Luciferase-peptide conjugate

Abstract

To provide a novel probe based on luminescence, emitting at a luminescence intensity dependent on the concentration of a target substance.SOLUTION: A luciferase-peptide conjugate comprises: a luciferase; a first peptide connected to the N-terminal side of the luciferase via a first linker; and a second peptide connected to the C-terminal side of the luciferase via a second linker, wherein the luciferase has an amino acid sequence having at least 85% sequence identity to the amino acid sequence shown in SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3, and can emit light, and the first peptide and the second peptide are capable of forming a complex with a target substance.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a luciferase-peptide conjugate in which luciferase is linked to a peptide. [Background technology]

[0002] Labeled probes have been used to observe intracellular phenomena, and fluorescence is widely used as a label because of the wide range of wavelengths available.

[0003] Patent Document 1 discloses a technology for selectively detecting the interaction between dopamine type 1 receptor (DRD1) and dopamine using a fluorescent protein. More specifically, it discloses that a fusion protein in which the amino acid sequence of the third intracellular loop of DRD1 is replaced with the amino acid sequence of a fluorescent protein (cpmApple) selectively reacts with dopamine and changes in fluorescence intensity depending on the dopamine concentration. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Molecular Brain,2021,14:173 Summary of the Invention [Problem to be solved by the invention]

[0005] Fluorescence measurement is not suitable for quantitative analysis because the measured fluorescence intensity is a relative value. On the other hand, luminescence measurement is a method suitable for quantitative analysis because the measured luminescence intensity correlates with the number of molecules (the number of molecules that emit photons). However, no probes whose luminescence intensity changes depending on the concentration of the target substance have been known so far.

[0006] In view of the above circumstances, the present invention aims to provide a novel probe that utilizes luminescence and emits light with a luminescence intensity corresponding to the concentration of a target substance. Another object of the present invention is to provide a method for measuring the concentration of a target substance in a test sample using the probe. [Means for solving the problem]

[0007] The present inventors selected a specific luciferase as a luminescent label and discovered that a luciferase-peptide conjugate in which a first peptide and a second peptide are linked to the N-terminus and C-terminus of the luciferase, respectively, emits light with a luminescence intensity that corresponds to the concentration of a target substance. Here, the first peptide and the second peptide are peptides that can form a complex (a complex of the first peptide, the second peptide, and the target substance) together with the target substance. The present invention is based on this finding.

[0008] The present invention relates to a luciferase-peptide conjugate comprising luciferase, a first peptide linked to the N-terminus of the luciferase via a first linking part, and a second peptide linked to the C-terminus of the luciferase via a second linking part, wherein the luciferase has an amino acid sequence having 85% or more sequence identity with the amino acid sequence shown in SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3 and is capable of emitting light, and the first peptide and the second peptide are capable of forming a complex with a target substance.

[0009] The luciferase-peptide conjugate of the present invention is configured by selecting a specific luciferase and further linking a first peptide and a second peptide, which can form a complex (a complex of a first peptide, a second peptide, and the target substance) together with a target substance, to the N-terminus and C-terminus, respectively, of the luciferase, thereby emitting light with an intensity that corresponds to the concentration of the target substance.

[0010] The luciferase-peptide conjugate may be such that the first peptide has an amino acid sequence that has 85% or more sequence identity to the amino acid sequence from the N-terminus to the first, third or fifth transmembrane site of a G protein-coupled receptor and is capable of interacting with a ligand of the G protein-coupled receptor, and the second peptide has an amino acid sequence that has 85% or more sequence identity to the amino acid sequence from the second, fourth or sixth transmembrane site to the seventh transmembrane site of the G protein-coupled receptor, respectively, and is capable of interacting with a ligand of the G protein-coupled receptor. By configuring the first peptide and the second peptide in this way, it is possible to emit light with a luminescence intensity that corresponds to the concentration of a target substance, using a ligand of a G protein-coupled receptor as the target substance.

[0011] The luciferase-peptide conjugate may be such that the first peptide has an amino acid sequence that has 85% or more sequence identity with the amino acid sequence from the first transmembrane site to the fifth transmembrane site of the dopamine type 1 receptor (DRD1) and is capable of interacting with dopamine, and the second peptide has an amino acid sequence that has 85% or more sequence identity with the amino acid sequence from the sixth transmembrane site to the seventh transmembrane site of the dopamine type 1 receptor (DRD1) and is capable of interacting with dopamine. By configuring the first peptide and the second peptide in this way, it is possible to emit light with a luminescence intensity that corresponds to the concentration of dopamine as a target substance.

[0012] The luciferase-peptide conjugate may be such that the first peptide has an amino acid sequence that has 85% or more sequence identity with the amino acid sequence of FK506 binding protein (FKBP) and is capable of interacting with rapamycin, and the second peptide has an amino acid sequence that has 85% or more sequence identity with the amino acid sequence of the rapamycin binding domain fragment (FRB) of the target protein of rapamycin (TOR) and is capable of interacting with rapamycin. By configuring the first peptide and the second peptide in this way, it is possible to emit light with a luminescence intensity according to the concentration of a target substance, using rapamycin as the target substance.

[0013] The luciferase-peptide conjugate may be such that the first peptide and / or the second peptide further comprises an amino acid sequence of a fluorescent protein. With this configuration, the luminescence intensity of luciferase can be normalized using the measurement result of the fluorescence from the fluorescent protein. Furthermore, normalization can eliminate the influence of the variation in the number of molecules of the luciferase-peptide conjugate in the test sample (e.g., test cell) on the measurement result.

[0014] In the luciferase-peptide conjugate, the first linking part and / or the second linking part may be a peptide linker, or both the first linking part and the second linking part may be peptide linkers. When both the first linking part and the second linking part are peptide linkers, the luciferase-peptide conjugate can be expressed as a fusion protein (chimeric protein), which makes it easy to produce the luciferase-peptide conjugate or introduce it into a cell (test cell).

[0015] The peptide linker may be 6 amino acid residues or less. Since the luciferase-peptide conjugate has a tendency that the shorter the peptide linker, the greater the change in luminescence intensity depending on the concentration of the target substance. By configuring the luciferase-peptide conjugate in this way, the concentration of the target substance can be easily measured.

[0016] The present invention also relates to a nucleic acid comprising a base sequence encoding the luciferase-peptide conjugate and a regulatory sequence operably linked to the base sequence. The nucleic acid may be in the form of a vector (expression vector).

[0017] The nucleic acid may further include a base sequence that expresses a fluorescent protein. With this configuration, the luciferase-peptide conjugate and the fluorescent protein can be expressed from the nucleic acid. Furthermore, the fluorescent protein can be used to normalize the luminescence intensity of luciferase, thereby eliminating the influence of the variation in the number of molecules of the luciferase-peptide conjugate caused by the difference in gene expression level in the test sample (e.g., test cell) on the measurement result.

[0018] The present invention also relates to a method for measuring the concentration of a substance of interest in a test sample, the method comprising the steps of adding the luciferase-peptide conjugate to the test sample and measuring luminescence from the luciferase-peptide conjugate.

[0019] The present invention also relates to a method for measuring the concentration of a target substance in a test cell, the method comprising the steps of introducing the nucleic acid into the test cell and measuring luminescence from the luciferase-peptide conjugate.

[0020] In the above-mentioned method, when the luciferase-peptide conjugate contains an amino acid sequence of a fluorescent protein and / or when the nucleic acid contains a base sequence expressing a fluorescent protein, in addition to the step of measuring luminescence, the method may further comprise the steps of measuring fluorescence from the fluorescent protein and normalizing the measured luminescence with the measured fluorescence. By configuring in this way, it is possible to eliminate the influence of variation in the number of luciferase-peptide conjugate molecules in a test sample (e.g., test cells) on the measurement results.

[0021] The present invention includes, for example, the following inventions. [1] The nucleic acid sequence comprises a luciferase, a first peptide linked to the N-terminus of the luciferase via a first linking part, and a second peptide linked to the C-terminus of the luciferase via a second linking part, The luciferase has an amino acid sequence having 85% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3, and is capable of emitting light; A luciferase-peptide conjugate, wherein the first peptide and the second peptide are capable of forming a complex with a substance of interest. [2] the first peptide has an amino acid sequence that has 85% or more sequence identity to the amino acid sequence from the N-terminus to the first, third or fifth transmembrane site of a G protein-coupled receptor, and is capable of interacting with a ligand of the G protein-coupled receptor; The conjugate according to [1], wherein the second peptide has an amino acid sequence that has 85% or more sequence identity to the amino acid sequence from the second, fourth, or sixth transmembrane site to the seventh transmembrane site of the G protein-coupled receptor, respectively, and is capable of interacting with a ligand of the G protein-coupled receptor. [3] the first peptide has an amino acid sequence having 85% or more sequence identity with the amino acid sequence from the first transmembrane domain to the fifth transmembrane domain of the dopamine type 1 receptor (DRD1) and is capable of interacting with dopamine; The conjugate according to [1] or [2], wherein the second peptide has an amino acid sequence that has 85% or more sequence identity with the amino acid sequence from the sixth transmembrane site to the seventh transmembrane site of the dopamine type 1 receptor (DRD1) and is capable of interacting with dopamine. [4] The first peptide has an amino acid sequence having 85% or more sequence identity with the amino acid sequence of FK506 binding protein (FKBP) and is capable of interacting with rapamycin; The conjugate described in [1], wherein the second peptide has an amino acid sequence that has 85% or more sequence identity with the amino acid sequence of the rapamycin binding domain fragment (FRB) of the target of rapamycin (TOR) and is capable of interacting with rapamycin. [5] The conjugate according to any one of [1] to [4], wherein the first peptide and / or the second peptide further comprises an amino acid sequence of a fluorescent protein. [6] The conjugate according to any one of [1] to [5], wherein the first linking part and / or the second linking part is a peptide linker. [7] The conjugate according to [6], wherein the peptide linker is 6 amino acid residues or less. [8] The linker according to any one of [1] to [7], wherein the first linking part and the second linking part are both peptide linkers. [9] A nucleic acid comprising a base sequence encoding the conjugate described in [8] and a regulatory sequence operably linked to the base sequence.

[10] The nucleic acid according to [9], further comprising a base sequence for expressing a fluorescent protein.

[11] 1. A method for measuring a concentration of a target substance in a test sample, comprising: adding the conjugate according to any one of [1] to [8] to a test sample; and measuring luminescence from said conjugate.

[12] 1. A method for measuring a concentration of a target substance in a test sample, comprising: adding the conjugate according to any one of [1] to [8] to a test sample; measuring luminescence from the conjugate; Measuring the fluorescence from the fluorescent protein; normalizing the measured luminescence to the measured fluorescence; The method, wherein the conjugate is such that the first peptide and / or the second peptide further comprises the amino acid sequence of a fluorescent protein.

[13] 1. A method for measuring a concentration of a target substance in a test cell, comprising: Introducing the nucleic acid according to [9] or

[10] into a test cell; and measuring luminescence from said conjugate.

[14] 1. A method for measuring a concentration of a target substance in a test cell, comprising:

[10] introducing the nucleic acid according to the present invention into a test cell; measuring luminescence from the conjugate; Measuring the fluorescence from the fluorescent protein; and normalizing the measured luminescence to the measured fluorescence. Effect of the Invention

[0022] According to the present invention, it is possible to provide a novel probe that utilizes luminescence and emits light with a luminescence intensity corresponding to the concentration of a target substance. According to the present invention, it is also possible to provide a method for measuring the concentration of a target substance in a test sample using the probe. [Brief description of the drawings]

[0023] [Figure 1] 1 is a graph showing the evaluation results of the luciferase-peptide conjugate produced in Test Example 1. [Diagram 2] 1 is a graph showing the evaluation results of the luciferase-peptide conjugate produced in Test Example 1. [Diagram 3] 1 is a graph showing the evaluation results of the luciferase-peptide conjugate produced in Test Example 1. [Figure 4] 1 is a graph showing the evaluation results of the luciferase-peptide conjugate produced in Test Example 2. [Diagram 5] 1 shows a vector map of the expression vector constructed in Test Example 1. [Figure 6]1 shows a vector map of the expression vector constructed in Test Example 2. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0024] Hereinafter, an embodiment of the present invention will be described in detail. However, the present invention is not limited to the following embodiment.

[0025] [Luciferase-peptide conjugate] The luciferase-peptide conjugate (also simply referred to as "conjugate" in this specification) according to this embodiment comprises luciferase, a first peptide linked to the N-terminus of luciferase via a first linking part, and a second peptide linked to the C-terminus of luciferase via a second linking part. In this conjugate, luciferase has an amino acid sequence that has 85% or more sequence identity with the amino acid sequence shown in SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3 and is capable of emitting light, and the first peptide and the second peptide are capable of forming a complex (a complex of the first peptide, the second peptide, and the target substance) together with a target substance.

[0026] In the luciferase-peptide conjugate according to the present embodiment, the first peptide and / or the second peptide are linked to luciferase, so that the three-dimensional structure of luciferase is changed and luminescence (metabolism of the luminescent substrate) is suppressed. In the luciferase-peptide conjugate according to the present embodiment, when a target substance is present, it is presumed that the first peptide and / or the second peptide interact with the target substance (for example, association of the first peptide with the target substance, association of the second peptide with the target substance, formation of a (ternary) complex by the first peptide, the second peptide, and the target substance) to cause a further change in the three-dimensional structure of luciferase, thereby (re)activating luminescence (metabolism of the luminescent substrate). In this case, it is presumed that if the amount of the target substance present is large, the number of molecules of the luciferase-peptide conjugate that interact with the first peptide and / or the second peptide with the target substance also increases, and thus luminescence is emitted with a luminescence intensity according to the concentration of the target substance. The first peptide, the second peptide, and the target substance do not necessarily need to form a (ternary) complex. It is presumed that the association of the first peptide with the target substance and / or the association of the second peptide with the target substance also causes further changes in the three-dimensional structure of luciferase, activating luminescence (metabolism of the luminescent substrate).

[0027] (Substances) There is no particular limitation on the target substance, and it may be a low molecular weight compound or a high molecular weight compound. The target substance may be a peptide (protein).

[0028] (Luciferase) The amino acid sequence shown in SEQ ID NO: 1 is the amino acid sequence of a 19 kDa protein (KAZ) constituting Oplophorus luciferase derived from Oplophorus gracilirostris. KAZ oxidizes coelenterazine with oxygen molecules to produce blue light.

[0029] The amino acid sequence shown in SEQ ID NO: 2 is the amino acid sequence of a mutant of KAZ (eKAZ). eKAZ has enhanced luminescence activity, and it is known that the luminescence activity is also enhanced by using coelenterazine, a derivative of coelenterazine, as a substrate.

[0030] The amino acid sequence shown in SEQ ID NO: 3 is the amino acid sequence of a mutant of KAZ (nanoKAZ). It is known that nanoKAZ also has enhanced luminescence activity, and that its luminescence activity is also enhanced by using coelenterazine, a derivative of coelenterazine, as a substrate. It has also been shown that nanoKAZ is secreted into the culture medium even in the absence of a secretory signal peptide.

[0031] [Table 1]

[0032] The luciferase contained in the luciferase-peptide conjugate of this embodiment may be one having an amino acid sequence that has 85% or more sequence identity to the amino acid sequence shown in SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3, may be one having an amino acid sequence that has 90% or more sequence identity, may be one having an amino acid sequence that has 95% or more sequence identity, may be one having an amino acid sequence that has 97% or more sequence identity, may be one having an amino acid sequence that has 98% or more sequence identity, may be one having an amino acid sequence that has 99% or more sequence identity, or may be the amino acid sequence shown in SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3.

[0033] The luciferase contained in the luciferase-peptide conjugate according to this embodiment may have an amino acid sequence in which amino acid residues have been substituted, deleted or added relative to the amino acid sequence shown in SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3. There is no particular limitation on the number of amino acid residues to be substituted, deleted or added, and may be, for example, 1 to 25, 1 to 20, 1 to 17, 1 to 15, 1 to 12, 1 to 10, 1 to 7, 1 to 5, or 1 to 3.

[0034] The luciferase contained in the luciferase-peptide conjugate of this embodiment is capable of emitting light. For example, the luciferase may emit light by oxidation reaction using coelenterazine or a coelenterazine derivative (e.g., coelenterazine h) as a substrate.

[0035] (First peptide and second peptide) The first peptide and the second peptide can form a complex (a complex of the first peptide, the second peptide, and the target substance) together with the target substance. The first peptide and the second peptide may be selected depending on the target substance.

[0036] When the target substance can form a complex with two peptides, the first peptide and the second peptide may be the two peptides, respectively. The first peptide and the second peptide may be peptide fragments (i.e., parts of the two peptides) that include a site that interacts with the target substance. The two peptides may be the same type of peptide or different types of peptides.

[0037] Examples of combinations in which a target substance can form a complex with two peptides include a combination of FK506 binding protein (FKBP), a rapamycin binding domain fragment (FRB) of the target protein for rapamycin (TOR), and rapamycin, a combination of DmrC, DmrA, and their ligands (e.g., A / C heterodimerizer), and a combination of a G protein-coupled receptor (heterodimer) and its ligand. Note that these specific examples do not limit the amino acid sequences of the first peptide and the second peptide to the amino acid sequences of the peptides shown in these specific examples, and the amino acid sequences may be changed as long as they can interact with the target substance. For example, the first peptide and the second peptide may have an amino acid sequence having 85% or more sequence identity to the amino acid sequences of the peptides shown in these specific examples. The above-mentioned sequence identity may be 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100%. The first peptide and the second peptide may have an amino acid sequence in which amino acid residues have been substituted, deleted or added relative to the amino acid sequences of the peptides shown in these specific examples. The number of substituted, deleted or added amino acid residues is not particularly limited, and may be, for example, 1 to 15, 1 to 12, 1 to 10, 1 to 7, 1 to 5, or 1 to 3.

[0038] A detailed description will be given of a case where the combination of the first peptide, the second peptide, and the target substance is a combination of FK506 binding protein (FKBP), a rapamycin-binding domain fragment (FRB) of the target protein of rapamycin (TOR), and rapamycin.

[0039] As a specific example of the first peptide and the second peptide, for example, the first peptide may be a peptide having an amino acid sequence having 85% or more sequence identity with the amino acid sequence of FKBP, and the second peptide may be a peptide having an amino acid sequence having 85% or more sequence identity with the amino acid sequence of FRB, the first peptide may be a peptide having an amino acid sequence having 85% or more sequence identity with the amino acid sequence from the 1st amino acid residue to the 109th amino acid residue of the amino acid sequence shown in SEQ ID NO: 5, and the second peptide may be a peptide having an amino acid sequence having 85% or more sequence identity with the amino acid sequence from the 292nd amino acid residue to the 388th amino acid residue of the amino acid sequence shown in SEQ ID NO: 5. These first peptide and second peptide may be capable of interacting with rapamycin. In addition, the above-mentioned sequence identity may be 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100%. The first peptide and the second peptide may have an amino acid sequence in which an amino acid residue is substituted, deleted or added with respect to the amino acid sequence of FKBP, the amino acid sequence of FRB, the amino acid sequence from the 1st amino acid residue to the 109th amino acid residue of the amino acid sequence shown in SEQ ID NO: 5, or the amino acid sequence from the 292nd amino acid residue to the 388th amino acid residue of the amino acid sequence shown in SEQ ID NO: 5. The number of amino acid residues to be substituted, deleted or added is not particularly limited, and may be, for example, 1 to 15, 1 to 12, 1 to 10, 1 to 7, 1 to 5, or 1 to 3. Furthermore, the first peptide and the second peptide may be those in which the above-mentioned specific examples are reversed.

[0040] When the target substance can form a complex with a certain peptide, the first peptide may be a peptide fragment of the peptide that includes a site that interacts with the target substance, and the second peptide may be a peptide fragment of the peptide that includes a site that interacts with the target substance (preferably a site different from that of the first peptide).

[0041] Examples of combinations in which a target substance can form a complex with a certain peptide include, for example, a combination of a G protein-coupled receptor and its ligand, a combination of an insulin receptor and insulin, a combination of a Notch receptor and its ligand (e.g., Delta or Serrate / Jagged), a combination of a RAS kinase and a RAF kinase, a combination of a BRAF kinase and a CRAF kinase, a combination of HER2 / Neu (ErbB2) and a HER family (e.g., HER1, HER3, HER4), a combination of BCR / ABL, a combination of N-Myc and Max, a combination of N-Myc, Max and DNA, a combination of G proteins (e.g., a combination of Gα and Gβ, a combination of Gβ and Gγ, a combination of Gγ and Gα), etc. When the target substance is a peptide (protein), the peptide as the target substance and the peptide contained in the luciferase-peptide conjugate may be interchanged. Note that these specific examples do not limit the amino acid sequences of the first peptide and the second peptide to the amino acid sequences of the peptides shown in these specific examples, and the amino acid sequences may be changed as long as they can interact with the target substance. For example, the first peptide and the second peptide may have an amino acid sequence having 85% or more sequence identity to the amino acid sequences of the peptides shown in these specific examples. The above-mentioned sequence identity may be 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100%. The first peptide and the second peptide may have an amino acid sequence in which amino acid residues are substituted, deleted, or added to the amino acid sequences of the peptides shown in these specific examples. There is no particular limit to the number of amino acid residues to be substituted, deleted, or added, and may be, for example, 1 to 15, 1 to 12, 1 to 10, 1 to 7, 1 to 5, or 1 to 3.

[0042] Examples of combinations of G protein-coupled receptors and their ligands include a combination of dopamine receptors (DRD) and dopamine, a combination of histamine receptors and histamine, a combination of angiotensin receptors and angiotensin, a combination of serotonin receptors and serotonin, and a combination of metabotropic glutamate receptors (mGluR) and umami components (e.g., glutamic acid). Examples of combinations of G protein-coupled receptors (heterodimers) and their ligands include a combination of sweet receptors (T1R2 and T1R3) and sweet components (e.g., sucrose, glucose), and a combination of umami receptors (T1R1 and T1E3) and umami components (e.g., glutamic acid). In addition, when there are subtypes of G protein-coupled receptors, any of them may be used. For example, the dopamine receptor (DRD) may be a dopamine type 1 receptor (DRD1), a dopamine type 2 receptor (DRD2), or the like.

[0043] A G protein-coupled receptor is a seven-transmembrane membrane protein having seven transmembrane sites (hereinafter, referred to as the first transmembrane site, the second transmembrane site, ..., the seventh transmembrane site, in order from the N-terminus). In the case of a G protein-coupled receptor, for example, the first peptide may be a peptide fragment containing a site that interacts with a ligand of the G protein-coupled receptor, and the second peptide may be a peptide fragment (preferably a peptide fragment different from the first peptide) containing a site that interacts with a ligand of the G protein-coupled receptor. Specific examples of the first peptide and the second peptide may include a combination in which the first peptide is a peptide having an amino acid sequence from the first transmembrane site to the fifth transmembrane site of the G protein-coupled receptor, and the second peptide is a peptide having an amino acid sequence from the sixth transmembrane site to the seventh transmembrane site of the G protein-coupled receptor. In this case, the luciferase-peptide conjugate corresponds to one containing the amino acid sequence of luciferase at the position of the third intracellular loop of the G protein-coupled receptor. The selection of the first peptide and the second peptide is not limited to the above specific example, and may be selected to correspond to one containing the amino acid sequence of luciferase at the position of the first intracellular loop of a G protein-coupled receptor (for example, a combination in which the first peptide is a peptide having an amino acid sequence from the N-terminus to the first transmembrane site of a G protein-coupled receptor, and the second peptide is a peptide having an amino acid sequence from the second transmembrane site to the seventh transmembrane site of a G protein-coupled receptor), or may be selected to correspond to one containing the amino acid sequence of luciferase at the position of the second intracellular loop of a G protein-coupled receptor (for example, a combination in which the first peptide is a peptide having an amino acid sequence from the first transmembrane site to the third transmembrane site of a G protein-coupled receptor, and the second peptide is a peptide having an amino acid sequence from the fourth transmembrane site to the seventh transmembrane site of a G protein-coupled receptor). In addition, the first peptide and the second peptide may have an amino acid sequence having 85% or more sequence identity to the amino acid sequence of a G protein-coupled receptor.The sequence identity may be 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100%. The first peptide and the second peptide may have an amino acid sequence in which an amino acid residue is substituted, deleted, or added to the amino acid sequence of the G protein-coupled receptor. The number of amino acid residues to be substituted, deleted, or added is not particularly limited, and may be, for example, 1 to 15, 1 to 12, 1 to 10, 1 to 7, 1 to 5, or 1 to 3. The first peptide and the second peptide may be the reverse of the above specific examples.

[0044] As a specific example of a combination of a G protein-coupled receptor and its ligand, a combination of a first peptide, a second peptide, and a target substance is a combination of dopamine type 1 receptor (DRD1) and dopamine. DRD1 is a seven-transmembrane protein and has seven transmembrane sites (hereinafter, they are referred to as the first transmembrane site, the second transmembrane site, ..., the seventh transmembrane site, in order from the N-terminus). In this embodiment, for example, the first peptide can be a peptide fragment containing a site of DRD1 that interacts with dopamine, and the second peptide can be a peptide fragment (preferably a peptide fragment different from the first peptide) containing a site of DRD1 that interacts with dopamine.

[0045] Specific examples of the first peptide and the second peptide may be, for example, a combination in which the first peptide is a peptide having an amino acid sequence having 85% or more sequence identity with the amino acid sequence from the first transmembrane site to the fifth transmembrane site of DRD1, and the second peptide is a peptide having an amino acid sequence having 85% or more sequence identity with the amino acid sequence from the sixth transmembrane site to the seventh transmembrane site of DRD1, or the first peptide may be a peptide having an amino acid sequence having 85% or more sequence identity with the amino acid sequence from the 25th amino acid residue to the 247th amino acid residue of the amino acid sequence shown in SEQ ID NO: 8, and the second peptide may be a peptide having an amino acid sequence having 85% or more sequence identity with the amino acid sequence from the 430th amino acid residue to the 611th amino acid residue of the amino acid sequence shown in SEQ ID NO: 8. These first peptide and second peptide may be capable of interacting with dopamine. The sequence identity may be 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100%. The first peptide and the second peptide may have an amino acid sequence in which an amino acid residue is substituted, deleted, or added with respect to the amino acid sequence from the first transmembrane site to the fifth transmembrane site of DRD1, the amino acid sequence from the sixth transmembrane site to the seventh transmembrane site of DRD1, the amino acid sequence from the 25th amino acid residue to the 247th amino acid residue of the amino acid sequence shown in SEQ ID NO: 8, or the amino acid sequence from the 430th amino acid residue to the 611th amino acid residue of the amino acid sequence shown in SEQ ID NO: 8. The number of amino acid residues to be substituted, deleted, or added is not particularly limited, and may be, for example, 1 to 15, 1 to 12, 1 to 10, 1 to 7, 1 to 5, or 1 to 3. Furthermore, the first peptide and the second peptide may be reversed from the above embodiments.

[0046] (First connecting portion and second connecting portion) The first linking part links the first peptide to the N-terminus of luciferase. The mode of linking may be any mode that can maintain the bond between luciferase and the first peptide under physiological conditions, and may be, for example, a covalent bond, a hydrogen bond, an ionic bond, a bond based on intermolecular forces, or the like.

[0047] The first linking part is not particularly limited as long as it can link the N-terminus of luciferase to the first peptide, and specific examples include linkers such as a peptide linker, polyethylene glycol, and polypropylene glycol.

[0048] When the first linking part is a peptide linker, the luciferase and the first peptide are connected via the peptide linker to form a single polypeptide. The amino acid sequence and the number of amino acid residues (length) of the peptide linker can be appropriately set depending on the types of the first and second polypeptides, the use of the luciferase-peptide conjugate, etc.

[0049] The shorter the linker, the more pronounced the change in luminescence intensity depending on the concentration of the target substance, and therefore the number (length) of amino acid residues of the peptide linker may be, for example, 1 to 15, 1 to 12, 1 to 10, 1 to 9, 1 to 8, 1 to 7, or 1 to 6. As described above, in the luciferase-peptide conjugate according to this embodiment, when the target substance is present, the first peptide and / or the second peptide interacts with the target substance (for example, the association of the first peptide with the target substance, the association of the second peptide with the target substance, or the formation of a (ternary) complex by the first peptide, the second peptide, and the target substance), which is presumed to cause a further change in the three-dimensional structure of luciferase and (re)activate luminescence (metabolism of the luminescent substrate). It is considered that the shorter the linker, the easier it is to transmit the structural change of the first peptide due to the interaction between the first peptide and the target substance, and the easier it is to cause a structural change of luciferase.

[0050] Specific examples of peptide linkers include SGGGGS (SEQ ID NO: 9), GGGGS (SEQ ID NO: 10), GGGSGGGGSGGGGGS (SEQ ID NO: 11), SGGGGSGGGGSGGGGS (SEQ ID NO: 12), and EAAAK (SEQ ID NO: 13).

[0051] The second linking part links the second peptide to the C-terminus of luciferase. The mode of linking may be any mode that can maintain the bond between luciferase and the second peptide under physiological conditions, and may be, for example, a covalent bond, a hydrogen bond, an ionic bond, a bond based on intermolecular forces, or the like.

[0052] The second linking part is not particularly limited as long as it can link the C-terminus of luciferase to the second peptide, and specific examples include linkers such as a peptide linker, polyethylene glycol, and polypropylene glycol.

[0053] When the second linking part is a peptide linker, the luciferase and the second peptide are connected via the peptide linker to form a single polypeptide. The amino acid sequence and the number of amino acid residues (length) of the peptide linker can be appropriately set depending on the types of the first and second polypeptides, the use of the luciferase-peptide conjugate, etc.

[0054] The shorter the linker, the more pronounced the change in luminescence intensity depending on the concentration of the target substance, and this is more pronounced in the second linking portion. Therefore, the number (length) of amino acid residues of the peptide linker may be, for example, 1 to 10, 1 to 9, 1 to 8, 1 to 7, or 1 to 6. As described above, in the luciferase-peptide conjugate according to this embodiment, when the target substance is present, the first peptide and / or the second peptide interacts with the target substance (for example, the association of the first peptide with the target substance, the association of the second peptide with the target substance, or the formation of a (ternary) complex by the first peptide, the second peptide, and the target substance), which is presumed to cause a further change in the three-dimensional structure of luciferase and (re)activate luminescence (metabolism of the luminescent substrate). It is considered that the shorter the linker, the easier it is to transmit the structural change of the second peptide due to the interaction between the second peptide and the target substance, and the easier it is to cause a structural change in luciferase. Furthermore, since the C-terminal side of the luciferase contained in the luciferase-peptide conjugate of this embodiment is considered to contribute greatly to luminescence (metabolism of the luminescent substrate), it is presumed that the shorter the linker in the second linking portion, the easier it is to (re)activate luminescence (metabolism of the luminescent substrate).

[0055] Specific examples of peptide linkers include SGGGGS (SEQ ID NO: 9), GGGGS (SEQ ID NO: 10), GGGSGGGGSGGGGGS (SEQ ID NO: 11), SGGGGSGGGGSGGGGS (SEQ ID NO: 12), and EAAAK (SEQ ID NO: 13).

[0056] In the luciferase-peptide conjugate according to this embodiment, at least one of the first peptide and the second peptide may further contain an amino acid sequence of a fluorescent protein. By containing the amino acid sequence of a fluorescent protein, the fluorescence emitted by the fluorescent protein can be used as an internal standard, and the luminescence intensity of luciferase can be normalized. The position at which the amino acid sequence of the fluorescent protein is bound is not particularly limited as long as it does not prevent the target substance, the first peptide, and the second peptide from forming a complex. The amino acid sequence of the fluorescent protein may be directly bound to at least one of the first peptide and the second peptide, or may be bound via a linker similar to the first or second linking part.

[0057] The fluorescent protein is not particularly limited, and examples thereof include green fluorescent proteins (eg, GFP, EGFP), blue fluorescent proteins (eg, BFP), and red fluorescent proteins (eg, RFP, mCherry, cpmApple).

[0058] (Method of producing luciferase-peptide conjugate) The luciferase-peptide conjugate according to this embodiment can be obtained, for example, by preparing luciferase, a first peptide, and a second peptide, respectively, and then linking the luciferase and the first peptide, and the luciferase and the second peptide, via the above-mentioned linker. In this case, the luciferase, the first peptide, and the second peptide may each be prepared, for example, by chemical synthesis, or may be prepared by extraction and purification from a host transformed with an expression vector expressing each of them.

[0059] When both the first peptide and the second peptide are peptide linkers, the luciferase-peptide conjugate becomes a fusion protein, and can be prepared by a method commonly used for protein production. In this case, the luciferase-peptide conjugate (luciferase-peptide fusion protein) can be prepared by expressing the luciferase-peptide fusion protein in the test cell by introducing the luciferase-peptide conjugate (luciferase-peptide fusion protein) into the test cell in the form of a nucleic acid described below, without preparing the luciferase-peptide conjugate in advance.

[0060] [Nucleic acid] The nucleic acid according to this embodiment comprises a base sequence encoding a luciferase-peptide conjugate (luciferase-peptide fusion protein) and a regulatory sequence operably linked to the base sequence.

[0061] The regulatory sequence is a sequence (e.g., a promoter, enhancer, ribosome binding sequence, transcription termination sequence, etc.) that controls the expression of the luciferase-peptide fusion protein in a host (e.g., a test cell, etc.), and can be selected appropriately depending on the type of host.

[0062] The nucleic acid according to the present embodiment can also be used as an expression vector. The type of expression vector can be appropriately selected according to the type of host, such as a plasmid vector, a virus vector, a cosmid vector, a fosmid vector, or an artificial chromosome vector.

[0063] The host may be any prokaryotic or eukaryotic organism, such as yeast, filamentous fungi, insect cells, animal cells and plant cells.

[0064] The nucleic acid according to this embodiment may further include a base sequence that expresses a fluorescent protein. The base sequence that expresses a fluorescent protein includes, for example, a base sequence that encodes a fluorescent protein and a regulatory sequence operably linked to the base sequence. When the nucleic acid according to this embodiment includes a base sequence that expresses a fluorescent protein, the fluorescence emitted by the fluorescent protein can be used as an internal standard, and the luminescence intensity by luciferase can be normalized. There is no particular limitation on the type of fluorescent protein, and examples thereof include the fluorescent proteins described above.

[0065] The nucleic acid according to this embodiment may further include a selection marker for selecting a transformed host (e.g., a test cell, etc.) Examples of the selection marker include, but are not limited to, antibiotic resistance genes such as puromycin resistance gene, neomycin resistance gene, and hygromycin resistance gene.

[0066] The nucleic acid according to this embodiment can be produced according to a conventional method.

[0067] Methods of using luciferase-peptide conjugates The luciferase-peptide conjugate according to this embodiment can be used to measure the concentration of a target substance in a test sample. The luciferase-peptide conjugate according to this embodiment may be added to a test sample as a luciferase-peptide conjugate, or may be added to a test sample as the above-mentioned nucleic acid. In the latter case, the luciferase-peptide conjugate is expressed from the nucleic acid in the test sample, so that the luciferase-peptide conjugate is present (added) in the test sample. The latter is particularly advantageous when the test sample is a cell (test cell).

[0068] A method for measuring the concentration of a target substance in a test sample according to one embodiment includes the steps of adding a luciferase-peptide conjugate according to this embodiment to the test sample (addition step) and measuring luminescence from the luciferase-peptide conjugate in the test sample (luminescence measurement step 1). The luciferase-peptide conjugate according to this embodiment emits light with a luminescence intensity according to the concentration of the target substance in the test sample, so that the concentration of the target substance in the test sample can be measured by the steps described above.

[0069] In the addition step, the luciferase-peptide conjugate according to the present embodiment is added to the test sample. In the addition step, the conjugate itself may be added to the test sample, or, if the test sample is in an environment in which a protein can be expressed from the nucleic acid, the above-mentioned nucleic acid may be added to the test sample. The amount of the conjugate added in the addition step may be appropriately set depending on the type of the test sample, the (expected) concentration of the target substance, and the like.

[0070] In the luminescence measurement step 1, luminescence from the luciferase-peptide conjugate in the test sample is measured. The luminescence measurement can be performed by a method commonly used for measuring luminescence from luciferase. Specifically, for example, a suitable luminescence substrate (luciferin) according to the type of luciferase in the luciferase-peptide conjugate according to the present embodiment is added, and the luminescence intensity can be measured using a luminescence measuring device (for example, SpectraMax i3x multimode microplate reader, manufactured by Molecular Devices). In the luciferase-peptide conjugate according to the present embodiment, for example, coelenterazine and coelenterazine h can be used as the luminescence substrate. The luminescence measurement can be performed, for example, at a temperature of 25°C or higher and 40°C or lower, for a measurement time of 30 minutes with an interval of 3 to 5 minutes.

[0071] The measurement method according to this embodiment may further include a step of measuring the fluorescence from the fluorescent protein (fluorescence measurement step 1) and a step of normalizing the measured luminescence with the measured fluorescence (normalization step 1), which makes it possible to eliminate the influence of variations in the state of the test sample (e.g., the proliferation rate of the test cells, the protein expression activity of the test cells, etc.) on the measured value.

[0072] Fluorescence measurement step 1 and normalization step 1 can be performed when the luciferase-peptide conjugate of this embodiment contains the amino acid sequence of a fluorescent protein, and when the nucleic acid of this embodiment contains a base sequence that expresses a fluorescent protein.

[0073] In the fluorescence measurement step 1, the fluorescence from the fluorescent protein is measured. The fluorescence can be measured by a method commonly used for measuring the fluorescence of fluorescent proteins. Specifically, for example, the fluorescence intensity can be measured using a fluorescence measuring device at an appropriate excitation wavelength and observation wavelength according to the type of fluorescent protein.

[0074] In the normalization step 1, the measured luminescence is normalized by the measured fluorescence. In the normalization step 1, for example, the measured luminescence intensity may be divided by the measured fluorescence intensity to calculate the normalized luminescence intensity.

[0075] A method for measuring the concentration of a target substance in a test cell according to another embodiment includes a step of introducing the nucleic acid according to this embodiment into the test cell (introduction step) and a step of measuring luminescence from the luciferase-peptide conjugate in the test sample (luminescence measurement step 2). The method according to this embodiment is applied when the test sample is a cell (test cell).

[0076] In the introduction step, the nucleic acid according to the present embodiment is introduced into the test cell. The introduction method is not particularly limited, and any method commonly used for cell transformation can be applied. Specific examples include electroporation and lipofection.

[0077] The measurement method according to the present embodiment may further include a step of selecting test cells into which the nucleic acid has been introduced after the introduction step. The selection of test cells into which the nucleic acid has been introduced can be performed using a selection marker contained in the nucleic acid. For example, when the nucleic acid according to the present embodiment contains an antibiotic resistance gene as a selection marker, the test cells into which the nucleic acid has been introduced can be selected by culturing the test cells after the introduction step in a medium containing the corresponding antibiotic.

[0078] Luminescence from the luciferase-peptide conjugate in the test sample is measured in the luminescence measurement step 2. The luminescence measurement step 2 can be carried out in the same manner as the luminescence measurement step 1.

[0079] The measurement method according to this embodiment may further include a step of measuring the fluorescence from the fluorescent protein (fluorescence measurement step 2) and a step of normalizing the measured luminescence with the measured fluorescence (normalization step 2), which makes it possible to eliminate the influence of variability in the state of the test cells (e.g., the proliferation rate of the test cells, the protein expression activity of the test cells, the amount of nucleic acid introduced, etc.) on the measured value.

[0080] The fluorescence measurement step 2 and the normalization step 2 can be performed when the luciferase-peptide conjugate of this embodiment contains the amino acid sequence of a fluorescent protein, and when the nucleic acid of this embodiment contains a base sequence that expresses a fluorescent protein.

[0081] Fluorescence from the fluorescent protein is measured in the fluorescence measurement step 2. The fluorescence measurement step 2 can be performed in the same manner as the fluorescence measurement step 1.

[0082] In normalization step 2, the measured luminescence is normalized by the measured fluorescence. Normalization step 2 can be performed similarly to normalization step 1. EXAMPLES

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

[0084] Test Example 1: Production and evaluation of luciferase-peptide conjugate (1) (Test Example 1-1: Preparation of luciferase-peptide conjugate) Luciferase-peptide conjugates (luciferase-peptide fusion proteins) having the amino acid sequences shown in SEQ ID NOs: 4 to 7 were designed.

[0085] The amino acid sequence shown in SEQ ID NO: 4 is FK506 binding protein (FKBP) to which eKAZ (SEQ ID NO: 2) is directly linked at the C-terminus (FKBP-eKAZ).

[0086] The amino acid sequence shown in SEQ ID NO:5 is such that eKAZ is linked to the C-terminus of FKBP via a linker (SEQ ID NO:9: SGGGGS), and the rapamycin-binding domain fragment (FRB) of mTOR is further linked to the C-terminus of eKAZ via a linker (SEQ ID NO:10: GGGGS) (FKBP-SGGGGS-eKAZ-GGGGS-FRB).

[0087] The amino acid sequence shown in SEQ ID NO:6 is such that eKAZ is linked to the C-terminus of FKBP via a linker (SEQ ID NO:9), and FRB is further linked to the C-terminus of eKAZ via a linker (SEQ ID NO:12: SGGGGSGGGGSGGGGS) (FKBP-SGGGGS-eKAZ-SGGGGSGGGGSGGGGS-FRB).

[0088] The amino acid sequence shown in SEQ ID NO: 7 is such that eKAZ is linked to the C-terminus of FKBP via a linker (SEQ ID NO: 12), and FRB is further linked to the C-terminus of eKAZ via a linker (SEQ ID NO: 12) (FKBP-SGGGGSGGGGSGGGGS-eKAZ-SGGGGSGGGGSGGGGS-FRB).

[0089] [Table 2]

[0090] Nucleic acids (DNA) encoding the amino acid sequences shown in SEQ ID NOs: 4 to 7 were synthesized, and expression vectors capable of expressing luciferase-peptide fusion proteins were constructed. Figure 5 shows a vector map of the constructed expression vectors. Table 3 shows the definitions of the abbreviations in Figure 5.

[0091] [Table 3]

[0092] (Test Example 1-2: Evaluation of luciferase-peptide conjugate) The expression vector prepared in Test Example 1-1 was used to transform a CHO-K1 cell line (distributed by JCRB, JCRB9018), a 3T3-L1 cell line (distributed by JCRB, JCRB9014), or a HeLa cell line (distributed by JCRB, JCRB9004), and the luciferase-peptide conjugate was evaluated.

[0093] (CHO-K1 cell line) The medium used was Ham's F12 medium (Fujifilm Wako Pure Chemical Industries, Ltd.) supplemented with 10% FBS. CHO-K1 cell line was precultured in a T-25 flask, and after it became confluent, the cells were harvested (approximately 3 × 10 6 Next, the collected cells were transformed with each expression vector prepared in Test Example 1-1 using 4D-Nucleofector (manufactured by LONZA). The transformed cells were cultured and grown in a medium containing puromycin. After growth, the cells were collected and placed in a 96-well plate (manufactured by ThermoFisher) at 3 × 10 4The cells were seeded at 100 cells / well. After culturing and growing in the wells for 1 day, rapamycin was added to each well (8 wells for each concentration) and cultured overnight. Next, serum (FBS)-free medium containing 5 μM coelenterazine h (Fujifilm Wako Pure Chemical Industries, Ltd.) was added to each well (final concentration 2.5 μM), and the 96-well plate was set in a SpectraMax i3x multimode microplate reader (Molecular Devices, Ltd.) to measure luminescence. Luminescence was measured 7 times in total, including immediately after the start, at intervals of about 5 minutes until 30 minutes later. After the luminescence measurement was completed, the fluorescence (excitation wavelength 545 nm, observation wavelength 610 nm) of the fluorescent protein (mCherry) was measured. The average value of the luminescence intensity for each interval and each well was used as the representative value, and the maximum value was used as the luminescence intensity for each concentration. In addition, in order to eliminate the influence of variations in culture and variations in the expression level of cells, the luminescence intensity was normalized by dividing it by the fluorescence intensity of mCherry.

[0094] FIG. 1 is a graph showing the evaluation results of luciferase-peptide conjugates in CHO-K1 cell lines. FIG. 1(A) is a graph plotting luminescence intensity (RLU) per well against rapamycin concentration when 1.1eKAZ is used as the luciferase-peptide conjugate. FIG. 1(B) is a graph plotting luminescence intensity (RLU) per well against rapamycin concentration when 1.3eKAZ is used as the luciferase-peptide conjugate. FIG. 1(C) is a graph plotting luminescence intensity (RLU) per well against rapamycin concentration when 3.3eKAZ is used as the luciferase-peptide conjugate.

[0095] As shown in FIG. 1, when the rapamycin concentration was extremely low (for example, 0.2 μM), almost no luminescence was observed from luciferase. This is thought to mean that the three-dimensional structure of luciferase changed due to the linkage of FKBP to the N-terminus and FRB to the C-terminus, and luminescence (metabolism of the luminescent substrate) became impossible. On the other hand, as the rapamycin concentration increased, the luminescence intensity increased in a concentration-dependent manner. Specifically, the amount of change in luminescence intensity dependent on the concentration of rapamycin was greater in the order of (1) 1.1eKAZ, (2) 1.3eKAZ, and (3) 3.3eKAZ. 1.1eKAZ, 1.3eKAZ, and 3.3eKAZ differ in the length of the linker binding luciferase to FKBP and the length of the linker binding luciferase to FRB. The shorter the length of these linkers, the more significant the change in luminescence intensity dependent on the concentration of rapamycin. In particular, the effect of the length of the linker connecting luciferase and FRB (the linker on the C-terminal side of luciferase) was remarkable.

[0096] FKBP and FRB can each interact (associate) with rapamycin, and can form a (ternary) complex (complex of FKBP, FRB, and rapamycin) with rapamycin. When FKBP and / or FRB interact with rapamycin (for example, association of FKBP with rapamycin, association of FRB with rapamycin, formation of a (ternary) complex by FKBP, FRB, and rapamycin), it is considered that the conformation of luciferase to which they are linked changes due to structural changes of FKBP, structural changes of FRB, and / or the proximity of FKBP and FRB, and the like, and luminescence (metabolism of luminescence substrate) is (re)activated. At that time, it is presumed that the shorter the length of the linker, the easier it is to transmit the structural changes of FKBP, structural changes of FRB, and / or structural changes due to the proximity of FKBP and FRB to luciferase, and the greater the change in luminescence intensity.

[0097] (3T3-L1 cell line) Except for changing the medium to DMEM low glucose (Hanai Scientific) supplemented with 10% FBS, the evaluation was carried out in the same manner as for the CHO-K1 cell line.

[0098] FIG. 2 is a graph showing the evaluation results of luciferase-peptide conjugates in 3T3-L1 cell lines. Next to each graph, a photograph showing an example of luminescence in a 96-well plate is shown. The same concentration of rapamycin was added to 8 wells in each row (row numbers 1 to 12). The rapamycin concentration in row number 12 is 1 μM. Next, the rapamycin concentration in each row is a 2-fold dilution series from row number 12 to row number 2. No rapamycin was added to row number 1. FIG. 2(A) is a graph plotting the luminescence intensity (RLU) per well against row number when 0eKAZ was used as the luciferase-peptide conjugate. FIG. 2(B) is a graph plotting the luminescence intensity (RLU) per well against row number when 1.1eKAZ was used as the luciferase-peptide conjugate. Figure 2(C) is a graph plotting the luminescence intensity (RLU) per well against the row number when 1.3eKAZ was used as the luciferase-peptide conjugate, and Figure 2(D) is a graph plotting the luminescence intensity (RLU) per well against the row number when 3.3eKAZ was used as the luciferase-peptide conjugate.

[0099] As shown in Figure 2, the same tendency was observed as in the case of the CHO-K1 cell line. Furthermore, 0eKAZ, in which FKBP was directly linked to the N-terminus of luciferase (eKAZ), luminescence was observed regardless of the rapamycin concentration. This suggests that the C-terminus of luciferase (eKAZ) is closely involved in luminescence (metabolism of the luminescence substrate). This is also consistent with the significant effect of the length of the linker connecting luciferase and FRB (the linker on the C-terminus side of luciferase) on the dose-dependent change in luminescence intensity.

[0100] (Hela cell line) Except for changing the medium to DMEM low glucose (Hanai Scientific) supplemented with 10% FBS, the evaluation was carried out in the same manner as for the CHO-K1 cell line.

[0101] 3 is a graph showing the results of evaluating luciferase-peptide conjugates in HeLa cell lines. Similar trends were observed as in the CHO-K1 and 3T3-L1 cell lines.

[0102] [Test Example 2: Production and evaluation of luciferase-peptide conjugate (2)] (Test Example 2-1: Preparation of luciferase-peptide conjugate) A luciferase-peptide conjugate (luciferase-peptide fusion protein) having the amino acid sequence shown in SEQ ID NO:8 was designed.

[0103] The amino acid sequence shown in SEQ ID NO: 8 is an amino acid sequence including the N-terminus to the 5th transmembrane site from the N-terminus of dopamine type 1 receptor (DRD1) (a part of the N-terminus of DRD1) linked to the C-terminus of eKAZ via a linker (SEQ ID NO: 13: EAAAK), and further linked to the C-terminus of eKAZ via a linker (SEQ ID NO: 9) an amino acid sequence including the 6th transmembrane site from the N-terminus of DRD1 to the C-terminus of DRD1 (a part of the C-terminus of DRD1), and a fluorescent protein (mCherry) linked to the C-terminus of the amino acid sequence (DRD1_eKAZ(EAAAK-SGGGGS)). DRD1_eKAZ(EAAAK-SGGGGS) corresponds to a sequence in which the amino acid sequence of the 3rd intracellular loop of DRD1 has been replaced with the amino acid sequence of luciferase (eKAZ).

[0104] [Table 4]

[0105] A nucleic acid (DNA) encoding the amino acid sequence shown in SEQ ID NO:8 was synthesized, and an expression vector capable of expressing a luciferase-peptide fusion protein was constructed. Figure 6 shows a vector map of the constructed expression vector. The definitions of the abbreviations in Figure 6 are shown in Table 5.

[0106] [Table 5]

[0107] (Test Example 2-2: Evaluation of luciferase-peptide conjugate) CHO-K1 cells were precultured in a T-25 flask and harvested after reaching confluence (approximately 3 × 10 6 Next, the collected cells were transformed with the expression vector prepared in Test Example 2-1 using 4D-Nucleofector (manufactured by LONZA). The transformed cells were cultured and grown in a medium containing puromycin. After growth, the cells were collected and placed in a 96-well plate (manufactured by ThermoFisher) at 3 × 10 4 The cells were seeded at 1000 cells / well. After culturing and growing in the wells for 1-2 days, dopamine was added to each well (8 wells for each concentration) and cultured overnight. Next, serum (FBS)-free medium containing 5 μM coelenterazine h (Fujifilm Wako Pure Chemical Industries, Ltd.) was added to each well (final concentration 2.5 μM), and the 96-well plate was set in a SpectraMax i3x multimode microplate reader (Molecular Devices, Ltd.) to measure luminescence. Luminescence was measured for 30 minutes at intervals of about 3 minutes. After the luminescence measurement was completed, the fluorescence of the fluorescent protein (mCherry) (excitation wavelength 545 nm, observation wavelength 610 nm) was measured. The average value of the luminescence intensity for each interval and each well was used as the representative value, and the maximum value was used as the luminescence intensity for each concentration. In order to eliminate the effects of variations in culture and variations in the expression level of cells, the luminescence intensity was normalized by dividing it by the fluorescence intensity of mCherry. The 3T3-L1 cell line and HeLa cell line were also evaluated in a similar manner.

[0108] 4 is a graph showing the results of evaluation of luciferase-peptide conjugates in a CHO-K1 cell line, plotting luminescence unit (RLU) per unit cell against dopamine concentration.

[0109] As shown in FIG. 4, when the dopamine concentration was low (e.g., 0.1 μM), almost no luminescence was observed from luciferase. This is considered to mean that by linking a part of the N-terminal side of DRD1 to the N-terminus and a part of the C-terminal side of DRD1 and a fluorescent protein to the C-terminus, the three-dimensional structure of luciferase changed and it was no longer possible to emit light (metabolism of the luminescent substrate). On the other hand, as the dopamine concentration increased, the luminescence intensity increased in a concentration-dependent manner. The part of the N-terminus side of DRD1 and the part of the C-terminus side of DRD1 can each interact (associate) with dopamine and can also form a (ternary) complex with dopamine. When a portion of the N-terminus of DRD1 and / or a portion of the C-terminus of DRD1 interacts with dopamine (for example, the association of a portion of the N-terminus of DRD1 with dopamine, the association of a portion of the C-terminus of DRD1 with dopamine, or the formation of a (ternary) complex between a portion of the N-terminus of DRD1, a portion of the C-terminus of DRD1, and dopamine), the portion of the N-terminus of DRD1 undergoes a structural change, the portion of the C-terminus of DRD1 undergoes a structural change, and / or the portion of the N-terminus of DRD1 and the portion of the C-terminus of DRD1 come into close proximity, which is thought to change the three-dimensional structure of luciferase to which they are linked, thereby (re)activating luminescence (metabolism of the luminescent substrate).

Claims

1. The nucleic acid sequence comprises a luciferase, a first peptide linked to the N-terminus of the luciferase via a first linking part, and a second peptide linked to the C-terminus of the luciferase via a second linking part, The luciferase has an amino acid sequence having 85% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3, and is capable of emitting light; A luciferase-peptide conjugate, wherein the first peptide and the second peptide are capable of forming a complex with a substance of interest.

2. the first peptide has an amino acid sequence that has 85% or more sequence identity to the amino acid sequence from the N-terminus to the first, third or fifth transmembrane site of a G protein-coupled receptor, and is capable of interacting with a ligand of the G protein-coupled receptor; The conjugate of claim 1, wherein the second peptide has an amino acid sequence that has 85% or more sequence identity to the amino acid sequence from the second, fourth, or sixth transmembrane domain to the seventh transmembrane domain of the G protein-coupled receptor, and is capable of interacting with a ligand of the G protein-coupled receptor.

3. the first peptide has an amino acid sequence having 85% or more sequence identity with the amino acid sequence from the first transmembrane domain to the fifth transmembrane domain of the dopamine type 1 receptor (DRD1) and is capable of interacting with dopamine; The conjugate according to claim 1, wherein the second peptide has an amino acid sequence having 85% or more sequence identity with the amino acid sequence from the sixth transmembrane domain to the seventh transmembrane domain of the dopamine type 1 receptor (DRD1) and is capable of interacting with dopamine.

4. The conjugate of claim 2 , wherein the first peptide and / or the second peptide further comprises an amino acid sequence of a fluorescent protein.

5. the first peptide has an amino acid sequence having 85% or more sequence identity with the amino acid sequence of FK506 binding protein (FKBP) and is capable of interacting with rapamycin; The conjugate of claim 1, wherein the second peptide has an amino acid sequence that has 85% or more sequence identity with the amino acid sequence of the rapamycin binding domain fragment (FRB) of the rapamycin target protein (TOR) and is capable of interacting with rapamycin.

6. The conjugate according to any one of claims 1 to 5, wherein the first linking portion and the second linking portion are both peptide linkers.

7. The conjugate of claim 6 , wherein the peptide linker is 6 amino acid residues or less.

8. A nucleic acid comprising a base sequence encoding the conjugate according to claim 6 and a regulatory sequence operably linked to said base sequence.

9. The nucleic acid of claim 8, further comprising a base sequence that expresses a fluorescent protein.

10. 1. A method for measuring a concentration of a target substance in a test sample, comprising: Adding the conjugate according to any one of claims 1 to 5 to a test sample; and measuring luminescence from said conjugate.

11. 1. A method for measuring a concentration of a target substance in a test cell, comprising: Introducing the nucleic acid according to claim 8 into a test cell; and measuring luminescence from said conjugate.

12. 1. A method for measuring a concentration of a target substance in a test cell, comprising: Introducing the nucleic acid according to claim 9 into a test cell; measuring luminescence from the conjugate; Measuring the fluorescence from the fluorescent protein; and normalizing the measured luminescence to the measured fluorescence.

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