLLNRLAIIAE (SEQ ID NO: 34) (iii) RLRFLLQRLAIIAE (SEQ ID NO: 35) (iv) RLRFLLSRLAIIAE (SEQ ID NO: 36) (v) RLRFLLTRLAIIAE (SEQ ID NO: 37) (vi) RLRILLGRIAIIAE (SEQ ID NO: 50) (vii) RLRILLGRYAIIAE (SEQ ID NO: 51) (viii) RLRILLGRFAIIAE (SEQ ID NO: 52) (ix) RLRFLLGRIAIIAF (SEQ ID NO: 60) (x) The peptide according to any one of (1) to (4), comprising an amino acid sequence in which one to several amino acids are substituted, deleted, inserted or added in any one of the amino acid sequences (i) to (ix).(6) The peptide according to (5), comprising any of the following amino acid sequences (i) to (x): (i) RLRFLLGRLAIIAE (SEQ ID NO: 18) (ii) RLRFLLNRLAIIAE (SEQ ID NO: 34) (iii) RLRFLLQRLAIIAE (SEQ ID NO: 35) (vii) RLRILLGRYAIIAE (SEQ ID NO: 51) (viii) RLRILLGRFAIIAE (SEQ ID NO: 52) (x) an amino acid sequence in which one to several amino acids have been substituted, deleted, inserted or added in any of the amino acid sequences of (i), (ii), (iii), (vii) or (viii). (7) The peptide according to any of (1) to (6), having 50 or less amino acid residues. (8) The peptide according to any of (1) to (7), which forms a secondary structure. (9) A peptide comprising a plurality of peptides according to any one of (1) to (8) linked together, which catalyzes a luminescent reaction due to the oxidation of luciferin. (10) A peptide comprising the following amino acid sequence: (c) KFWARLRFLLHRLAIIAE (SEQ ID NO: 1), or (d) an amino acid sequence in which one to several amino acids have been substituted, deleted, inserted, or added in the amino acid sequence shown in SEQ ID NO: 1. (11) The peptide according to (10), which comprises the amino acid sequence in which one to four amino acids have been deleted from the N-terminus of the amino acid sequence shown in SEQ ID NO: 1. (12) The peptide according to (10) or (11), which has 50 or less amino acid residues. (13) The peptide according to any one of (10) to (12), which forms a secondary structure. (14) A peptide comprising a plurality of peptides according to any one of (10) to (13) linked together, which catalyzes a luminescent reaction due to the oxidation of luciferin. (15) A polynucleotide encoding the peptide according to any one of (1) to (14). (16) An expression vector comprising the polynucleotide according to (15). (17) A cell into which the expression vector according to (16) has been introduced. (18) A kit for bioluminescence analysis comprising the peptide according to any one of (1) to (14), a polynucleotide encoding the peptide, an expression vector comprising the polynucleotide, or a cell into which the expression vector has been introduced.(19) A bioluminescence analysis method comprising using luciferin, and the peptide according to any one of (1) to (14), a polynucleotide encoding the peptide, an expression vector containing the polynucleotide, or a cell into which the expression vector has been introduced. (20) A method for producing the peptide according to any one of (1) to (14), comprising culturing the cell according to claim 17 and obtaining the peptide according to any one of (1) to (14) from the culture.

[0007] The peptides of the present invention that catalyze the luciferin oxidation luminescence reaction are short peptides, so they have little or no artifactual effect on biological phenomena. Therefore, biological phenomena can be analyzed more accurately than with conventional luciferin-luciferase combinations. Like conventional luciferases, the peptides of the present invention can be used to luminescently label target molecules and tissues using genetic engineering techniques, but because they are low-molecular-weight probes, they can also luminescently label target samples simply by spraying them.

[0008] Figure 1 shows the CD spectra of KE-18, PS-16, and PS-16SS at pH 7.4. Figure 2 shows the CD spectra of KE-18 measured at different pHs or buffers. Figure 3(i) shows the CD spectra of KE-18 and its C-terminal truncated peptides (10 mM PB buffer, pH 7.4). Figure 3(ii) shows the CD spectra of KE-18 and its N-terminal truncated peptides (10 mM PB buffer, pH 7.4). Figure 3(iii) shows the luminescence signals of Cypridina luciferin and KE-18 C-terminal truncated peptides and KE-18 N-terminal truncated peptides. Figure 3(iv) shows the luminescence spectra of Cypridina luciferin and Cypridina luciferase, KE-18, and RE-14. Figure 4 shows the luminescence signals of Cypridina luciferin and peptides in which each amino acid in RE-14 is substituted with glycine. The secondary structure of each peptide is also shown in the figure. Figure 5 shows the luminescence signals from Cypridina luciferin and a peptide in which the amino acid at position 7 of RE-14 has been substituted with another amino acid. The secondary structure of each peptide is also shown in the figure. Figure 6 shows the luminescence signals from Cypridina luciferin and a peptide in which the amino acids at positions 4 and 9 of RE14-G7 have been substituted with other amino acids. The secondary structure of each peptide is also shown in the figure.

[0009] In a first aspect, the present invention provides a peptide that catalyzes a luminescent reaction by oxidizing luciferin, comprising the following amino acid sequence: (a) RLRFLLHRLAIIAE (SEQ ID NO: 9), or (b) an amino acid sequence in which one to several amino acids have been substituted, deleted, inserted, or added in the amino acid sequence shown in SEQ ID NO: 9.

[0010] As used herein, a peptide refers to a molecule in which multiple amino acid residues are linked via peptide bonds. As used herein, a short peptide refers to a peptide of about 70 amino acid residues or less, preferably 50 amino acid residues or less, for example, about 30 residues or less, about 25 residues or less, or about 20 residues or less. The amino acids constituting the peptides of the present invention are usually L-amino acids, but may also be D-amino acids. The amino acids constituting the peptides of the present invention may be amino acids that constitute normal proteins, or may be amino acids that do not constitute normal proteins. The amino acids constituting the peptides of the present invention may also be modified.

[0011] The peptides of the present invention can be produced by known methods. For example, the peptides of the present invention can be produced using known solid-phase synthesis methods. Alternatively, the polynucleotide encoding the peptide of the present invention can be incorporated into a vector, which is then introduced into host cells, and the host cells can be cultured to obtain the peptide of the present invention. Such genetic engineering techniques are useful for producing peptides of the present invention linked in tandem.

[0012] The luciferin oxidation luminescence reaction is known, and is a reaction in which the substrate luciferin is oxidized to an excited state and generates photons when returning to the ground state. The enzyme luciferase catalyzes this oxidation. Therefore, the peptide of the present invention can be called a luciferase. The catalytic activity of the peptide for the luciferin oxidation luminescence reaction can be measured by known means and methods. For example, the peptide may be reacted with luciferin, and the light generated may be measured using a luminometer or plate reader.

[0013] Luciferins with various structures derived from various organisms are known. Examples include firefly luciferin, bacterial luciferin, dinoflagellate luciferin, sea firefly luciferin, and coelenterazine. The peptide of the present invention can use luciferins having an imidazopyrazinone ring (imidazo[1,2-a]pyrazin-3(7H)-one) as a substrate. Also imidazo[1,2-b]pyridazin-3(5H)-one, imidazo[2,1-f][1,2,4]triazin-7(1H)-one, imidazo[1,2-a]pyridin-3-ol, imidazo[1,2-a]quinoxalin-1(5H)-one, benzo[f]imidazo[1,2-a]quinoxalin-3(11H)-one, imidazo[1',2':1,6]pyrazino[2,3-c]quinolin-3(11H)-one, 5,11-dihydro-3H-chromeno[4,3-e]imidazo[1,2-a]pyrazin-3-one (Glwadys Gagnot et al. al., Chem. Eur. J. 2021, 27, 2112-2123.) can also be used as a substrate. Examples of luciferins having an imidazopyrazinone ring as the mother skeleton include, but are not limited to, coelenterazine, Cypridina luciferin, and analogs thereof. The luciferin that serves as the substrate for the peptide of the present invention may be naturally occurring, a derivative thereof, or a synthetic product.

[0014] The peptide of the first aspect described above comprises an amino acid sequence consisting of the 14 amino acid residues set forth in SEQ ID NO: 9, or a variant thereof. In this specification, a peptide comprising a certain amino acid sequence refers to not only a peptide comprising that amino acid sequence, but also a peptide consisting of that amino acid sequence.

[0015] The mutant amino acid sequence of SEQ ID NO: 9 is an amino acid sequence in which one to several amino acids have been substituted, deleted, inserted or added in the amino acid sequence of SEQ ID NO: 9. In this specification, several means 2, 3, 4, 5, 6, 7, 8 or 9 amino acids.

[0016] Preferred examples of mutant amino acid sequences of SEQ ID NO: 9 include amino acid sequences in which one or more amino acids at positions 1, 4, 5, 6, 7, 8, 9, 10, 13, and 14 are substituted.

[0017] An amino acid in the peptide of the present invention may be substituted with any amino acid, preferably with an amino acid having similar properties and / or structure. For example, the amino acids in the following brackets may be substituted for each other: (G, A), (K, R, H), (D, E), (N, Q), (S, T, Y), (C, M), (F, W, Y, H), (V, L, I).

[0018] More preferred examples of the mutant amino acid sequence of SEQ ID NO: 9 include amino acid sequences in which one or more amino acids at positions 4, 7 and 9 are substituted.

[0019] Luciferin luminescence activity may be increased by substituting the amino acid (H) at position 7 of the amino acid sequence of SEQ ID NO: 9 with another amino acid. Luciferin luminescence activity may be increased by substituting the amino acid at position 7 of the amino acid sequence of SEQ ID NO: 9 with glycine, asparagine, glutamine, serine, or threonine. Preferably, luciferin luminescence activity may be increased by substituting the amino acid at position 7 of the amino acid sequence of SEQ ID NO: 9 with glycine, asparagine, or glutamine. More preferably, luciferin luminescence activity may be increased by substituting the amino acid at position 7 of the amino acid sequence of SEQ ID NO: 9 with glutamine.

[0020] Luciferin luminescence activity may be enhanced by substituting the amino acid (F) at position 4 and / or the amino acid (L) at position 9 of the amino acid sequence of SEQ ID NO: 9 with other amino acids. Amino acids at these positions may contribute to the formation of a β-sheet structure of the peptide. Luciferin luminescence activity may be increased when the peptide forms a secondary structure such as a β-sheet structure or an α-helical structure (this point will be explained later). Threonine, isoleucine, phenylalanine, and tryptophan may contribute to the formation of a β-sheet structure of proteins and peptides. Luciferin luminescence activity may be enhanced by substituting the amino acid at position 4 of the amino acid sequence of SEQ ID NO: 9 with or without isoleucine and / or substituting the amino acid at position 9 with tyrosine, phenylalanine, or isoleucine. Preferably, luciferin luminescence activity may be enhanced by substituting the amino acid at position 4 of the amino acid sequence of SEQ ID NO: 9 with isoleucine and / or substituting the amino acid at position 9 with tyrosine or phenylalanine.

[0021] Preferred specific examples of the mutant amino acid sequence of SEQ ID NO: 9 include the following amino acid sequences: (i) RLRFLLGRLAIIAE (SEQ ID NO: 18) (ii) RLRFLLNRLAIIAE (SEQ ID NO: 34) (iii) RLRFLLQRLAIIAE (SEQ ID NO: 35) (iv) RLRFLLSRLAIIAE (SEQ ID NO: 36) (v) RLRFLLTRLAIIAE (SEQ ID NO: 37) (vi) RLRILLGRIAIIAE (SEQ ID NO: 50) (vii) RLRILLGRYAIIAE (SEQ ID NO: 51) (viii) RLRILLGRFAIIAE (SEQ ID NO: 52) (ix) RLRFLLGRIAIIAE (SEQ ID NO: 60). In the above amino acid sequence, one to several amino acids may be substituted, deleted, inserted or added.

[0022] More preferred specific examples of the mutant amino acid sequence of SEQ ID NO: 9 include the following amino acid sequences: (i) RLRFLLGRLAIIAE (SEQ ID NO: 18) (ii) RLRFLLNRLAIIAE (SEQ ID NO: 34) (iii) RLRFLLQRLAIIAE (SEQ ID NO: 35) (vii) RLRILLGRYAIIAE (SEQ ID NO: 51) (viii) RLRILLGRFAIIAE (SEQ ID NO: 52) In the above amino acid sequences, one to several amino acids may be substituted, deleted, inserted or added.

[0023] The length of the peptide of the first aspect is not particularly limited, but is preferably about 70 residues or less, more preferably about 50 residues or less, and may be, for example, about 30 residues or less, about 25 residues or less, or about 20 residues or less.

[0024] The luminescence signal tends to increase when a peptide of the first embodiment that forms a secondary structure is used. In particular, the luminescence signal tends to increase when a peptide that forms a β-sheet structure under neutral to basic conditions is used. Therefore, it can be said that a peptide of the first embodiment that forms a β-sheet structure under neutral to basic conditions is preferred. The neutral to basic conditions may be a pH of about 7 to about 9, such as pH 7, pH 7.4, pH 8, pH 8.5, or pH 9.

[0025] In a second aspect, the present invention provides a peptide comprising a plurality of the peptides of the first aspect linked in tandem, which catalyzes the luminescent reaction of luciferin oxidation. Here, "plurality" means two or more, and may be, for example, about 2 to about 10, about 10 to about 20, about 20 to about 40, or about 50 to about 100. The peptides may be linked directly via a chemical bond such as a peptide bond, or via a known spacer.

[0026] In a third aspect, the present invention provides a peptide that catalyzes a luminescent reaction by oxidizing luciferin, comprising the following amino acid sequence: (e) KFWARLRFLLHRLAIIAE (SEQ ID NO: 1), or (f) an amino acid sequence in which one to several amino acids have been substituted, deleted, inserted or added in the amino acid sequence shown in SEQ ID NO: 1.

[0027] A specific example of the peptide of the third aspect is a peptide containing an amino acid sequence in which 1 to 4 amino acids are deleted from the N-terminus of the amino acid sequence shown in SEQ ID NO: 1.

[0028] The length of the peptide of the third aspect is not particularly limited, but is preferably about 70 residues or less, more preferably about 50 residues or less, and may be, for example, about 30 residues or less, about 25 residues or less, or about 20 residues or less.

[0029] Among the peptides of the third aspect, those that form a secondary structure tend to increase the luminescence signal. For example, the peptide shown in SEQ ID NO: 1 forms an α-helix structure under neutral to basic conditions and a β-sheet structure under acidic conditions. Furthermore, for example, an N-terminal deletion peptide (up to four amino acids) of the peptide shown in SEQ ID NO: 1 forms a β-sheet structure under acidic to basic conditions. The luminescence signal tends to increase when these peptides are used. The acidic conditions may be a pH of about 5 to about 6.5, such as pH 5, pH 5.5, pH 6, or pH 6.5. Neutral to basic conditions are as described above.

[0030] In a fourth aspect, the present invention provides a peptide comprising a plurality of peptides according to the third aspect linked in tandem, the peptide catalyzing the luminescent reaction of luciferin oxidation. Here, "plurality" means two or more, and may be, for example, about 2 to about 10, about 10 to about 20, about 20 to about 40, or about 50 to about 100. The peptides may be linked directly via a chemical bond such as a peptide bond, or via a known spacer.

[0031] In a further aspect, the present invention provides a polynucleotide encoding the peptide of any one of the first to fourth aspects. The polynucleotide is usually a DNA encoding the peptide, but may also be a DNA complementary to the DNA, an RNA complementary to the DNA, or an RNA complementary to the RNA.

[0032] In a further aspect, the present invention provides an expression vector containing the above-mentioned polynucleotide. Such an expression vector can be introduced into a host cell by known methods. Thus, in a further aspect, the present invention provides a cell into which the above-mentioned expression vector has been introduced.

[0033] The peptides, polynucleotides, vectors, and cells of the present invention described above are useful as described below. Therefore, a further aspect of the present invention relates to uses of the peptides of the present invention. The uses of the peptides, polynucleotides, vectors, and cells of the present invention are not limited to the uses described below.

[0034] The peptide of the present invention can be used as a reporter protein, like conventional luciferase, to measure the transcription activity of promoters and the like.

[0035] The peptides of the present invention can be used as luminescent detection markers. By fusing the peptides of the present invention to a target protein by chemical or genetic engineering, they can be used in in vitro diagnostic techniques such as immunoassays, or as imaging probes for visualizing the intracellular dynamics of the target protein.

[0036] Polynucleotides encoding the peptides of the present invention can be used as reporter genes. For example, by introducing an expression vector containing a polynucleotide encoding the peptides of the present invention into cells, or by introducing a polynucleotide encoding the peptides of the present invention into cells by techniques such as microinjection, any organelle, protein, or other molecule within the cell can be labeled with the peptides of the present invention. This allows the dynamics and amounts of target molecules to be visualized and analyzed.

[0037] Furthermore, because the peptides of the present invention are small molecules, they can be used to label target tissues, cells, or molecules simply by spraying them on target tissues or cells, just like conventional small molecule probes. For example, by modifying (fusing) the peptides of the present invention with a tag that has high affinity for the target molecule, the dynamics of the target molecule can be visualized simply by spraying this tag-fused peptide of the present invention. Because this method does not use genetic engineering technology, it is expected to become widespread as an extremely non-invasive imaging technique.

[0038] Therefore, in a further aspect, the present invention provides a kit for bioluminescence analysis, which comprises the peptide of the present invention, a polynucleotide encoding the peptide, an expression vector containing the polynucleotide, or a cell into which the expression vector has been introduced.

[0039] Bioluminescence analysis can be carried out using the peptide of the present invention, a polynucleotide encoding the peptide, an expression vector containing the polynucleotide, a cell into which the expression vector has been introduced, or a kit of the present invention.

[0040] Two or more peptides, polynucleotides, vectors, cells or kits of the present invention may be used in a bioluminescence assay.

[0041] The peptides, polynucleotides, vectors, cells, or kits of the present invention may be used in combination with other analytical techniques. For example, the peptides of the present invention may be modified with organic fluorescent dyes or fluorescent proteins and introduced into cells, allowing the dynamics and expression of target molecules to be detected by both luminescence and fluorescence. Using such methods, a large amount of information can be obtained, enabling more sophisticated and multifaceted analysis of biological phenomena.

[0042] Therefore, the present invention provides a bioluminescence analysis method comprising using luciferin and a peptide of the present invention, a polynucleotide encoding the peptide, an expression vector containing the polynucleotide, or a cell into which the expression vector has been introduced.

[0043] In a further aspect, the present invention provides a method for producing the peptide of any one of the first to fourth aspects, comprising culturing cells into which the expression vector has been introduced and obtaining the peptide of any one of the first to fourth aspects from the culture. The host cells used in this production method may be any cell, for example, bacteria such as Escherichia coli or Bacillus subtilis, filamentous fungi such as Aspergillus oryzae or Neurospora crassa, actinomycetes, plant cells, or animal cells. The host cells can be selected and the culture conditions can be set taking into consideration the type of peptide of interest, the amount of peptide required, etc.

[0044] Unless otherwise specified, terms used herein have the meanings commonly understood in the fields of biology, biochemistry, chemistry, medicine, pharmacology, etc. In this specification, when a number is preceded by "approximately," it means a range of ±20%, preferably ±10%. In this specification, the amino acid sequence of a peptide is represented with the amino-terminal amino acid at the left end and the carboxyl-terminal amino acid at the right end. In this specification, amino acid sequences are represented using the known three-letter or one-letter notation.

[0045] The present invention will be described in more detail and specifically below with reference to examples, but the description of the examples is not intended to limit the scope of the present invention.

[0046] 1. Design and Properties of the Peptide of the Present Invention Peptides incorporating key amino acids that are significantly involved in the enzymatic reaction of human serum albumin (HSA) (Florian Hollfelder et al., J. Am. Chem. Soc. 2000, 122, 1022-1029) were obtained by solid-phase synthesis. The obtained peptides were examined for their ability to catalyze luminescence reactions, and for the correlation between luminescence catalytic ability and amino acid sequence and structure.

[0047] The enzymatic reaction of HSA proceeds at drug binding site 1 or 2 as the reaction field. In the present invention, peptides incorporating important amino acids constituting each site were designed and synthesized. The following three peptides were synthesized: KFWARLRFLLHRLAIIAE (linear peptide KE-18, SEQ ID NO: 1) PLINCFRYLVCAELRS (linear peptide PS16, SEQ ID NO: 2) PLINCFRYLVCAELRS (cyclic peptide PS16SS having a disulfide bond between Cys4 and Cys11, SEQ ID NO: 3)

[0048] For each peptide, the luminescence activity of the substrate coelenterazine and its analogues was confirmed using a plate reader (Promega, GloMax).As a result, it was found that peptide KE-18 (18 amino acids, approximately 2 kDa), which incorporates the amino acids that constitute HSA drug binding site 1, exhibits a luminescence reaction with coelenterazine.The circular dichroism spectrum (CD spectrum) was measured using a circular dichroism spectrometer (JASCO Corporation, J-1100) (Figure 1), and it was found that KE-18 formed an α-helix structure, and for the peptides that did not exhibit luminescence activity, no secondary structure such as α-helix was confirmed.These results are different from the conventional bioluminescence research that requires the three-dimensional structure (tertiary-quaternary structure) of luciferase in the reaction field, and are the first to discover that even secondary structure peptides can catalyze the oxidation luminescence reaction of luciferin.

[0049] 2. Conditions for the Reaction of the Peptide of the Present Invention with Luciferin (Hydrophobicity) It is believed that the hydrophobic nature of the luminescence field of imidazopyrazine and other imidazopyrazine compounds enhances the efficiency of singlet excitation, resulting in high luminescence intensity. It has been reported that cyclodextrin, which has a hydrophobic cavity inside the molecule, oxidizes Cypridina luciferin, another imidazopyrazine compound, to emit light. (Yoshiaki Toya, Nippon Nogeikagaku Kaishi, 1992, 66, 742-747)

[0050] Therefore, KE-18, PS-16, PS16SS, cyclodextrin (α, β, γ) and coelenterazine derivative: [In the formula, R １ -R ４ are as shown in Table 1.] and the luminescence intensity was measured. The coelenterazine derivatives shown in Table 1 are those disclosed in Ryo Nishihara et al., Chem. Commun. 2015, 51, 391-394, Ryo Nishihara et al., Sci. Rep. 2017, 7, 8 pages, Masahiro Abe et al., ChemBioChem, 2019, 20, 1870, Ryo Nishihara et al., Theranostics, 2019, 9, 2646-2661, Ryo Nishihara et al., Bioconjugate Chem. Synthesis methods are described in JP2020,31,2679-2684, JP2018-158896A, JP2020-046137, and PCT / JP2021 / 010852. 50 μM of the peptide or cyclodextrin was reacted with 20 μM of a coelenterazine derivative or a commercially available product (Prolume Purple, Prolume Purple II, Prolume Purple III, Prolume Purple IV, Prolume Purple V, e-Coelenterazine, Coelenterazine-f, e-Coelenterazine-f, manufactured by Nano Light Technology; Coelenterazine, Coelenterazine-h, manufactured by Fujifilm Wako Pure Chemical Industries; and Cypridina Luciferin, manufactured by ATTO) in 10 mM PBS, pH 7.4, for 60 seconds. KE-18, developed by the present inventors, showed significantly higher luminescence activity (S / N ratio) than cyclodextrin (Table 2: S / N ratio for the peptide of the present invention, Table 3: S / N ratio for cyclodextrin). These results indicate that not only a simple hydrophobic environment but also the appropriate amino acid arrangement and higher-order structure of the peptide are necessary for efficient luminescence reactions of imidazopyrazinone compounds.

[0051] 3. Conditions for Reaction of the Peptide of the Present Invention with Luciferin (Peptide Structure) Based on KE-18, whose luminescence activity was confirmed, the conditions for forming a luminescence reaction field (peptide length, amino acid sequence, and structure) were investigated. Specifically, peptides were designed and synthesized in which two amino acids were deleted from the N-terminus or C-terminus of KE-18. CD spectrum analysis revealed that the secondary structure of KE-18 was pH-dependent, with a β-sheet structure on the acidic side (pH 6) and an α-helix structure on the neutral to basic side (pH 7-8) (Figure 2). Therefore, the pH dependence of the CD spectrum and luminescence activity of the deleted peptides was comprehensively investigated.

[0052] The results are shown in Figure 3. The C-terminal deleted peptides either did not form a secondary structure or a random coil structure was confirmed (Figure 3(i)), but no luminescence signal was observed with either luciferin (Figure 3(iii), only the results with Cypridina luciferin are shown). On the other hand, for the N-terminal deleted peptides, secondary structure was confirmed up to four amino acids deleted (WE-16 and RE-14), and both were found to form a β-sheet structure independently of pH in acidic to basic environments (Figure 3(ii)). Furthermore, WE-16 and RE-14, which formed a secondary structure, showed luminescence signals with either luciferin, but RE-12 and LE-10, which did not show any observable structure, did not show any luminescence signal, just like the C-terminal deleted peptides (Figure 3(iii), only the results with Cypridina luciferin are shown). Interestingly, the luminescence signal of the deletion peptide RE-14 (14 amino acids, approximately 1.7 kDa) was generally higher than that of KE-18 (Figure 3(iii)). Under the same conditions (pH 7.4), KE-18 forms an α-helix structure, while RE-14 forms a β-sheet structure. This indicates that the luminescence reaction of imidazopyrazinone compounds proceeds efficiently with peptides that form β-sheet structures. Next, the luminescence spectra were evaluated using an ATTO LumiFL. The results showed that the luminescence spectra of Cypridina luciferin were highly dependent on the secondary structure of the peptide, and were all shifted to longer wavelengths than Cypridina luciferase (CLuc) (Figure 3(iv): λmax[CLuc]: 471 nm, λmax[KE-18]: 498 nm, λmax[RE-14]: 528 nm). Cypridina luciferin exhibits solvatochromism, whereby the emission wavelength shifts depending on the surrounding microenvironment (Osamu Shimomura, Bioluminescence: Chemical Principles and Methods, 2012, 66). The spectral changes in Figure 3(iv) suggest that the microenvironment (structure, polarity, hydrophobicity, etc.) of the luminescence reaction field differs depending on the luciferase and peptide species.

[0053] 4. Conditions for the Reaction of the Peptide of the Present Invention with Luciferin (Amino Acid Sequence of the Peptide) Next, we confirmed the amino acids required for the luminescent reaction field. Using the shortest peptide (RE-14) required as a luciferin reaction field as a standard, we synthesized a new peptide in which one residue was substituted with G (glycine), and confirmed the correlation between the structure and luminescent activity.

[0054] The results are shown in Figure 4. The peptides that emitted light formed a β-sheet structure, as in the results shown in Section 3. On the other hand, a peptide (RE-14G5) was also found that formed a β-sheet structure but failed to produce a detectable luminescence signal, suggesting that not only the structure but also the amino acid sequence is necessary for the luminescence reaction. Since the luminescence intensity of the RE14 peptide was relatively maintained even when R at position 1, L at position 6, H at position 7, A at position 10, and A at position 13 were substituted with G (RE14-G1, RE14-G6, RE14-G7, RE-14G10, RE14-G13), mutations at these sites may further improve luminescence intensity. In particular, the G substitution at H7 showed an approximately three-fold improvement in luminescence intensity compared to the reference peptide (RE-14), and the seventh mutation was found to be advantageous for developing peptide probes with higher luminescence intensity.

[0055] 1. Luminescence activity of mutants in which the amino acid at position 7 of RE-14 was substituted with various amino acids In the above experiment, a mutant (RE14-G7) in which position 7 of the reference peptide RE14 was substituted with glycine improved luminescence intensity. Therefore, various amino acids were introduced at position 7 of RE14 (Table 6), and their effect on luminescence intensity was examined. As in the above experiment, Cypridina luciferin was used as the luminescence substrate.

[0056] The results are shown in Figure 5. RE14-Q7, in which the amino acid at position 7 of the reference peptide RE14 was substituted with Q (glutamine), exhibited a luminescence intensity approximately 1.2 times that of RE14-G7. RE14-N7, in which the amino acid at position 7 of the reference peptide RE14 was substituted with N (asparagine), exhibited almost the same luminescence intensity as RE14-G7. RE14-S7 and ER14-T7, in which the amino acid at position 7 of the reference peptide RE14 was substituted with S (serine) and T (threonine), respectively, also exhibited luminescence intensities greater than that of the reference peptide RE14.

[0057] 2. Luminescence activity of mutants in which the amino acids at positions 4 and 9 of RE-14 were substituted with T, I, F, or W. The RE14-G7 peptide, which has high luminescence intensity, forms a β-sheet structure based on circular dichroism (CD) spectra. Therefore, we tested whether introducing amino acids that tend to form a sheet structure (T, I, F, W) (Nature, 1994, 367, 660-663) into positions 4 and 9 of RE14-G7 (Table 7) would improve luminescence intensity. As in the above experiments, Cypridina luciferin was used as the luminescent substrate.

[0058] The results are shown in Figure 6. RE14-G7-IY, in which position 4 is substituted with I (isoleucine) and position 9 with Y (tyrosine), showed a 1.6-fold improvement in luminescence intensity compared to RE14-G7, while RE14-G7-IF, in which position 4 is substituted with I (isoleucine) and position 9 with F (phenylalanine), showed a 1.3-fold improvement in luminescence intensity compared to RE14-G7. RE14-G7-II, in which position 4 is substituted with I and position 9 with I, and RE14-G7-FI, in which position 4 is not substituted but position 9 is substituted with I, also showed a luminescence intensity higher than that of the reference peptide RE14 (the luminescence intensity of RE14 is approximately one-third that of RE14-G7).

[0059] The peptides of the present invention that catalyze the luciferin oxidation luminescence reaction are short peptides, and therefore have little or no artifactual effects on biological phenomena. Therefore, biological phenomena can be analyzed more accurately than with conventional luciferin-luciferase combinations. Therefore, the present invention is useful in fields such as biology, biochemistry, chemistry, medicine, and pharmacology, and can be applied specifically to analytical reagents, diagnostic agents, etc.

[0060] Free Text of Sequence Listing Each of the amino acid sequences of SEQ ID NOs: 1 to 68 represents the amino acid sequence of a peptide that the present inventors designed and synthesized as a peptide that catalyzes the luciferin oxidation luminescence reaction.

Claims

1. A peptide that catalyzes the luminescent reaction of luciferin oxidation, comprising the following amino acid sequence: (a) RLRFLLHRLAIIAE (SEQ ID NO: 9), or (b) the amino acid sequence shown in (a) in which one to several amino acids have been substituted, deleted, inserted, or added.

2. The peptide according to claim 1, comprising an amino acid sequence in which one or more amino acids at positions 1, 4, 5, 6, 7, 8, 9, 10, 13 and 14 in the amino acid sequence shown in SEQ ID NO: 9 have been substituted.

3. The peptide according to claim 1 or 2, which comprises an amino acid sequence in which one or more amino acids at positions 4, 7 and 9 in the amino acid sequence shown in SEQ ID NO: 9 have been substituted.

4. The peptide according to claim 3, comprising the amino acid sequence set forth in SEQ ID NO: 9, in which the amino acid at position 4 is not substituted or is substituted with isoleucine, and / or the amino acid at position 7 is substituted with glycine, asparagine, glutamine, serine or threonine, and / or the amino acid at position 9 is substituted with tyrosine, phenylalanine or isoleucine.

5. Any of the following amino acid sequences (i) to (x): (i) RLRFLLGRLAIIAE (SEQ ID NO: 18) (ii) RLRFLLNRLAIIAE (SEQ ID NO: 34) (iii) RLRFLLQRLAIIAE (SEQ ID NO: 35) (iv) RLRFLLSRLAIIAF (SEQ ID NO: 36) (v) RLRFLLTRLAIIAE (SEQ ID NO: 37) (vi) RLRILLGRIAIIAE (SEQ ID NO: 50) (vii) RLRILLGRYAIIAE (SEQ ID NO: 51) (viii) RLRILLGRFAIIAE (SEQ ID NO: 52) (ix) RLRFLLGRIAIIAE (SEQ ID NO: 60) (x) The peptide according to any one of claims 1 to 4, comprising an amino acid sequence in which one to several amino acids are substituted, deleted, inserted or added in any of the amino acid sequences (i) to (ix).

6. The peptide according to claim 5, comprising any of the following amino acid sequences (i) to (x): (i) RLRFLLGRLAIIAE (SEQ ID NO: 18) (ii) RLRFLLNRLAIIAE (SEQ ID NO: 34) (iii) RLRFLLQRLAIIAE (SEQ ID NO: 35) (vii) RLRILLGRYAIIAE (SEQ ID NO: 51) (viii) RLRILLGRFAIIAF (SEQ ID NO: 52) (x) an amino acid sequence in which one to several amino acids have been substituted, deleted, inserted or added in any of the amino acid sequences of (i), (ii), (iii), (vii) or (viii).

7. A peptide according to any one of claims 1 to 6, having 50 or less amino acid residues.

8. A peptide according to any one of claims 1 to 7, which forms a secondary structure.

9. A peptide comprising a plurality of peptides linked together according to any one of claims 1 to 8, which catalyzes the luminescent reaction of luciferin oxidation.

10. A peptide that catalyzes the luminescent reaction of luciferin oxidation, comprising the following amino acid sequence: (c) KFWARLRFLLHRLAIIAE (SEQ ID NO: 1), or (d) an amino acid sequence in which one to several amino acids have been substituted, deleted, inserted, or added in the amino acid sequence shown in SEQ ID NO:

1.

11. The peptide according to claim 10, which comprises an amino acid sequence in which 1 to 4 amino acids are deleted from the N-terminus of the amino acid sequence shown in SEQ ID NO:

1.

12. The peptide according to claim 10 or 11, which has 50 or less amino acid residues.

13. The peptide according to any one of claims 10 to 12, which forms a secondary structure.

14. A peptide comprising a plurality of peptides linked together according to any one of claims 10 to 13, which catalyzes the luminescent reaction of luciferin oxidation.

15. A polynucleotide encoding the peptide according to any one of claims 1 to 14.

16. An expression vector comprising the polynucleotide of claim 15.

17. A cell into which the expression vector according to claim 16 has been introduced.

18. A kit for bioluminescence analysis comprising the peptide according to any one of claims 1 to 14, a polynucleotide encoding said peptide, an expression vector containing said polynucleotide, or a cell into which said expression vector has been introduced.

19. A method for bioluminescence analysis, comprising using luciferin, a peptide according to any one of claims 1 to 14, a polynucleotide encoding said peptide, an expression vector containing said polynucleotide, or a cell into which said expression vector has been introduced.

20. A method for producing a peptide according to any one of claims 1 to 14, comprising culturing the cell according to claim 17 and obtaining the peptide according to any one of claims 1 to 14 from the culture.

Images