WO2017094885A1 - Ligand fluorescent sensor protein and use thereof - Google Patents

Abstract

The present invention involves a ligand fluorescent sensor protein that specifically responds to ligands, leading to a change in fluorescence properties. The ligand fluorescent sensor protein includes a first fluorescent protein domain, an N terminal-side linker, a ligand-bonding domain, a C terminal-side linker, and a second fluorescent protein domain. The fluorescent protein used in the ligand fluorescent sensor protein has a β-barrel structure. The first fluorescent protein domain includes a β1–β3 β-sheet region from the N-terminal of the fluorescent protein, an α-helix region continuing therefrom, and a β4–β6 β-sheet region. The second fluorescent protein domain includes a β7–β11 β-sheet region of the same fluorescent protein as the first fluorescent protein domain. The N-terminal-side linker and the C-terminal-side linker are each independently a polypeptide comprising one or a plurality of amino acids.

Description

Ligand fluorescent sensor protein and its use

The present invention relates to ligand fluorescent sensor proteins and uses thereof.
The present application is filed in Japanese Patent Application No. 2015-237524 filed in Japan on December 4, 2015 and US Patent No. 62 / 340,533 filed provisionally in the United States on May 24, 2016. Claims priority and incorporates the contents here.

Fluorescent bioimaging is a new research technique that observes the inside of living tissues or organs and analyzes the dynamics of living cells and molecules in real time. Recent technological innovations in optical instruments and fluorescence imaging have made it possible to visualize the movement of living cells and molecules by applying fluorescent bioimaging to a wide variety of life phenomena in various tissues or organs. It was.

In fluorescence bioimaging, the movement of a ligand in a living tissue or organ can be detected by using a receptor to which a molecule (ligand) present in a cell specifically binds.
Specifically, as a receptor, for example, an antibody that specifically binds to a molecule in a living body, an antigen for an antibody present in the living body, a substrate for a specific enzyme, an enzyme for a specific substrate, or ATP, cAMP, or cGMP By using a fluorescent sensor protein having a binding domain of a molecule in a living body, etc., it is possible to visualize and analyze the concentration distribution and temporal change (spatiotemporal dynamics) of a ligand in a cell.

Fluorescent sensor proteins are broadly classified into monochromatic types, ratio types, and FRET (fluorescence resonance energy transfer) types. The monochromatic type is a type in which the fluorescence intensity at a single wavelength specified for each fluorescent sensor protein changes according to the ligand concentration. The ratio type is a type in which the relative ratio of fluorescence intensities at two wavelengths specified for each fluorescent sensor protein changes according to the ligand concentration. In the FRET type, when one fragment of the fusion protein containing two different fluorescent protein fragments is in an excited state, if the distance from the other fragment is close according to the ligand concentration, the energy from the excited fragment becomes the other A type in which the relative ratio between the fluorescence intensity of the former fragment and the fluorescence intensity of the latter fragment changes depending on the ligand concentration by using the Forster resonance energy transfer phenomenon that causes non-radiative transition to the fragment. It is.
For example, to date, monochromatic (for example, see Non-Patent Document 1), FRET (for example, Non-Patent Document 2), and ratio-type (for example, Non-Patent Documents 3 and 4) ATP fluorescent sensor proteins have been reported. Has been.

However, conventional fluorescent sensor proteins have several problems. First, an expensive dedicated microscope is required for analysis using a FRET or ratio type sensor. Further, since a wide range of fluorescence wavelengths is used for fluorescence detection, it is not suitable for multicolor analysis. In other words, the dynamics of various ligands cannot be analyzed simultaneously. Furthermore, it is impossible to visualize and analyze the dynamics of ligands of different organelles simultaneously.

On the other hand, a fluorescence sensor based on a monochromatic fluorescent protein that excites at one wavelength and observes fluorescence at one wavelength may overcome these problems. However, the monochromatic sensor reported so far (see, for example, Non-Patent Document 1) has a dynamic range, that is, a change in fluorescence intensity by the presence or absence of ATP is only a little less than 8%, and an intracellular ATP sensor. As a practical use level was not reached. Thereafter, there is no report on a monochromatic ATP fluorescent protein sensor having a higher dynamic range than the report.
In addition, there is no report of a monochromatic fluorescent protein sensor with a high dynamic range capable of detecting a desired ligand without being limited to ATP.

The present invention has been made in view of the above circumstances, and provides a highly sensitive ligand fluorescent sensor protein regardless of the type of ligand to be detected.

That is, the present invention includes the following aspects.
[1] A ligand fluorescence sensor protein whose fluorescence characteristics change in response to a ligand specifically, wherein the ligand fluorescence sensor protein comprises a first fluorescence protein domain, an N-terminal linker, a ligand binding domain, A fluorescent protein comprising a C-terminal linker and a second fluorescent protein domain, wherein the fluorescent protein used in the ligand fluorescent sensor protein has a β-barrel structure, and the first fluorescent protein domain is the N-terminal of the fluorescent protein To β1 to β3 β sheet region followed by α helix region and β4 to β6 β sheet region, wherein the second fluorescent protein domain is the same as the first fluorescent protein domain. A β-sheet region of β7 to β11 of the protein, the N-terminal linker and the C-terminal The ligand fluorescent sensor protein, wherein each side linker is a polypeptide consisting of one or several amino acids independently.
[2] The ligand fluorescent sensor protein according to [1], wherein the fluorescent protein is BFP, GFP, Ciline, or mA Apple.
[3] The ligand fluorescent sensor protein includes a first fluorescent protein domain, an N-terminal linker, a ligand binding domain, a C-terminal linker, and a second fluorescent protein domain from the N-terminus toward the C-terminus. And a ligand fluorescent sensor protein according to [1] or [2], wherein the ligand fluorescent sensor protein comprises a polypeptide formed by directly connecting peptide bonds in this order.
[4] The ligand fluorescent sensor protein includes two ligand binding domains, and from the N-terminus toward the C-terminus, the first ligand-binding domain, the N-terminal linker, the second fluorescent protein domain, The ligand fluorescence according to [1] or [2], comprising a polypeptide in which one fluorescent protein domain, a C-terminal linker, and a second ligand-binding domain are directly linked by a peptide bond in this order Sensor protein.
[5] The first fluorescent protein domain includes any of the following polypeptides (B1) to (B3), and the second fluorescent protein domain includes any of the following (C1) to (C3): The ligand fluorescent sensor protein according to [1] or [2], wherein the first fluorescent protein domain and the second fluorescent protein domain are derived from the same fluorescent protein.
(B1) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 1, 3, 5, or 7,
(B2) the amino acid sequence represented by SEQ ID NO: 1, 3, 5, or 7, comprising an amino acid sequence in which one or several amino acids are deleted, inserted, substituted or added, and the second fluorescence A polypeptide that forms a β-barrel structure with a protein domain and fluoresces,
(B3) including an amino acid sequence having an identity of 80% or more with the amino acid sequence represented by SEQ ID NO: 1, 3, 5, or 7, and forming a β barrel structure with the second fluorescent protein domain, A fluorescent polypeptide,
(C1) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 2, 4, 6, or 8,
(C2) the amino acid sequence represented by SEQ ID NO: 2, 4, 6, or 8, comprising an amino acid sequence in which one or several amino acids are deleted, inserted, substituted or added, and the first fluorescence A polypeptide that forms a β-barrel structure with a protein domain and fluoresces,
(C3) comprising an amino acid sequence having 80% or more identity with the amino acid sequence represented by SEQ ID NO: 2, 4, 6, or 8, and forming a β barrel structure with the first fluorescent protein domain, Fluorescent polypeptide [6] The ligand according to any one of [1] to [5], wherein the ligand is a nucleotide or a derivative thereof, a nucleic acid, a sugar chain, a protein, a lipid complex, or a low molecular compound. Fluorescent sensor protein.
[7] The ligand fluorescent sensor protein according to [6], wherein the nucleotide or derivative thereof is ATP, cAMP, or cGMP.
[8] The ligand fluorescent sensor protein according to [6], wherein the protein is an antigen or an antibody.
[9] The ligand fluorescent sensor protein according to any one of [1] to [8], which comprises any of the following polypeptides (D1) to (D3).
(D1) a polypeptide comprising the amino acid sequence represented by any of SEQ ID NOs: 9 to 14,
(D2) an amino acid sequence represented by any one of SEQ ID NOs: 9 to 14, including an amino acid sequence in which one or several amino acids are deleted, inserted, substituted or added, and the polypeptide (D1) Polypeptides having the same ability to bind to a ligand and fluorescent properties;
(D3) Ligand binding ability and fluorescence, which include the amino acid sequence of 80% or more identity with the amino acid sequence represented by any of SEQ ID NOs: 9 to 14, and are identical to the polypeptide (D1) Polypeptide having properties [10] The ligand fluorescent sensor protein according to any one of [1] to [9], further comprising an organelle localization signal peptide.
[11] The ligand fluorescent sensor protein according to [10], wherein the organelle localization signal peptide is a mitochondrial localization signal peptide.
[12] The ligand fluorescent sensor protein according to [10], wherein the organelle localization signal peptide is a nuclear localization signal peptide.
[13] The ligand fluorescent sensor protein according to any one of [1] to [12], further comprising a cell membrane permeable peptide.
[14] A polynucleotide encoding the ligand fluorescent sensor protein according to any one of [1] to [13].
[15] An expression vector comprising the polynucleotide according to [14].
[16] A cell comprising at least one ligand fluorescent sensor protein according to any one of [1] to [13].
[17] A cell comprising a chromosome containing at least one polynucleotide encoding the ligand fluorescent sensor protein according to any one of [1] to [13].
[18] A cell comprising at least one expression vector according to [15].
[19] A non-human organism comprising the cell according to any one of [16] to [18].
[20] The ligand fluorescent sensor protein according to any one of [1] to [13], the polynucleotide according to [14], the expression vector according to [15], any of [16] to [18] A kit for measuring a ligand concentration, comprising at least one selected from the group consisting of the cell according to any one of the above and the non-human organism according to [19].
[21] Calibration for preparing a calibration curve by contacting a fluorescent fluorescence sensor protein according to any one of [1] to [13] with a standard solution containing a ligand of a known concentration to measure fluorescence intensity. Line creation process,
A fluorescence measurement step of measuring fluorescence intensity by contacting the ligand fluorescence sensor protein with a solution containing a ligand of unknown concentration;
In the calibration curve creating step, based on the created calibration curve, a concentration determining step for determining a ligand concentration with respect to the fluorescence intensity measured in the fluorescence measuring step;
A method for determining a ligand concentration in a test sample.
[22] A method for detecting a change in ligand concentration over time in a living cell, comprising the step of measuring fluorescence intensity over time using the cell according to any one of [16] to [18] .
[23] A method for detecting a change in ligand concentration over time in a living non-human organism, comprising the step of measuring fluorescence intensity over time using the non-human organism according to [19].
[24] An ATP fluorescent sensor protein whose fluorescence characteristics change in response to ATP concentration specifically, wherein the ATP fluorescent sensor protein includes a first fluorescent protein domain and an NTP from the N-terminus toward the C-terminus. A polypeptide comprising a terminal linker, an ATP-binding domain, a C-terminal linker, and a second fluorescent protein domain directly linked by a peptide bond in this order, the polypeptide comprising: A β-sheet region of β1-β3, followed by an α-helical region, and a β-sheet region of β4-β6, from the N-terminus of the fluorescent protein BFP, Citriline or mA Apple, the second fluorescent protein domain is A β-sheet region of β7 to β11 of the same fluorescent protein as that of one fluorescent protein domain, Inn consists ε subunit of F ₀ F ₁ -ATP synthase, ATP fluorescent N-terminal linker and C-terminal linker, respectively, which is a one or a polypeptide consisting of a few amino acids Sensor protein.
[25] (A11) The ATP-binding domain is the amino acid sequence of SEQ ID NO: 15, and the first and second fluorescent protein domains are the amino acid sequences of SEQ ID NOS: 1 and 2, respectively, The linker is a MaLion B polypeptide that is the amino acid sequence of SEQ ID NOs: 16 and 17, respectively;
(A12) The ATP-binding domain, the first fluorescent protein domain, the second fluorescent protein domain, the N-terminal linker, and the C-terminal linker are each independently SEQ ID NOs: 15, 1, 2, 16, and 17 amino acid sequence, or an amino acid sequence in which one or several amino acids are deleted, substituted, or added to the amino acid sequences of SEQ ID NOs: 15, 1, 2, 16, and 17, and MaLion B poly A MaLion B polypeptide having the same ATP binding ability and fluorescence properties as the peptide (A11);
(B11) The ATP-binding domain is the amino acid sequence of SEQ ID NO: 15, the first and second fluorescent protein domains are the amino acid sequences of SEQ ID NOs: 5 and 6, respectively, and the N-terminal and C-terminal linkers are MaLion G polypeptide, which is the amino acid sequence of WRG (Trp-Arg-Gly) and SEQ ID NO: 18, respectively,
(B12) The ATP binding domain, the first fluorescent protein domain, the second fluorescent protein domain, the N-terminal side linker and the C-terminal side linker are each independently SEQ ID NO: 15, 5, 6, WRG (Trp- Arg-Gly) and the amino acid sequence of SEQ ID NO: 18, or SEQ ID NOs: 15, 5, 6, WRG (Trp-Arg-Gly) and the amino acid sequence of SEQ ID NO: 18 have one or several amino acids deleted, A MaLion G polypeptide that is a substituted or added amino acid sequence and has the same ATP binding ability and fluorescence characteristics as the MaLion G polypeptide (B11);
(C11) The ATP binding domain is the amino acid sequence of SEQ ID NO: 15, the first and second fluorescent protein domains are the amino acid sequences of SEQ ID NOs: 7 and 8, respectively, and the N-terminal and C-terminal linkers are MaLion R polypeptide, which is the amino acid sequence of SEQ ID NO: 19 and PEE (Pro-Glu-Glu), respectively
(C12) The ATP binding domain, the first fluorescent protein domain, the second fluorescent protein domain, the N-terminal linker and the C-terminal linker are SEQ ID NOs: 15, 7, 8, 19, and PEE (Pro -Glu-Glu) amino acid sequence or SEQ ID NOs: 15, 7, 8, 19 and amino acid sequence of PEE (Pro-Glu-Glu) with one or several amino acids deleted, substituted or added [24] comprising at least one polypeptide selected from the group consisting of MaLion R polypeptides that are sequences and have the same ATP binding ability and fluorescence properties as MaLion R polypeptides (C11) ATP fluorescence sensor protein.
[26] (A21) a MaLion B polypeptide consisting of the amino acid sequence of SEQ ID NO: 9,
(A22) The amino acid sequence of SEQ ID NO: 9 consists of an amino acid sequence in which one or several amino acids are deleted, substituted or added to the amino acid sequence excluding the amino acid sequences of SEQ ID NOS: 16 and 17, and MaLion B A MaLion B polypeptide having the same ATP binding ability and fluorescence properties as the polypeptide (A21);
(B21) a MaLion G polypeptide consisting of the amino acid sequence of SEQ ID NO: 10,
(B22) one of amino acid sequences excluding the amino acid sequence of WRG (Trp-Arg-Gly) at positions 146 to 148 of SEQ ID NO: 10 and the amino acid sequence of SEQ ID NO: 18 among the amino acid sequences of SEQ ID NO: 10 or MaLion G polypeptide consisting of an amino acid sequence in which several amino acids are deleted, substituted or added, and having the same ATP binding ability and fluorescence characteristics as MaLion G polypeptide (B21);
(C21) a MaLion R polypeptide consisting of the amino acid sequence of SEQ ID NO: 11,
(C22) One of the amino acid sequences of SEQ ID NO: 11 excluding the amino acid sequence of PEE (Pro-Glu-Glu) at positions 288 to 290 of SEQ ID NO: 11 and the amino acid sequence of SEQ ID NO: 19 or It is selected from the group consisting of an MaLion R polypeptide consisting of an amino acid sequence in which several amino acids are deleted, substituted or added, and having the same ATP binding ability and fluorescence characteristics as MaLion R polypeptide (C21). The ATP fluorescence sensor protein according to [24] or [25], comprising at least one polypeptide.
[27] A fluorescent composition comprising the ATP fluorescent sensor protein according to any one of [24] to [26].
[28] The fluorescent composition according to [27], wherein the ATP fluorescent sensor protein is immobilized on a solid support.
[29] The fluorescent composition according to [28], wherein the ATP fluorescent sensor protein is at least two types of ATP fluorescent sensor proteins having different pairs of first and second fluorescent protein domains.

According to the present invention, a highly sensitive ligand fluorescent sensor protein can be provided regardless of the type of ligand to be detected.

The ratio of the fluorescence intensity in the presence of 10 mM ATP and the fluorescence intensity in the absence of ATP of the 19 candidate proteins of the MaLion B series ATP fluorescence sensor in Example 1 (hereinafter sometimes referred to as “dynamic range”) .) Histogram. The histogram of the dynamic range of 27 candidate proteins of the AL fluorescence sensor of MaLion G series in Example 1. MaLion R Series ATP fluorescence sensor of the histogram of the candidate protein 47 amino F / _{F 0} in the first embodiment. Excitation spectrum diagrams of MaLion B, G, and R in Example 2 in the presence and absence of 10 mM ATP. The fluorescence spectrum figure of MaLion B, G, and R in the presence and absence of 10 mM ATP in Example 2. The fluorescence spectrum figure of negMaLion B, G, and R in the presence and absence of 10 mM ATP in Example 2. The graph which shows the ATP density | concentration dependence change of the fluorescence intensity of MaLion B, G, and R in Example 2. FIG. The graph which shows the ligand specificity of the fluorescence intensity of MaLion B, G, and R in Example 2. FIG. The graph which shows the pH dependence change of the fluorescence intensity in the presence of ATP of MaLion B in Example 2, or ATP absence, and a dynamic range. The graph which shows the pH dependence change of the fluorescence intensity in the presence of ATP of MaLion G in Example 2, or ATP absence, and a dynamic range. The graph which shows the pH dependence change of the fluorescence intensity in the presence of ATP of MaLion R in Example 2, or ATP absence, and a dynamic range. The graph which shows the change of the fluorescence after administering NaF which inhibits glycolytic ATP production to the HeLa cell in which MaLion G in Example 4 was expressed. The graph which shows the change of the fluorescence after administering NaF which inhibits glycolytic ATP production to the HeLa cell in which MaLion R in Example 4 was expressed. The graph which shows the change of the fluorescence after administering NaF which inhibits glycolytic ATP production to the HeLa cell in which MaLion B in Example 4 was expressed. The graph which shows the change of the fluorescence after administering NaF which inhibits glycolytic ATP production to the HeLa cell in which negMaLion G was expressed in Example 4. FIG. The graph which shows the change of the fluorescence after administering NaF which inhibits glycolytic ATP production to the HeLa cell in which negMaLionR was expressed in Example 4. FIG. The graph which shows the change of the fluorescence after administering NaF which inhibits glycolytic ATP production to HeLa cell in which negMaLion B was expressed in Example 4. For MaLion G, R and B and negMaLion G, R and B in Example 4, the average value and standard deviation of normalized fluorescence intensity 25 minutes after the start of fluorescence measurement of each fluorescent protein expressed in HeLa cells A bar graph showing After simultaneously transfecting HeLa cells with the MaLion G expression vector and the mito-MaLion R expression vector in Example 4, fluorescence microscopy in the same field at two wavelengths corresponding to two different ATP fluorescent sensor proteins The graph which showed the change of the fluorescence after administering oligomycin 3 minutes after the imaging start. Fluorescence microscope images of B-GECO (Ca ²⁺ ions, blue), Flamindo 2 (cAMP, green) and mito-MaLion R (ATP, red) of differentiated WT-1 cells with the same visual field in Example 4. The number on the upper left of each frame represents the time (minutes) after the observation starts. The arrow indicates that 1 μM isopreterenol was added between the first frame and the second frame (5 minutes after the start of observation). The arrow indicates that 1 μM isopreterenol was added between the first frame and the second frame (5 minutes after the start of observation). Time course of normalized fluorescence intensity of B-GECO (Ca ²⁺ ions, blue), Flamindo 2 (cAMP, green) and mito-MaLion R (ATP, red) of differentiated WT-1 cells of the same visual field in Example 4 A graph showing the change. The arrow indicates that 1 μM isopreterenol was added 5 minutes after the start of observation. The graph which shows the simultaneous measurement result of the time-dependent change of the cytoplasm of the nematode pharyngeal muscle in Example 4, and the ATP density | concentration of a mitochondria. The arrow indicates that M9 buffer containing 0.5% 1-phenoxy-2-propanol as an anesthetic was administered 5 minutes after the start of observation. The graph which shows the simultaneous measurement result of the time-dependent change of the cytoplasm of the nematode pharyngeal muscle in Example 4, and the ATP density | concentration of a mitochondria. The arrow indicates that M9 buffer of the control experiment was administered 5 minutes after the start of observation. The fluorescence spectrum figure of cGull in 100 microM cGMP presence and absence in Example 5. FIG. Changes in fluorescence from the start of fluorescence microscopy imaging to 20 minutes after administration of 8-Br-cGMP (1 mM) or SNP (300 μM) of nitric oxide donor to HeLa cells expressing cGull in Example 5 An image showing. Changes in fluorescence from the start of fluorescence microscopy imaging to 20 minutes after administration of 8-Br-cGMP (1 mM) or SNP (300 μM) of nitric oxide donor to HeLa cells expressing cGull in Example 5 Graph showing. The fluorescence spectrum figure of Pink Flamindo in the presence and absence of 100 μM cAMP in Example 6. From HeLa cells expressing Pink Flamindo in Example 6 to Forskolin (100 μM) as an adenylate cyclase activator or IBMX (500 μM) as a phosphodiesterase inhibitor until 20 minutes after the start of fluorescence microscopic imaging. Image showing changes in fluorescence. From HeLa cells expressing Pink Flamindo in Example 6 to Forskolin (100 μM) as an adenylate cyclase activator or IBMX (500 μM) as a phosphodiesterase inhibitor until 20 minutes after the start of fluorescence microscopic imaging. The graph which shows the change of fluorescence. FIG. 6 is an excitation / fluorescence spectrum diagram of gBGP in the presence and absence of 100 μM BGP7C in Example 7. The graph which shows the ligand specificity and the BGP7C density | concentration dependence change of the fluorescence intensity of gBGP in Example 7. FIG. The image which shows the fluorescence in the HeLa cell which expressed gBGP or GFP in Example 7, and cell membrane localization PMmCherry-BGP7C or cell membrane localization PMmCherry. The image which shows the fluorescence in the HeLa cell which expressed gBGP or GFP in Example 7, and nuclear localization NLSmCherry-BGP7C or nuclear localization NLSmCherry. The fluorescence spectrum figure of gHSA in the presence and absence of 2 μM HSA in Example 8.

The technical scope of the present invention is limited only by the description of the scope of claims. Modifications of the present invention, for example, addition, deletion, and replacement of the configuration requirements of the present invention can be made on the condition that the gist of the present invention is not deviated.

As used herein, “protein”, “peptide”, “oligopeptide” or “polypeptide” is a compound in which two or more amino acids are linked by peptide bonds. “Protein”, “peptide”, “oligopeptide” or “polypeptide” includes amido groups, alkyl groups including methyl groups, phosphate groups, sugar chains, and / or ester bonds and other covalent modifications. There is a case. In addition, “protein”, “peptide”, “oligopeptide” or “polypeptide” is a metal ion, coenzyme, allosteric ligand or other atom, ion, atomic group, or other “protein”, “peptide”, “ When `` oligopeptides '' or `` polypeptides '', biopolymers such as sugars, lipids, nucleic acids, etc., polystyrene, polyethylene, polyvinyl, polyester or other synthetic polymers are bound or associated by covalent or non-covalent bonds There is.

In the present invention, “an amino acid sequence in which one or several amino acids are deleted, substituted or added” to a specific amino acid sequence is an amino acid sequence in which n or less amino acids have been deleted, substituted or added. Say. Here, n is an integer not exceeding 10% of the total number of amino acids in the specific amino acid sequence. That is, an amino acid sequence in which one or more and n or less amino acids are deleted, substituted or added to a specific amino acid sequence. Similarly, the N-terminus or C-terminus of any domain of the present invention is linked to the N-terminus or C-terminus of a particular protein or protein domain by “adding one to several additional amino acids via peptide bonds. "The N-terminus or C-terminus of any domain of the present invention is defined by adding one or more amino acids to the N-terminus or C-terminus of the specific protein or protein domain by peptide bonds. It is connected. The N-terminal or C-terminal of any domain of the present invention is “the first to several amino acid residues” from the N-terminal or C-terminal of a specific protein or protein domain. The N-terminus or C-terminus of any of the domains refers to the first to n-th amino acid residues from the N-terminus or C-terminus of the specific protein or protein domain.

≪Ligand fluorescence sensor protein≫
In one embodiment, the present invention relates to a ligand fluorescent sensor protein whose fluorescence characteristics change in response to a ligand specifically, wherein the ligand fluorescent sensor protein includes a first fluorescent protein domain, an N-terminal linker, , A ligand binding domain, a C-terminal linker, and a second fluorescent protein domain, wherein the fluorescent protein used in the ligand fluorescent sensor protein has a β barrel structure, and the first fluorescent protein domain is A β-sheet region of β1 to β3, an α-helix region following this, and a β-sheet region of β4 to β6 from the N-terminus of the fluorescent protein, wherein the second fluorescent protein domain is the first fluorescent protein A β-sheet region of β7 to β11 of the fluorescent protein identical to the domain, and the N-terminal The side linker and the C-terminal side linker each independently provide a ligand fluorescent sensor protein which is a polypeptide consisting of one or several amino acids.

According to the ligand fluorescent sensor protein of this embodiment, the ligand can be detected with high sensitivity regardless of the type of ligand to be detected.

The ligand fluorescence sensor protein of this embodiment changes its fluorescence characteristics in response to the ligand concentration specifically. The fluorescence characteristics include, but are not limited to, fluorescence intensity. The fluorescence intensity is limited by using a fluorescence optical instrument such as a fluorescence microscope or a fluorescence microspectrophotometer, and the spectrum of light from a predetermined excitation light source is limited to a certain wavelength band by an excitation light filter. A biological sample containing a protein or ligand fluorescent sensor negative control protein is irradiated, and the fluorescence is limited by a fluorescent filter and captured by a cooled CCD camera. A specific visual field or a part of the obtained image is captured. It is expressed quantitatively based on the luminance value of the pixels in the area.

(Ligand)
The “ligand” in the present specification means a substance that specifically binds to a specific receptor (receptor). Specific examples of the ligand include, but are not limited to, nucleotides or derivatives thereof, nucleic acids, sugar chains, proteins, lipid complexes, low molecular compounds, and the like.
The ligand fluorescent sensor protein of the present embodiment includes a receptor for the specific ligand described above, and can easily detect the distribution of the ligand concentration and the change with time in the living body with high sensitivity.

Examples of the nucleotide or derivative thereof include adenosine triphosphate (ATP), adenosine diphosphate (ADP), adenosine monophosphate (AMP), cyclic AMP (cAMP), guanosine triphosphate (GTP), and guanosine diphosphate. Examples thereof include phosphoric acid (GDP), guanosine monophosphate (GMP), cyclic GMP (cGMP), and the like, but are not limited thereto. Among these, ATP, cAMP, and cGMP are preferable as nucleotides or derivatives thereof.

ATP is an energy source for chemical reactions, transport and muscle contraction, plays a role in signal transmission, and is widely involved in the maintenance of life systems. When ATP production is reduced or ATP is consumed without being consumed, the cell loses its energy balance and disease occurs.
Moreover, cAMP is known to act as a second messenger in intracellular signal transduction during hormone transmission such as glucagon and adrenaline.
CGMP regulates ion channel conductivity, glycogen degradation, cell apoptosis, and the like. It is also involved in smooth muscle relaxation. In addition, it plays the role of a second messenger in optical transmission of eyes.
Therefore, in this embodiment, by using a ligand fluorescent sensor protein whose ligand is ATP, cAMP, or cGMP (that is, ATP fluorescent sensor protein, cAMP fluorescent sensor protein, or cGMP fluorescent sensor protein), various ligands in the cell Concentration analysis and temporal changes (spatio-temporal dynamics) can be visualized and analyzed to elucidate various life phenomena and disease onset mechanisms.

Examples of the protein include, but are not limited to, antigens, antibodies, enzymes, and the like. Among them, the protein is preferably an antigen or an antibody.
When the ligand is various biomolecules, by using an antibody against a specific biomolecule as a receptor, the concentration distribution and temporal change of the biomolecule in the living body can be detected easily and accurately regardless of the type of biomolecule. Can do.
Further, when the ligand is an antibody present in the living body, for example, by using various allergic substances as receptors, the presence of antibodies against allergic substances in humans or non-human animals can be detected easily and accurately.

Antibody includes antibody fragments. Antibody fragments include Fab, Fab ′, F (ab ′) 2, variable region fragment (Fv), disulfide bond Fv, single chain Fv (scFv), sc (Fv) 2, diabody, multispecific antibody, And polymers thereof.

Examples of the lipid complex include, but are not limited to, lipoprotein and DNA-lipid complex.

Examples of the low molecular weight compound include, but are not limited to, hydrogen ions, calcium, chlorine, oxygen and other ions, glucose, redox substances, and the like.

(Constitution)
Polypeptide domains and linkers included in the ligand fluorescent sensor protein and the ligand fluorescent sensor negative control protein of the present embodiment are as follows. First, a domain that specifically binds to a ligand is referred to as a “ligand binding domain”. The invertebrate-derived fluorescent protein disrupted by insertion or circular permutation and the amino-terminal domain and carboxyl-terminal domain of the fluorescent protein variant are hereinafter referred to as “first fluorescent protein domain” and “second fluorescent protein”. The fluorescent protein domain. A polypeptide linker linked to the amino terminus of the ligand binding domain or the first fluorescent protein domain is referred to as “N-terminal linker”, and a polypeptide linker linked to the carboxyl terminus of the ligand binding domain or the second fluorescent protein domain is referred to as “C-terminal”. Side linker ".

The ligand fluorescent sensor protein of this embodiment may contain one or two ligand binding domains.
In the ligand fluorescent sensor protein of the present embodiment, when one ligand binding domain is included, the first fluorescent protein domain, the N-terminal linker, the ligand binding domain, and the C-terminal side from the N-terminus toward the C-terminus It is preferable to include a polypeptide in which the linker and the second fluorescent protein domain are directly linked by a peptide bond in this order.

In the ligand fluorescent sensor protein of the present embodiment, when two ligand binding domains are included, from the N terminus toward the C terminus, the first ligand binding domain, the N terminal linker, the second fluorescent protein domain, It is preferable that the first fluorescent protein domain, the C-terminal linker, and the second ligand-binding domain include a polypeptide formed by directly connecting them in this order with peptide bonds. Further, a polypeptide linker may be further provided between the second fluorescent protein domain and the first fluorescent protein domain.
For example, when the ligand binding domain is an antibody, since the molecular structure is large, the heavy chain (first ligand binding domain) and the light chain (second ligand binding domain) of the antibody are combined as shown in the Examples below. By adopting a separate configuration, it is possible to prevent steric hindrance between the antibody and the fluorescent protein from being placed in the ligand fluorescent sensor protein.

Fluorescent protein The ligand fluorescent sensor protein and the ligand fluorescent sensor negative control protein of the present embodiment are artificially modified proteins based on a fluorescent protein variant derived from an invertebrate and a ligand binding domain of the ligand binding protein. Fluorescent proteins derived from invertebrates include green fluorescent protein isolated from Aequorea victoria belonging to the hydrozoa discovered by Dr. Atsushi Shimomura and others, and reef-building coral Discosoma sp. Red fluorescent protein DsRed isolated from but not limited to.

The invertebrate-derived fluorescent protein used for the ligand fluorescent sensor protein and the ligand fluorescent sensor negative control protein of the present embodiment and the polypeptide contained in the modified fluorescent protein typically have 11 β sheet structure regions. And one α helix structure region, and the α helix structure region may be referred to as the third and fourth β sheet regions from the N-terminus (hereinafter, referred to as “β3” and “β4”, respectively). Other β sheets are also expressed according to these). When the polypeptide folds, a barrel-shaped three-dimensional structure is formed. That is, the 11 β-sheet structure regions are oriented antiparallel to form a side surface of the barrel, and the α helix structure region is formed from one end of the barrel where the β-sheet structure region forming the side surface of the barrel ends. A polypeptide portion that extends through the interior of the barrel to the opposite end and leads to the next β-sheet structure region. A chromophore of an invertebrate-derived fluorescent protein and a variant of the fluorescent protein comprises two amino acid residues of the α helix region and two β sheet regions (β4 and β11 opposite to each other across the α helix). ), Each of which is formed with one amino acid residue. When an invertebrate-derived fluorescent protein and a variant of the fluorescent protein are folded to form a barrel-shaped three-dimensional structure, the four amino acid residues react spontaneously between the side chains to form a multi-membered ring. Forming a chromophore that emits fluorescence. Since the invertebrate-derived fluorescent protein and the chromophore of the modified fluorescent protein are protected from water molecules and other external environments of the fluorescent protein by the barrel-like three-dimensional structure, they can emit fluorescence at any time. . In addition, the chromophore is protected from the external environment of water molecules and other proteins by the barrel-like three-dimensional structure, and thus is less susceptible to changes in pH and other external environments.

The first fluorescent protein domain and the second fluorescent protein domain of the polypeptide included in the ligand fluorescent sensor protein and the ligand fluorescent sensor negative control protein of the present embodiment are either a specific fluorescent protein derived from any invertebrate or its It includes N-terminal and C-terminal polypeptides of the variant. Examples of the fluorescent protein used in the present embodiment include, but are not limited to, BFP, GFP, Citrine, mAapple, and the like. BFP, GFP, Citrine, and mA Apple are Wachter, R. (Biochemistry 2960, 9759-9765 (1997)), Chalfie, M. et al. (Science 263 (5148), 802-805 (1994)), Griesbeck, O. et al. (J. Biol. Chem. 276, 29188-29194 (2001)), and Shaner, N., et al. It has been reported to C et al. (Nat. Methods 5, 545-551 (2008)). In addition, BFP and mA Apple are Zhao, Y. et al. (Science. 333, 1888-1891 (2011)), amino acid substitution mutations were introduced in several places (SEQ ID NOs: 1, 2, 7 and 8).

Typically, the first fluorescent protein domain included in the ligand fluorescent sensor protein of this embodiment and the ligand fluorescent sensor negative control protein is a specific fluorescent protein derived from invertebrates or a variant thereof (for example, BFP, GFP). And β-sheet region of β1 to β3, followed by α-helical region, and β-sheet region of β4-β6. However, in some cases, one to several amino acids may be additionally linked by peptide bonds to the N-terminus or C-terminus of the first fluorescent protein domain. That is, the N-terminus of the first fluorescent protein domain may be the N-terminus of a specific fluorescent protein derived from invertebrates or a variant thereof (for example, BFP, GFP, Citriline, or mAapple). In addition, one to several amino acids may be additionally connected to the N-terminus by a peptide bond. Alternatively, the N-terminal of the first fluorescent protein domain is the first to several amino acids from the N-terminal of a specific fluorescent protein derived from invertebrates or a variant thereof (eg, BFP, GFP, Citriline, or mAapple). May be a residue.
In addition, the C-terminus of the first fluorescent protein domain may be any amino acid residue between β6 and β7. Alternatively, there may be a case where one to several amino acids are further linked by a peptide bond.

Typically, the second fluorescent protein domain contained in the ligand fluorescent sensor protein and the ligand fluorescent sensor negative control protein of the present embodiment is the β sheet region of β7 to β11 of the same fluorescent protein as the first fluorescent protein domain. including. The N-terminus of the second fluorescent protein domain may be the N-terminus of β7. Further, in some cases, one to several amino acids are additionally linked to the N-terminus of β7 by a peptide bond. Alternatively, the N-terminus of the second fluorescent protein domain may be the first to several amino acid residues from the N-terminus of β7.
In addition, the C-terminus of the second fluorescent protein domain may be the C-terminus of a specific fluorescent protein derived from invertebrates or a variant thereof (for example, BFP, GFP, Citriline, or mAple). Alternatively, one to several additional amino acids may be linked to this by a peptide bond.

Specific examples of the first fluorescent protein domain and the second fluorescent protein domain include polypeptides shown in the following (B1) and (C1).
(B1) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 1, 3, 5, or 7,
(C1) A polypeptide comprising the amino acid sequence represented by SEQ ID NO: 2, 4, 6, or 8.

The amino acid sequence represented by SEQ ID NO: 1, 3, 5, or 7 in (B1) above is the β sheet region of β1-β3 of BFP, GFP, Ciline, or mAple, followed by the α helix region, It is an amino acid sequence composed of β4 to β6 β-sheet regions.
In addition, the amino acid sequence represented by SEQ ID NO: 2, 4, 6, or 8 in (C1) above is an amino acid sequence consisting of a β sheet region of β7 to β11 of BFP, GFP, Citriline, or mApple, respectively.

In the present embodiment, the first fluorescent protein domain and the second fluorescent protein domain are the same as the polypeptides of (B1) and (C1) described above as functionally equivalent polypeptides of (B2) and (C2) below. Contains the polypeptide.
(B2) the amino acid sequence represented by SEQ ID NO: 1, 3, 5, or 7, comprising an amino acid sequence in which one or several amino acids are deleted, inserted, substituted or added, and the second fluorescence A polypeptide that forms a β-barrel structure with a protein domain and fluoresces,
(C2) the amino acid sequence represented by SEQ ID NO: 2, 4, 6, or 8, comprising an amino acid sequence in which one or several amino acids are deleted, inserted, substituted or added, and the first fluorescence A polypeptide that forms a β-barrel structure with a protein domain and emits fluorescence.

Here, the number of amino acids that may be deleted, substituted, or added is preferably 1 to 15, more preferably 1 to 10, and particularly preferably 1 to 5.

In the present embodiment, the first fluorescent protein domain and the second fluorescent protein domain are the same as the polypeptides of (B1) and (C1) described above as functionally equivalent polypeptides of the following (B3) and (C3). Contains the polypeptide.
(B3) including an amino acid sequence having an identity of 80% or more with the amino acid sequence represented by SEQ ID NO: 1, 3, 5, or 7, and forming a β barrel structure with the second fluorescent protein domain, A fluorescent polypeptide,
(C3) comprising an amino acid sequence having 80% or more identity with the amino acid sequence represented by SEQ ID NO: 2, 4, 6, or 8, and forming a β barrel structure with the first fluorescent protein domain, A fluorescent polypeptide.

In order to be functionally equivalent to the polypeptides (B1) and (C1), they have 80% or more identity. Such identity is preferably 85% or more, more preferably 90% or more, still more preferably 95% or more, particularly preferably 98% or more, and most preferably 99% or more.

When BFP, GFP, Citrine, or mAapple is used as the fluorescent protein, the first fluorescent protein domain and the second fluorescent protein domain are “BFP-N domain”, “BFP-C domain”, “GFP-”, respectively. It may be referred to as “N domain” and “GFP-C domain”, “Citrine-N domain” and “Citrine-C domain”, or “mAapple-N domain” and “mAapple-C domain”. The amino acid sequences of the BFP-N domain and the BFP-C domain are listed in the sequence listing as SEQ ID NOs: 1 and 2. The amino acid sequences of the GFP-N domain and the GFP-C domain are listed in the sequence listing as SEQ ID NOs: 3 and 4. The amino acid sequences of the Citrine-N domain and the Citrine-C domain are listed as SEQ ID NOs: 5 and 6. The amino acid sequences of the mApple-N domain and the mApple-C domain are listed as SEQ ID NOs: 7 and 8.

-Ligand binding domain Using a fluorescent protein derived from an invertebrate and a modified version of the fluorescent protein, in order to create a fluorescent sensor protein whose fluorescence characteristics change in response to changes in the concentration of the ligand, An attempt is made to insert a part of a polypeptide of another protein (that is, a ligand binding domain) that binds to the ligand into the polypeptide linking the β sheet structural region. This is because it is expected that the fluorescence characteristics change because the barrel-like three-dimensional structure of the fluorescent protein is disturbed by the influence of the substance.

The ligand binding domain is not particularly limited as long as a specific ligand can bind specifically. For example, when the ligand is a nucleotide such as ATP, cAMP, or cGMP or a derivative thereof, a known binding domain of a nucleotide such as ATP, cAMP, or cGMP or a derivative thereof may be used as the ligand binding domain. For example, when the ligand is an arbitrary biomolecule, an antibody against the biomolecule may be used as the ligand binding domain. For example, when the ligand is an antibody, the antigen may be used as the ligand binding domain. Further, for example, when the ligand is a low molecular compound such as hydrogen ion, calcium, chlorine, oxygen or other ions, glucose, redox substance, etc., the low molecular compound receptor (specifically, When the ligand is glucose, a glucose binding site or the like in the glucose transporter may be used as the ligand binding domain.

More specifically, as the ligand binding domain, for example, when the ligand is ATP, examples of the ATP binding domain include the ε subunit of F ₀ F ₁ -ATP synthase. The ε subunit may be the ε subunit of bacterial F ₀ F ₁ -ATP synthase, or the ε subunit of Bacillus subtilis F ₀ F ₁ -ATP synthase. The N-terminus of the ATP binding domain may be the N-terminus of the ε subunit of F ₀ F ₁ -ATP synthase. Alternatively, the N-terminus of the ATP-binding domain may be the first or several amino acid residues from the N-terminus of the ε subunit of F ₀ F ₁ -ATP synthase. The C-terminus of the ATP binding domain may be the C-terminus of the ε subunit of F ₀ F ₁ -ATP synthase. Alternatively, the C terminus of the ATP binding domain may be the first or several amino acid residues from the C terminus of the ε subunit of F ₀ F ₁ -ATP synthase. The ATP binding domain may be the amino acid sequence of SEQ ID NO: 15.

In addition, for example, when the ligand is cAMP, examples of the cAMP binding domain include exchange factor activated by cAMP 1 (EPAC1). The EPAC1 may be derived from a human or a non-human animal. The N-terminus of the cAMP binding domain may be the N-terminus of EPAC1. Alternatively, the N-terminus of the cAMP-binding domain may be the first or several amino acid residues from the N-terminus of EPAC1. The C terminus of the cAMP binding domain may be the C terminus of EPAC1. Alternatively, the C-terminus of the cAMP binding domain may be the first or several amino acid residues from the C-terminus of EPAC1. The cAMP binding domain may be the amino acid sequence of SEQ ID NO: 20.

Also, for example, when the ligand is cGMP, examples of the cGMP binding domain include phosphodiesterase 5α (PDE5α). The PDE5α may be derived from a human or a non-human animal. The N-terminus of the cGMP binding domain may be the N-terminus of PDE5α. Alternatively, the N-terminus of the cGMP binding domain may be the first or several amino acid residues from the N-terminus of PDE5α. The C-terminus of the cGMP binding domain may be the C-terminus of PDE5α. Alternatively, the C-terminus of the cGMP binding domain may be the first or several amino acid residues from the C-terminus of PDE5α. The cGMP binding domain may be the amino acid sequence of SEQ ID NO: 21.

Also, for example, when the ligand is osteocalcin (bonecalcine (BGP)), examples of the BGP binding domain include an anti-BGP antibody. The anti-BGP antibody may be derived from a human or a non-human animal. When two BGP binding domains are included, the N-terminus of the first BGP binding domain may be the N-terminus of the heavy chain of the anti-BGP antibody, and the N-terminus of the second BGP binding domain may be the anti-BGP antibody's N-terminus. It may be the N-terminus of the light chain. Alternatively, the N-terminus of the first BGP binding domain may be the first or several amino acid residues from the N-terminus of the heavy chain of the anti-BGP antibody, and the N-terminus of the second BGP binding domain May be the first or several amino acid residues from the N-terminus of the light chain of the anti-BGP antibody. The C-terminus of the first BGP binding domain may be the C-terminus of the heavy chain of the anti-BGP antibody, and the N-terminus of the second BGP binding domain may be the C-terminus of the light chain of the anti-BGP antibody. Good. Alternatively, the C-terminus of the first BGP binding domain may be the first or several amino acid residues from the C-terminus of the heavy chain of the anti-BGP antibody, and the C-terminus of the second BGP binding domain May be the first or several amino acid residues from the C-terminus of the light chain of the anti-BGP antibody. The first BGP binding domain may be the amino acid sequence of SEQ ID NO: 22, and the second BGP binding domain may be the amino acid sequence of SEQ ID NO: 23.

Linker In the polypeptide included in the ligand fluorescent sensor protein and the ligand fluorescent sensor negative control protein of the present embodiment, the polypeptide binding domain is a polypeptide linker consisting of one to several amino acids on the N-terminal side and C-terminal side, respectively. (Hereinafter, they may be referred to as “N-terminal side linker” and “C-terminal side linker”, respectively.) Are inserted between the first and second fluorescent protein domains. Alternatively, the second and first fluorescent protein domains are inserted between the first and second ligand binding domains via their N-terminal linker and C-terminal linker.
The amino acid sequences of the N-terminal linker and the C-terminal linker are different for each ligand fluorescent sensor protein and ligand fluorescent sensor negative control protein of this embodiment. However, amino acids may allow conservative substitutions.

In the present specification, “conservative amino acid substitution” means that a certain amino acid residue is substituted with an amino acid residue having a side chain of similar properties. Depending on the side chain of the amino acid residue, a basic side chain (eg, lysine, arginine, histidine), an acidic side chain (eg, aspartic acid, glutamic acid), an uncharged polar side chain (eg, asparagine, glutamine, serine, threonine) , Tyrosine, cysteine), nonpolar side chains (eg glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-branched side chains (eg threonine, valine, isoleucine), aromatic side chains (For example, tyrosine, phenylalanine, tryptophan) and the like. A conservative amino acid substitution is preferably a substitution between amino acid residues within the same family.
Therefore, the amino acid sequence of the C-terminal linker of the ligand fluorescent sensor protein of this embodiment has the same ligand binding ability and fluorescence characteristics even if one or more amino acids differ from the amino acid sequence specified in the following examples. Such an amino acid sequence is also included in the amino acid sequence of the C-terminal linker of the ligand fluorescent sensor protein of the present embodiment.

(Suitable ligand fluorescent sensor protein)
Among the ATP fluorescent sensor protein and the ATP fluorescent sensor negative control protein in the present embodiment, those using the BFP-N domain and the BFP-C domain as the first fluorescent protein domain and the second fluorescent protein domain are referred to as “MaLion B series”. "
Among the ATP fluorescent sensor protein and the ATP fluorescent sensor negative control protein in the present embodiment, those using the Citrine-N domain and the Citrine-C domain as the first fluorescent protein domain and the second fluorescent protein domain are referred to as “MaLion G series. "
Among the ATP fluorescent sensor protein and the ATP fluorescent sensor negative control protein in the present embodiment, those using the mApple-N domain and the mApple-C domain as the first fluorescent protein domain and the second fluorescent protein domain are referred to as “MaLion R series”. "
Among the cGMP fluorescent sensor proteins in this embodiment, those using the Citrine-N domain and the Citrine-C domain as the first fluorescent protein domain and the second fluorescent protein domain are referred to as “cGull series”.
Among the cAMP fluorescent sensor proteins in the present embodiment, those using the mApple-N domain and the mApple-C domain as the first fluorescent protein domain and the second fluorescent protein domain are referred to as “Pink Flamen series”.
Among the BGP fluorescent sensor proteins in this embodiment, those using the GFP-N domain and the GFP-C domain as the first fluorescent protein domain and the second fluorescent protein domain are referred to as “gBGP series”.

As will be described in the following examples, the ligand fluorescent sensor protein and the ligand fluorescent sensor negative control protein of the present embodiment have a BFP-N domain and a BFP-C domain as the first and second fluorescent protein domains, GFP- Ε of Bacillus subtilis F ₀ F ₁ -ATP synthase is used as an ATP-binding domain using N domain and GFP-C domain, Citrine-N domain and Citrine-C domain, mAple-N domain and mApple-C domain Using subunits, PDE5α as cGMP binding domain, EPAC1 as cAMP binding domain, or anti-BGP antibody as BGP binding domain, using various polypeptide combinations as N-terminal linker and C-terminal linker, Polynucleotides encoding candidate molecules of MaLion B, G and R series protein, cGull series protein, Pink Flamindo series protein, and gBGP series protein are synthesized, incorporated into an E. coli expression vector, and expressed in E. coli. By analyzing the fluorescence characteristics, based on the ratio of the fluorescence intensity in the presence of the ligand and in the absence of the ligand (dynamic range), the candidate molecules with the highest dynamic range are determined as MaLion B, G and R series, cGull series, The turn-on ligand fluorescent sensor protein of the Pink Flamingo series and the BGP fluorescent sensor series (hereinafter referred to as “MaLion B”, “MaLion G”, “MaLion R”, “c”, respectively) ull ", was selected as the" Pink Flamindo ", and is sometimes referred to as" gBGP ".). Further, candidate molecules having the dynamic range closest to 1 were selected as MaLion B, G and R series ATP fluorescent sensor negative control proteins (referred to as “negMaLion B”, “negMaLion G” and “negMaLion R”, respectively).

Relationship between the amino acid sequence identified by SEQ ID No. in the sequence listing referred to in the present specification and claims, and the ligand fluorescent sensor protein and the ligand fluorescent sensor negative control protein of the present embodiment, or these domains and linkers Is as shown in the table below.

More specifically, examples of the ligand fluorescent sensor protein of the present embodiment include the following polypeptide (D1).
(D1) A polypeptide comprising the amino acid sequence represented by any of SEQ ID NOs: 9 to 14.

The amino acid sequence represented by SEQ ID NO: 9 in the above (D1) is [BFP-N domain]-[N-terminal side linker]-[ATP binding domain]-[C-terminal side linker in the direction from the amino terminus to the carboxyl terminus. ]-[BFP-C domain] in this order, a polypeptide domain and a polypeptide linker are linked.
The amino acid sequence represented by SEQ ID NO: 10 in the above (D1) is [Citrine-N domain]-[N-terminal side linker]-[ATP-binding domain]-[C-terminal side linker in the direction from the amino terminus to the carboxyl terminus. ]-[Citrine-C domain] in this order, a polypeptide domain and a polypeptide linker are linked.
The amino acid sequence represented by SEQ ID NO: 11 in the above (D1) has a [mAapple-N domain]-[N-terminal side linker]-[ATP binding domain]-[C-terminal side linker in the direction from the amino terminus to the carboxyl terminus. ]-[MApple-C domain] in this order, a polypeptide domain and a polypeptide linker are linked.
The amino acid sequence represented by SEQ ID NO: 12 in the above (D1) is [Citrine-N domain]-[N-terminal side linker]-[cGMP binding domain]-[C-terminal side linker in the direction from the amino terminus to the carboxyl terminus. ]-[Citrine-C domain] in this order, a polypeptide domain and a polypeptide linker are linked.
The amino acid sequence represented by SEQ ID NO: 13 in the above (D1) is [mapple-N domain]-[N terminal linker]-[cAMP binding domain]-[C terminal linker in the direction from the amino terminus to the carboxyl terminus. ]-[MApple-C domain] in this order, a polypeptide domain and a polypeptide linker are linked.
The amino acid sequence represented by SEQ ID NO: 14 in the above (D1) is [anti-BGP antibody heavy chain]-[N-terminal linker]-[GFP-C domain]-[peptide in the direction from the amino terminus to the carboxyl terminus. In this structure, a polypeptide domain and a polypeptide linker are linked in the order of linker]-[GFP-N domain]-[C-terminal side linker]-[light chain of anti-BGP antibody].

The ligand fluorescent sensor protein in the present embodiment contains the following polypeptide (D2) as a polypeptide functionally equivalent to the polypeptide (D1).
(D2) an amino acid sequence represented by any one of SEQ ID NOs: 9 to 14, including an amino acid sequence in which one or several amino acids are deleted, inserted, substituted or added, and the polypeptide (D1) Polypeptides having the same ability to bind to a ligand and fluorescent properties.

Here, the number of amino acids that may be deleted, substituted, or added is preferably 1 to 15, more preferably 1 to 10, and particularly preferably 1 to 5.

The ligand fluorescent sensor protein in the present embodiment contains the following polypeptide (D3) as a polypeptide functionally equivalent to the polypeptide (D1).
(D3) Ligand binding ability and fluorescence that include the amino acid sequence that is 80% or more identical to the amino acid sequence represented by any of SEQ ID NOs: 9 to 14 and that are the same as the polypeptide (D1) A polypeptide having properties.

In order to be functionally equivalent to the polypeptide (D1), it has 80% or more identity. Such identity is preferably 85% or more, more preferably 90% or more, still more preferably 95% or more, particularly preferably 98% or more, and most preferably 99% or more.

(Other configurations)
Cell membrane permeable peptide The ligand fluorescent sensor protein of the present embodiment or the ligand fluorescent sensor negative control protein of the present embodiment penetrates the cell membrane and reaches the inside of the cell. Literature: Zorko, M. and Langel, U., Adv. Drug Deliv. Rev. 57, p529-545, 2005.). Examples of the cell membrane-penetrating peptide include, but are not limited to, peptides consisting of amino acid sequences represented by SEQ ID NOs: 38 to 41, and the like.
The amino acid sequence represented by SEQ ID NO: 38 is the amino acid sequence from the 48th to the 60th amino acid sequence of the HIV-1 virus tat protein.
The amino acid sequence represented by SEQ ID NO: 39 is the amino acid sequence from the 339th to the 354th amino acid sequence (the amino acid sequence from the 43rd to the 58th amino acid of penetratin) of the Drosophila Antennapedia protein.
The peptide in which the C-terminal of the peptide having the amino acid sequence represented by SEQ ID NO: 40 is amidated can permeate the cell membrane and localize in the cell. This amino acid sequence is the amino acid sequence from the 616th to the 633th of the mouse VE cadherin precursor protein.
The amino acid sequence represented by SEQ ID NO: 41 is a 7-mer homooligomer of arginine.

Cell membrane localization peptide The ligand fluorescence sensor protein of this embodiment or the ligand fluorescence sensor negative control protein of this embodiment contains a cell membrane localization peptide well known in the art for localization on the cell membrane. You can go out.
Examples of the cell membrane localized peptide include, but are not limited to, a peptide consisting of the amino acid sequence represented by SEQ ID NO: 42.
The amino acid sequence represented by SEQ ID NO: 42 is disclosed in Zuber, M .; X. (Nature, vol. 341, p345-348, 1989.) is the amino acid sequence from the first to the 21st amino acid sequence of the cell membrane localization peptide derived from human neuroprotein GAP-43 (Neuromodulin).

Organelle localization signal peptide The ligand fluorescence sensor protein of this embodiment or the ligand fluorescence sensor negative control protein of this embodiment is an organelle localization signal that is well known in the art for localizing to a specific organelle. It may contain a peptide. Examples of the organelle include, but are not limited to, mitochondria, nucleus, nucleolus, endoplasmic reticulum, chloroplast, peroxisome, and bacterial periplasm.

Examples of the nuclear localization signal peptide include, but are not limited to, a peptide consisting of the amino acid sequence represented by SEQ ID NO: 43.
The amino acid sequence represented by SEQ ID NO: 43 is the amino acid sequence from the 126th to the 132nd amino acid sequence of the SV40 virus T antigen (Lanford, RE, et al., Cell, vol. 46, p575-582). 1986.).

Examples of the mitochondrial localization signal peptide include, but are not limited to, peptides consisting of the amino acid sequences represented by SEQ ID NOs: 44 and 45, and the like.
The amino acid sequence represented by SEQ ID NO: 44 is the amino acid sequence from the 2nd to the 29th amino acid sequence of the human cytochrome c oxidase subunit VIII-liver / heart type (8A subunit or 8-2 subunit). It is.
The amino acid sequence represented by SEQ ID NO: 45 is the 2nd to 24th amino acid sequences of chicken trispartate aminotransferase (Jausi, R. et al., J. Biol Chem., Vol. 260, p16060). -16063, 1985.).

Examples of the endoplasmic reticulum localization signal peptide include, but are not limited to, peptides consisting of the amino acid sequences represented by SEQ ID NOs: 46 and 47, and the like.
The amino acid sequence represented by SEQ ID NO: 46 is the first to 17th amino acid sequence of the human skeletal muscle sarcoplasmic reticulum high affinity calcium binding protein calreticulin (Fliegel, L. et al). , J Biol Chem., Vol. 264, no. 36, p21522-21528, 1989.).
The amino acid sequence represented by SEQ ID NO: 47 is the amino acid sequence from the 651st to the 654th (C-terminal) of the endoplasmic reticulum membrane localization signal peptide of rat grp78 protein (Munro, S. and Pelham HR). , Cell, vol. 48, no. 5, p899-907, 1987.).

Among them, the ligand fluorescent sensor protein of the present embodiment or the ligand fluorescent sensor negative control protein of the present embodiment preferably includes a nuclear localization signal peptide or a mitochondrial localization signal peptide.

<ATP fluorescence sensor protein>
In one embodiment, the present invention provides an ATP fluorescent sensor protein that changes its fluorescence characteristics in response to ATP concentration, wherein the ATP fluorescent sensor protein is a first protein from the N-terminus toward the C-terminus. A polypeptide comprising a fluorescent protein domain, an N-terminal linker, an ATP-binding domain, a C-terminal linker, and a second fluorescent protein domain directly linked by a peptide bond in this order. One fluorescent protein domain includes a β sheet region of β1 to β3, an α helix region following this, and a β sheet region of β4 to β6, from the N-terminus of the fluorescent protein BFP, Citriline or mAapple, The fluorescent protein domain is a β sheet region of β7 to β11 of the same fluorescent protein as the first fluorescent protein domain Includes, ATP fluorescence ATP-binding domain consists of ε-subunit of F ₀ F ₁ -ATP synthase, N-terminal linker and C-terminal linkers are each polypeptide consisting of one or several amino acids Provide a sensor protein.

According to the ATP fluorescence sensor protein of the present embodiment, the ratio of the fluorescence intensity in the presence of high concentration of ATP to the fluorescence intensity in the absence of ATP is sufficiently high under physiological conditions, Alternatively, pathological changes in ATP concentration can be detected. In addition, by using a plurality of types of ATP fluorescent sensor proteins of the present embodiment having different excitation wavelengths and emission wavelengths, each of them is localized in different organelles of the same cell, or is localized in different cells in the organism, and fluorescence microscope Detect optically the temporal and / or spatial change (spatio-temporal dynamics) of ATP concentration associated with physiological and / or pathological changes of the cells or organisms simultaneously or almost simultaneously in the same visual field Technology can be provided.

In the ATP fluorescence sensor protein of the present embodiment, the polypeptide is
(A11) The ATP binding domain is the amino acid sequence of SEQ ID NO: 15, the first and second fluorescent protein domains are the amino acid sequences of SEQ ID NOS: 1 and 2, respectively, and the N-terminal and C-terminal linkers are MaLion B polypeptides, which are the amino acid sequences of SEQ ID NOs: 16 and 17, respectively;
(A12) The ATP-binding domain, the first fluorescent protein domain, the second fluorescent protein domain, the N-terminal linker, and the C-terminal linker are each independently SEQ ID NOs: 15, 1, 2, 16, and 17 amino acid sequences or amino acid sequences in which one or several amino acids are deleted, substituted or added to the amino acid sequences of SEQ ID NOs: 13, 1, 2, 14, and 15, and MaLion B poly A MaLion B polypeptide having the same ATP binding ability and fluorescence properties as the peptide (A11);
(B11) The ATP-binding domain is the amino acid sequence of SEQ ID NO: 15, the first and second fluorescent protein domains are the amino acid sequences of SEQ ID NOs: 5 and 6, respectively, and the N-terminal and C-terminal linkers are MaLion G polypeptide, which is the amino acid sequence of WRG (Trp-Arg-Gly) and SEQ ID NO: 18, respectively,
(B12) The ATP binding domain, the first fluorescent protein domain, the second fluorescent protein domain, the N-terminal side linker and the C-terminal side linker are each independently SEQ ID NO: 15, 5, 6, WRG (Trp- Arg-Gly) and the amino acid sequence of SEQ ID NO: 18, or SEQ ID NOs: 15, 5, 6, WRG (Trp-Arg-Gly) and the amino acid sequence of SEQ ID NO: 18 have one or several amino acids deleted, A MaLion G polypeptide that is a substituted or added amino acid sequence and has the same ATP binding ability and fluorescence characteristics as the MaLion G polypeptide (B11);
(C11) The ATP binding domain is the amino acid sequence of SEQ ID NO: 15, the first and second fluorescent protein domains are the amino acid sequences of SEQ ID NOs: 7 and 8, respectively, and the N-terminal and C-terminal linkers are MaLion R polypeptide, which is the amino acid sequence of SEQ ID NO: 19 and PEE (Pro-Glu-Glu), respectively
(C12) The ATP binding domain, the first fluorescent protein domain, the second fluorescent protein domain, the N-terminal linker and the C-terminal linker are SEQ ID NOs: 15, 7, 8, 19, and PEE (Pro -Glu-Glu) amino acid sequence or SEQ ID NOs: 15, 7, 8, 19 and amino acid sequence of PEE (Pro-Glu-Glu) with one or several amino acids deleted, substituted or added It is preferably a sequence and includes at least one polypeptide selected from the group consisting of MaLion R polypeptides having the same ATP binding ability and fluorescence characteristics as MaLion R polypeptides (C11).

In the ATP fluorescence sensor protein of the present embodiment, the polypeptide is
(A21) a MaLion B polypeptide consisting of the amino acid sequence of SEQ ID NO: 9,
(A22) The amino acid sequence of SEQ ID NO: 9 consists of an amino acid sequence in which one or several amino acids are deleted, substituted or added to the amino acid sequence excluding the amino acid sequences of SEQ ID NOS: 16 and 17, and MaLion B A MaLion B polypeptide having the same ATP binding ability and fluorescence properties as the polypeptide (A21);
(B21) a MaLion G polypeptide consisting of the amino acid sequence of SEQ ID NO: 10,
(B22) one of amino acid sequences excluding the amino acid sequence of WRG (Trp-Arg-Gly) at positions 146 to 148 of SEQ ID NO: 10 and the amino acid sequence of SEQ ID NO: 18 among the amino acid sequences of SEQ ID NO: 10 or MaLion G polypeptide consisting of an amino acid sequence in which several amino acids are deleted, substituted or added, and having the same ATP binding ability and fluorescence characteristics as MaLion G polypeptide (B21);
(C21) a MaLion R polypeptide consisting of the amino acid sequence of SEQ ID NO: 11,
(C22) One of the amino acid sequences of SEQ ID NO: 11 excluding the amino acid sequence of PEE (Pro-Glu-Glu) at positions 288 to 290 of SEQ ID NO: 11 and the amino acid sequence of SEQ ID NO: 19 or It is selected from the group consisting of an MaLion R polypeptide consisting of an amino acid sequence in which several amino acids are deleted, substituted or added, and having the same ATP binding ability and fluorescence characteristics as MaLion R polypeptide (C21). More preferably, it comprises at least one polypeptide.

The ATP fluorescent sensor protein of this embodiment may further contain an organelle localization signal peptide. Examples of the organelle localization signal peptide include the same as those exemplified above (other configurations). Among them, the organelle localization signal peptide is preferably a nuclear localization signal peptide or a mitochondrial localization signal peptide.
The ATP fluorescent sensor protein of this embodiment may further contain a cell membrane permeable peptide.

<ATP fluorescence sensor negative control protein>
In one embodiment, the present invention provides ATP fluorescence sensor negative control proteins negMaLion B, G and R.

The ATP fluorescence sensor negative control protein of the present embodiment includes a first fluorescent protein domain, an N-terminal linker, an ATP-binding domain, a C-terminal linker, and a second fluorescence from the N-terminus toward the C-terminus. A polypeptide comprising a protein domain directly linked by a peptide bond in this order, the polypeptide comprising:
(A31) The ATP binding domain is the amino acid sequence of SEQ ID NO: 15, the first and second fluorescent protein domains are the amino acid sequences of SEQ ID NOS: 1 and 2, respectively, and the N-terminal and C-terminal linkers are A negMaLion B polypeptide, which is the amino acid sequence of SEQ ID NOs: 27 and 28, respectively;
(A32) The ATP binding domain, the first fluorescent protein domain, the second fluorescent protein domain, the N-terminal linker and the C-terminal linker are each independently SEQ ID NOs: 15, 1, 2, 27, and 28. Or an amino acid sequence in which one or several amino acids are deleted, substituted, or added to the amino acid sequences of SEQ ID NOs: 15, 1, 2, 27, and 28, and the negMaLion B polypeptide ( A negMaLion B polypeptide having the same ATP binding ability and fluorescence properties as A31);
(B31) The ATP-binding domain is the amino acid sequence of SEQ ID NO: 15, the first and second fluorescent protein domains are the amino acid sequences of SEQ ID NOs: 5 and 6, respectively, and the N-terminal and C-terminal linkers are Respectively, PRG (Pro-Arg-Gly) and the negMaLion G polypeptide which is the amino acid sequence of SEQ ID NO: 29;
(B32) The ATP-binding domain, the first fluorescent protein domain, the second fluorescent protein domain, the N-terminal side linker and the C-terminal side linker are each independently SEQ ID NO: 15, 5, 6, PRG (Pro- Arg-Gly) and the amino acid sequence of SEQ ID NO: 29, or SEQ ID NOs: 15, 5, 6, PRG (Pro-Arg-Gly) and the amino acid sequence of SEQ ID NO: 29 have one or several amino acids deleted, A polypeptide having a substituted or added amino acid sequence and having the same ATP binding ability and fluorescence characteristics as the negMaLion G polypeptide (B31);
(C31) The ATP binding domain is the amino acid sequence of SEQ ID NO: 15, the first and second fluorescent protein domains are the amino acid sequences of SEQ ID NOs: 7 and 8, respectively, and the N-terminal and C-terminal linkers are A negMaLion R polypeptide that is the amino acid sequence of SEQ ID NO: 30 and PEG (Pro-Glu-Gly), respectively;
(C32) The ATP-binding domain, the first fluorescent protein domain, the second fluorescent protein domain, the N-terminal side linker and the C-terminal side linker are each independently SEQ ID NO: 15, 7, 8, 30 and PEG One or several amino acids are deleted, substituted or added to the amino acid sequence of (Pro-Glu-Gly) or the amino acid sequences of SEQ ID NOs: 15, 7, 8, 30 and PEG (Pro-Glu-Gly). And at least one polypeptide selected from the group consisting of polypeptides having the same ATP binding ability and fluorescence characteristics as the negMaLion R polypeptide (C31).

The ATP fluorescence sensor negative control protein of this embodiment is
(A41) a negMaLion B polypeptide consisting of the amino acid sequence of SEQ ID NO: 24;
(A42) The amino acid sequence of SEQ ID NO: 24 consists of an amino acid sequence in which one or several amino acids are deleted, substituted or added to the amino acid sequence excluding the amino acid sequences of SEQ ID NO: 27 and 28, and negMaLion B A polypeptide having the same ATP binding ability and fluorescence characteristics as the polypeptide (A41);
(B41) a negMaLion G polypeptide consisting of the amino acid sequence of SEQ ID NO: 25;
(B42) Among the amino acid sequences of SEQ ID NO: 25, one or several amino acids are included in the amino acid sequence excluding SEQ ID NO: 29 and the amino acid sequence of PRG (Pro-Arg-Gly) at positions 146-148 of SEQ ID NO: 25 A negMaLion G polypeptide consisting of an amino acid sequence in which an amino acid is deleted, substituted or added, and having the same ATP binding ability and fluorescence characteristics as the negMaLion G polypeptide (B41);
(C41) a negMaLion R polypeptide consisting of the amino acid sequence of SEQ ID NO: 26;
(C42) Of the amino acid sequence of SEQ ID NO: 26, one amino acid sequence excluding the amino acid sequence of SEQ ID NO: 30 and the amino acid sequence of PEG (Pro-Glu-Gly) at positions 288-290 of SEQ ID NO: 26, or At least one selected from the group consisting of an amino acid sequence in which several amino acids have been deleted, substituted or added, and a polypeptide having the same ATP binding ability and fluorescence characteristics as the negMaLion R polypeptide (C41) More preferably, it comprises two polypeptides.

The ATP fluorescent sensor negative control protein of this embodiment may further contain an organelle localization signal peptide. Examples of the organelle localization signal peptide include the same as those exemplified above (other configurations). Among them, the organelle localization signal peptide is preferably a nuclear localization signal peptide or a mitochondrial localization signal peptide.
The ATP fluorescent sensor negative control protein of the present embodiment may further contain a cell membrane permeable peptide.

(Use)
-Fluorescent composition In one embodiment, the present invention provides a fluorescent composition comprising the ATP fluorescent sensor protein described above.

In the fluorescent composition of the present embodiment, the ATP fluorescent sensor protein described above may be immobilized on a solid support.

The shape of the solid support is not particularly limited, and examples thereof include a flat plate shape and a spherical shape.

Examples of the material of the solid support include silica, alumina, glass, metal and the like as inorganic substances. Moreover, a thermoplastic resin etc. are mentioned as an organic polymer substance.

More specifically, examples of the solid support include carriers (for example, magnetic carriers, affinity column purification carriers, etc.), cell culture substrates, preparations, microdevices, membranes, and the like. Examples of the substrate for cell culture include a multiwell plate, a petri dish and the like in which an arbitrary number of wells are arranged. Examples of the number of wells include 6, 12, 24, 96, 384, 1,536, etc., per plate.

In the fluorescent composition of the present embodiment, the ATP fluorescent sensor protein described above may be an ATP fluorescent sensor protein having at least two different excitation wavelengths and fluorescent wavelengths of the first and second fluorescent protein domain pairs.
As shown in the examples below, by using a plurality of types of ATP fluorescence sensor proteins having different fluorescence wavelengths, the ATP fluorescence sensor proteins are localized in different organelles, or are localized in different cells in the organism, Optical detection of temporal and / or spatial change (spatiotemporal dynamics) of ATP concentration associated with physiological and / or pathological changes of the cells or organisms detected simultaneously or nearly simultaneously in the same field of view of a fluorescence microscope Can be analyzed.

In addition, as a method of using the fluorescent composition of this embodiment, when the ATP fluorescent sensor protein is immobilized on the wall surface of the container, a solution containing ATP or a non-hydrolyzable ATP analog such as ATPγS Can be irradiated as an excitation light source with a long-wavelength ultraviolet light source that is slightly visible, such as black light. Alternatively, a solid support such as beads immobilizing the ATP fluorescent sensor protein can be suspended in a solution containing ATP or a non-hydrolyzable ATP analog and flowed through the container or flow path. For example, the container is shaped like a champagne glass, the containers are stacked in multiple layers, and the solution overflowing from the uppermost container is poured into the container in the layer immediately below by pouring the solution into the container on the top. The so-called champagne tower or trickle-down state in which the solution overflowing from the container of the layer immediately below is poured into the container of the layer below has a high visual effect by irradiating excitation light sources of various angles and timings. Fluorescent illumination can be performed.

≪Polynucleotide≫
In one embodiment, the present invention provides a polynucleotide encoding the above-described ligand fluorescent sensor protein.

Examples of the polynucleotide encoding the ligand fluorescent sensor protein include, for example, a polynucleotide comprising the base sequence represented by SEQ ID NO: 48 or 49, or 80% or more of the base sequence represented by SEQ ID NO: 48 or 49, for example, Examples thereof include a polynucleotide comprising a base sequence encoding a polypeptide having 85% or more, for example, 90% or more, for example, 95% or more, and having a ligand binding ability and fluorescence characteristics. The base sequence represented by SEQ ID NO: 48 or 49 is the base sequence of a polynucleotide encoding a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 12 or 13.

In the polynucleotide of this embodiment, codon selection may be optimized for expression in mammals, plants, and / or nematodes.

In the present specification, “the codon selection is optimized for expression in mammals, plants, and / or nematodes” means the frequency of codon usage in mammals, plants, and / or nematodes and / or Corresponding to the difference in the intracellular concentration of the aminoacyl-t-RNA molecular species, the nucleotide sequence of the polynucleotide encoding the desired protein whose codon was selected so as to have the highest translation efficiency in each biological species Means to design. Optimization of codon selection is described in, for example, Gustafsson, C.I. (Trends in biotechnology 22.7 (2004): 346-353) and the like.

≪Expression vector≫
In one embodiment, the present invention provides an expression vector comprising the polynucleotide described above.

The “expression vector” in the present specification refers to a polynucleotide encoding the protein, transcription of the polynucleotide, RNA splicing, RNA processing, RNA maturation, so that a desired protein can be expressed in a host cell. It means a vector containing a nucleic acid including, but not limited to, a promoter, enhancer, terminator and ribosome binding site necessary for translation, post-translational processing and other functions.

(Type of expression vector)
The expression vector for expression in mammals is not particularly limited. For example, plasmids derived from E. coli such as pBR322, pBR325, pUC12, and pUC13; plasmids derived from Bacillus subtilis such as pUB110, pTP5, and pC194; yeasts such as pSH19 and pSH15 Origin plasmids; bacteriophages such as λ phage; viruses such as adenoviruses, adeno-associated viruses, lentiviruses, vaccinia viruses, baculoviruses; and modified vectors thereof can be used.
The expression vector for expression in C. elegans is not particularly limited, and examples thereof include Okema, P. et al. G. And Andrew F. A pOK vector reported by (Development 120, 2175-2186 (1994)) can be used.
The expression vector for expression in plants is not particularly limited, and for example, viruses such as tobacco mosaic virus and cucumber mosaic virus, vectors obtained by modifying these viruses, and the like can be used.

(Structure of expression vector)
-Construction of an expression vector for expression in mammals An expression vector for expression in mammals may contain, for example, a promoter operably linked to a polynucleotide encoding the ligand fluorescent sensor protein.

As used herein, “operably linked” refers to a gene expression control sequence (for example, a promoter or a series of transcription factor binding sites) and a gene to be expressed (in this embodiment, the ligand fluorescent sensor protein). Functional linkage between the polynucleotide and the polynucleotide. Here, the “expression control sequence” means a sequence that directs transcription of a gene to be expressed (in this embodiment, the polynucleotide encoding the ligand fluorescent sensor protein).

The promoter is not particularly limited and can be appropriately determined according to, for example, the type of cell. Specific examples of the promoter include, for example, viral promoters, expression inducible promoters, tissue-specific promoters, promoters in which enhancer sequences and promoter sequences are fused, and the like. The promoter is preferably linked upstream (5 ′ side) of the polynucleotide encoding the ligand fluorescent sensor protein.

In the present specification, the “viral promoter” means a promoter derived from a virus. Examples of the virus to be derived include cytomegalovirus, moloney leukemia virus, JC virus, breast cancer virus, simian virus, retrovirus and the like.

In the present specification, “expression-inducible promoter” means a gene that is to be expressed when a specific stimulus such as a chemical agent or physical stress is applied (in the present embodiment, a gene encoding the ligand fluorescent sensor protein). A promoter capable of expressing nucleotides) and exhibiting no expression activity in the absence of stimulation. Examples of the expression-inducible promoter include TetO (tetracycline operator) promoter, metallothionein promoter, IPTG / lacI promoter system, ecdysone promoter system, and an inhibitory sequence for irreversibly deleting translation or transcription. lox stop lox "system and the like, but not limited to.

As used herein, “tissue-specific promoter” means a promoter having activity only in a specific tissue.

Examples of promoters in which enhancer sequences and promoter sequences are fused include, for example, the SRα promoter consisting of the promoter of the early gene of simian virus 40 (SV40) and the partial sequence of the long terminal repeat of human T cell leukemia virus 1, Examples thereof include a CAG promoter composed of a megalovirus immediate early (IE) gene enhancer and a chicken β-actin promoter. In particular, the CAG promoter is expressed by having a polyA signal site of the rabbit β-globin gene at the 3 ′ end of the gene to be expressed (in this embodiment, the polynucleotide encoding the ligand fluorescent sensor protein). The gene (in this embodiment, the polynucleotide encoding the ligand fluorescent sensor protein) can be overexpressed almost systemically.

In the expression vector for expression in mammals, for example, a polyadenylation signal necessary for polyadenylation of the 3 ′ end of mRNA can be operated downstream (3 ′ side) of the polynucleotide encoding the ligand fluorescent sensor protein. It may be connected to. Examples of the polyadenylation signal include polyadenylation signals contained in the above genes derived from viruses, various human or non-human animals, such as SV40 late gene or early gene, rabbit β globin gene, bovine growth hormone gene, human A3 Examples thereof include polyadenylation signals such as an adenosine receptor gene.

-Construction of expression vector for expression in plant Expression vectors for expression in plant include, for example, promoter / enhancer derived from cauliflower mosaic virus, 5 'untranslated region (translation enhancer region) derived from alcohol dehydrogenase gene, and Alternatively, a heat shock protein gene-derived terminator or the like may be included.

-Construction of expression vector for expression in nematode The expression vector for expression in nematode may contain, for example, a promoter derived from the Myo2 gene.

-Construction of other expression vectors The plasmid vector may contain, for example, a pBluescript vector or other modified E. coli host vectors, or a CMV promoter with high expression efficiency in mammals such as pcDNA3 vector. .

The above-described expression vector may further have, for example, a multicloning site, a splicing signal, a selection marker, an origin of replication, and the like.
Examples of the selection marker include a drug selection marker gene.
Specific examples of the drug selection marker gene include a neomycin resistance gene and a puromycin resistance gene. By inserting a drug selection marker gene, cells that have been introduced with the expression vector of the present embodiment described later are cultured using a medium containing the drug, and the cells into which the expression vector has been introduced are selected. can do.

≪Cells≫
By introducing the expression vector of the above-described ligand fluorescent sensor protein or the above-described ligand fluorescent sensor negative control protein into cells, the cell membrane permeation peptide contained in the above-described ligand fluorescent sensor protein or the above-described ligand fluorescent sensor negative control protein can be used. Alternatively, a reagent for delivering the protein into a cell can provide a cell in which the protein is temporarily or permanently present.

The biological species from which the cells are derived in this embodiment is not particularly limited, and examples thereof include Escherichia coli, Bacillus subtilis, yeast, insect cells, plant cells, animal cells (particularly mammalian cells), and the like. It is not limited to.
Examples of the mammal include, but are not limited to, human, mouse, rat, hamster, guinea pig, rabbit, dog, cat, horse, cow, sheep, pig, goat, marmoset, monkey and the like.

Specific examples of animal cells include, for example, germ cells (sperm, ova, etc.), somatic cells constituting the living body, stem cells, progenitor cells, cancer cells separated from the living body, separated from the living body, and acquired immortalizing ability. And cells that are stably maintained outside the body (cell lines), cells that have been isolated from the living body and artificially modified, and cells that have been isolated from the living body and have been artificially exchanged nuclei, etc. .

<First embodiment>
In one embodiment, the present invention provides a cell comprising at least one ligand fluorescent sensor protein as described above.

According to the cell of this embodiment, it is possible to detect a change in the concentration of the ligand in the living cell. In addition, by using cells containing multiple types of the above-mentioned ligand fluorescent sensor proteins with different excitation wavelengths and emission wavelengths, each cell can be localized in different organelles of the same cell and detected simultaneously or nearly simultaneously in the same field of view of the fluorescence microscope. Thus, it is possible to provide a technique for optically analyzing temporal and / or spatial changes (spatiotemporal dynamics) of various ligand concentrations associated with physiological and / or pathological changes of the cells.

(Cell production method)
Examples of the method for introducing the above-described ligand fluorescent sensor protein or the above-described ligand fluorescent sensor negative control protein into cells include a method using a reagent for delivering the protein into cells.
Examples of the reagent include N- [1- (2,3-dioleoxy) propyl] -N, N, N- (DOTMA), which is a cationic lipid, and dioleylphosphatidylethanolamine, which is a neutral lipid. (DOPE), 1,2-dioleoyl-3-trimethyl-ammonium-propane (DOTAP) 1,2-dioleoyl-3-dimethylammonium-propane (DODAP) and other lipid mixtures containing cationic lipids, etc. But are not limited to these.
These reagents include commercially available products such as Lipofectin (registered trademark), Lipofectamine (registered trademark), and Pierce (registered trademark) protein transfection reagent (both Thermo Fisher Scientific Inc.).
Examples of other reagents for delivering the above-described ligand fluorescent sensor protein or the above-described ligand fluorescent sensor negative control protein into cells include, for example, lipid-free X-fect ™ transfection reagent (Clontech Laboratories, Inc.) or the like may be used. This reagent can be delivered intracellularly when the ligand fluorescent sensor protein described above or the ligand fluorescent sensor negative control protein described above comprises a cell membrane permeable peptide.

The above-mentioned ligand fluorescent sensor protein contained in a cell may be one type or two or more types. When two or more types are included, it is preferable that each ligand fluorescent sensor protein has a different excitation wavelength and emission wavelength in order to easily detect in the same cell.

<Second embodiment>
In one embodiment, the present invention provides a cell having a chromosome comprising at least one polynucleotide encoding the above-described ligand fluorescent sensor protein.

According to the cell of this embodiment, it is possible to detect a change in the concentration of the ligand in the living cell. In addition, by using cells containing multiple types of the above-mentioned ligand fluorescent sensor proteins with different excitation wavelengths and emission wavelengths, each cell can be localized in different organelles of the same cell and detected simultaneously or nearly simultaneously in the same field of view of the fluorescence microscope. Thus, it is possible to provide a technique for optically analyzing temporal and / or spatial changes (spatiotemporal dynamics) of various ligand concentrations associated with physiological and / or pathological changes of the cells.

(Cell production method)
The cell of this embodiment can be produced using, for example, a known genome editing technique in which the above-described expression vector is introduced into a cell as a donor vector to induce homologous recombination repair (HDR).

As a method for introducing the above-described expression vector into a cell, a known gene transfer method can be used. Specifically, for example, a calcium phosphate method, an electroporation method, a lipofection method, an aggregation method, a microinjection method , Particle gun method, DEAE-dextran method and the like can be used.

As a known genome editing technique, for example, CRISPR-Cas9 system, TALEN system, Zn finger nuclease system and the like can be used. Alternatively, for example, by adding a sequence homologous to the site into which the polynucleotide encoding the ligand fluorescent sensor protein in the chromosome is to be inserted upstream and downstream of the polynucleotide encoding the ligand fluorescent sensor protein of the above-described expression vector. A method for inducing homologous recombination repair can be used.
In addition, by providing a drug selection marker to the polynucleotide encoding the ligand fluorescent sensor protein, cells in which the polynucleotide encoding the ligand fluorescent sensor protein has been introduced by drug selection can be efficiently selected.

When the cell is a plant cell, for example, a polynucleotide encoding a ligand fluorescent sensor protein is introduced into the plant cell using the Agrobacterium method, a DNA introduction method into an isolated protoplast, etc., and homologous recombination repair (HDR) ) Can be induced.
In addition, by providing a drug selection marker to the polynucleotide encoding the ligand fluorescent sensor protein, cells in which the polynucleotide encoding the ligand fluorescent sensor protein has been introduced by drug selection can be efficiently selected.

(Structure on chromosome)
The polynucleotide encoding the above-described ligand fluorescent sensor protein introduced into the chromosome may have, for example, a promoter upstream and a polyadenylation signal downstream.
Examples of the promoter and polyadenylation signal include those exemplified in the above-mentioned << expression vector >>.
In order to further increase the expression of a polynucleotide encoding a ligand fluorescent sensor protein, a splicing signal, an enhancer region, and a part of an intron of each gene are placed 5 ′ upstream of the promoter region, between the promoter region and the translation region, or between the translation region. You may connect 3 'downstream.
In addition, in order to efficiently select cells in which the polynucleotide encoding the ligand fluorescent sensor protein has been introduced, a selection marker (for example, a drug selection marker gene) is placed 5 ′ upstream of the promoter region, between the promoter region and the translation region. Alternatively, it may be linked 3 ′ downstream of the translation region.

The polynucleotide encoding the ligand fluorescent sensor protein introduced into the chromosome may be one type or two or more types. When two or more types are included, it is preferable that each ligand fluorescent sensor protein has a different excitation wavelength and emission wavelength in order to easily detect in the same cell.

In addition, when a polynucleotide encoding two or more types of ligand fluorescent sensor proteins is included, it may be on the same chromosome or on different chromosomes.
The gene locus into which the polynucleotide encoding the ligand fluorescent sensor protein is introduced is preferably a safe harbor locus.

The “safe harbor locus” in the present specification is a gene region that is constantly and stably expressed and a gene originally encoded in the region is deleted or altered. But it means an area where life can be maintained.
When a foreign DNA (in this embodiment, a polynucleotide encoding a ligand fluorescent sensor protein) is inserted into the safe harbor locus using the CRISPR system, it preferably has a PAM sequence in the vicinity.
Examples of the safe harbor locus include the Rosa26 locus and the AAVS1 locus.

<Third embodiment>
In one embodiment, the present invention provides a cell comprising at least one expression vector as described above.

According to the cell of this embodiment, it is possible to detect a change in the concentration of the ligand in the living cell. In addition, by using cells containing multiple types of the above-mentioned ligand fluorescent sensor proteins with different excitation wavelengths and emission wavelengths, each cell can be localized in different organelles of the same cell and detected simultaneously or nearly simultaneously in the same field of view of the fluorescence microscope. Thus, it is possible to provide a technique for optically analyzing temporal and / or spatial changes (spatiotemporal dynamics) of various ligand concentrations associated with physiological and / or pathological changes of the cells.

(Cell production method)
As a method for introducing the above-described expression vector into a cell, a known gene introduction method can be used, and examples thereof include the same methods as those exemplified in <Second Embodiment> above.

The introduced expression vector may be one type or two or more types. When two or more types are included, it is preferable that the excitation wavelength and emission wavelength of the ligand fluorescent sensor protein expressed from each expression vector are different in order to easily detect in the same cell.

≪Non-human organism≫
In one embodiment, the present invention provides a non-human organism comprising the cell described above.

According to the non-human organism of this embodiment, the ratio of the fluorescence intensity in the presence of a high concentration of ligand to the fluorescence intensity in the absence of ligand under physiological conditions is sufficiently high, Variations in the concentration of pathological ligands can be detected. In addition, by using a non-human organism containing cells expressing a plurality of types of ligand fluorescent sensor proteins having different excitation wavelengths and emission wavelengths in one or more locations in the organism, each is localized in different organelles of the same cell, Alternatively, it can be localized in different cells in the organism and detected simultaneously or nearly simultaneously in the same field of view of the fluorescence microscope, and the ligand concentration over time and / or associated with physiological and / or pathological changes in the non-human organism A technique for optically analyzing a spatial change (spatiotemporal dynamics) can be provided.

In addition, the “non-human organism” in the present specification may be any species other than human, and specifically includes those other than humans among those exemplified in the above “cells”.
The non-human organism of the present embodiment may be a non-human organism in which the above-described cells are transplanted, and a gene encoding a ligand fluorescent sensor protein is introduced into the germ cell line, and is inherited to the next generation. It may be a modified non-human organism.

<Method for producing non-human organism>
As a method for producing the non-human organism of this embodiment, for example, a method of introducing the above-mentioned cell surgically or non-surgically into the body of the non-human organism, a method of introducing the above-mentioned vector directly into the cell of the non-human organism. Etc.

Alternatively, when the non-human organism is a non-human mammal, for example, in <Second Embodiment> of the above <Cell>, a fertilized egg, embryonic stem cell, sperm or unfertilized egg of the non-human mammal is used as the cell. Introducing an expression vector comprising a polynucleotide encoding a ligand fluorescent sensor protein, and from an individual generated using these cells, the polynucleotide encoding the ligand fluorescent sensor protein is derived from all cells, including germ cells. It can also be produced by a method of selecting an individual integrated on a chromosome.

The presence of a polynucleotide encoding a ligand fluorescent sensor protein in the obtained non-human mammalian germ cells is confirmed by the fact that the resulting animal progeny have the transgene in all of the germ cells and somatic cells. can do. The selection of an individual can be performed by using a ligand fluorescence sensor for genomic DNA prepared from a part of the tissue constituting the individual, for example, blood tissue, epithelial tissue, connective tissue, cartilage tissue, bone tissue, muscle tissue, oral tissue or skeletal tissue. This is done by confirming the presence of a polynucleotide encoding the protein at the DNA level. The individual thus selected is usually a heterozygote having a polynucleotide encoding a ligand fluorescent sensor protein on one side of the homologous chromosome, so by crossing heterozygous individuals, A homozygous animal having a polynucleotide encoding a ligand fluorescent sensor protein in both homologous chromosomes can be obtained. By mating the homozygous male and female animals, all offspring become homozygotes that stably hold the polynucleotide encoding the ligand fluorescent sensor protein, so that non-human mammals can be used in normal breeding environments. Breeding can be passaged.

Alternatively, when the non-human organism is a plant, for example, the plant cell produced by the Agrobacterium method or the DNA introduction method into an isolated protoplast in <Second Embodiment> of the above <Cell> It can be produced by redifferentiating the whole genetically modified plant by a tissue culture method.
Alternatively, when the non-human organism is a plant, for example, an immature embryo is partially degraded with an enzyme, and an expression vector containing a polynucleotide encoding a ligand fluorescent sensor protein is introduced by electroporation or the like, It can be produced by redifferentiating an embryo cell in which a polynucleotide is inserted into a chromosome by a plant tissue culture method.

≪Ligand concentration measurement kit≫
In one embodiment, the present invention provides a ligand concentration measurement comprising at least one selected from the group consisting of the above-described ligand fluorescent sensor protein, the above-described polynucleotide, the above-described expression vector, the above-described cell, and the above-mentioned non-human organism. Provide kit.

According to the ligand concentration measurement kit of this embodiment, the ratio of the fluorescence intensity in the presence of a high concentration of ligand to the fluorescence intensity in the absence of the ligand is sufficiently high under physiological conditions, Variations in the concentration of physiological and / or pathological ligands can be detected.

In addition, for example, when the ligand is ATP, by using the ligand concentration measurement kit of this embodiment, there is ATP or glucose or other bioenergy sources that produce ATP when metabolized by the cells or organisms. Only when it can generate and detect fluorescence.
In addition, when the ligand is ATP, the conventional living cell detection technique using the luminescence reaction by luciferase has required to dissolve the cell membrane and to contact intracellular ATP with the enzyme luciferase and the substrate luciferin. However, since the living cell can be detected without destroying the cell and diffusing ATP by using the ligand concentration measurement kit of this embodiment, the living cell can be located anywhere in the spatial structure like the microbial flora. Can be detected.

When the ligand concentration measurement kit of this embodiment includes the above-described ligand fluorescence sensor protein, it may contain one type of ligand fluorescence sensor protein, and a plurality of types of ligand fluorescence sensor proteins having different excitation wavelengths and emission wavelengths. May be included.
Further, when the ligand concentration measurement kit of the present embodiment includes the above-described ligand fluorescence sensor protein, the ligand fluorescence sensor protein may be immobilized on a solid support.
Examples of the solid support include those similar to those described in the above << Ligand fluorescence sensor protein >>.

When the ligand concentration measurement kit of this embodiment includes the above-mentioned polynucleotide, it may contain one type of polynucleotide encoding the ligand fluorescence sensor protein, and the ligand fluorescence sensor protein having different excitation wavelength and emission wavelength. A plurality of kinds of encoding polynucleotides may be included.

When the ligand concentration measurement kit of this embodiment includes the above-described expression vector, it may include an expression vector containing a polynucleotide encoding one type of ligand fluorescent sensor protein, and the excitation wavelength and emission wavelength are different. A plurality of expression vectors containing a ligand fluorescent sensor protein may be included.

When the ligand concentration measurement kit of the present embodiment includes the above-described cells, it may include one type of cell or may include a plurality of types of cells.
In addition, the ligand concentration measurement kit of this embodiment may include one type of cell in which the ligand fluorescent sensor protein is expressed or introduced, and the ligand fluorescent sensor protein having different excitation wavelength and emission wavelength is expressed or introduced. Multiple types of cells may be included.
In addition, one type of ligand fluorescent sensor protein may be expressed or introduced in the cell, and a plurality of types of ligand fluorescent sensor proteins having different excitation wavelengths and emission wavelengths may be expressed or introduced.

When the ligand concentration measurement kit of the present embodiment includes the above-described non-human organism, it may include one type of non-human organism or may include a plurality of types of non-human organisms.
Further, the ligand concentration measurement kit of this embodiment may include one type of non-human organism in which the ligand fluorescent sensor protein is expressed or introduced, and the ligand fluorescent sensor protein having different excitation wavelength and emission wavelength is expressed or introduced. It may contain multiple types of non-human organisms.
In addition, one type of ligand fluorescent sensor protein may be expressed or introduced in the same cell of the non-human organism included in the ligand concentration measurement kit of this embodiment, and a plurality of types of ligands having different excitation wavelengths and emission wavelengths. A fluorescent sensor protein may be expressed or introduced.
In addition, one type of ligand fluorescent sensor protein may be expressed or introduced in different cells of the non-human organism included in the ligand concentration measurement kit of the present embodiment, and a plurality of types of ligands having different excitation wavelengths and emission wavelengths. A fluorescent sensor protein may be expressed or introduced.

In the present embodiment, when the above-described ligand fluorescent sensor protein is included, the ligand concentration measurement kit of the present embodiment may further include the reagent exemplified in <First Embodiment> of the above <Cell>. .

In the present embodiment, when the above-described expression vector is included, the ligand concentration measurement kit of the present embodiment may further include a transfection reagent for vector introduction.
The transfection reagent for vector introduction is selected from the group consisting of, for example, cationic polymers, cationic lipids, polyamine reagents, polyimine reagents, and calcium phosphate. Such transfection reagents include, for example, Effectene Transfection Reagent (cat. Transfection Reagent (301305, Qiagen, CA), PolyFect Transfection Reagent (301105, Qiagen, CA), LipofectAMINE 2000 Reagent (11668-019, Invitrogen Corporation, CA), JetPEI (× ) Conc. (101-30, Polyplus-translation, France), ExGen 500 (R0511, Fermentas Inc., MD), and the like, but are not limited thereto.

In the present embodiment, when the above-described cells are included, the ligand concentration measurement kit of the present embodiment may further include a cell culture medium.
The cell culture medium may be a basic medium containing components (inorganic salts, carbohydrates, hormones, essential amino acids, non-essential amino acids, vitamins) necessary for viable cell growth, and should be selected appropriately depending on the cell type. Can do.
Specific examples of the cell culture medium include, for example, LB medium, E. coli minimal medium (Davis medium), Escherichia coli minimal salt medium (MS), and the like; Bacillus subtilis minimal medium (Spizzen minimal medium) Bacillus subtilis culture medium, Bacillus subtilis transformation medium I, Bacillus subtilis transformation medium II, etc .; yeast minimal medium (YPD medium), yeast complete medium (YPAD medium), etc. Yeast culture medium; Murashige and Skoog medium (MS medium), B5 medium, Hyponex medium and other plant culture mediums; Grace insect medium, Schneider insect medium and other insect cell culture mediums; DMEM, Minimum Essential Medium (MEM) , RPMI-1640, Basal Medium Eagle (BME), Dulbecco's odified Eagle's Medium: Nutrient Mixture F-12 (DMEM / F-12), Glasgow Minimum Essential Medium (Glasgow MEM) for animal cell culture media, etc., and the like, but are not limited to.

The ligand concentration measurement kit of this embodiment may further include an excitation light source. The excitation light source may be appropriately selected according to the excitation wavelength of the fluorescent sensor protein.

≪Method for determining ligand concentration in test sample≫
In one embodiment, the present invention comprises a calibration curve creating step of contacting the above-described ligand fluorescence sensor protein with a standard solution containing a known concentration of a ligand, measuring fluorescence intensity, and creating a calibration curve; Measured in the fluorescence measurement step based on the calibration curve created in the fluorescence measurement step of measuring fluorescence intensity by bringing the sensor into contact with a solution containing a fluorescent sensor protein and a ligand of unknown concentration, and the calibration curve creation step And a concentration determining step for determining a ligand concentration with respect to the fluorescence intensity, and a method for determining a ligand concentration in a test sample.

According to the determination method of the ligand concentration in the test sample of the present embodiment, the ligand concentration in the test sample can be determined easily and accurately.
Each step of the method for determining the ligand concentration in the test sample of the present embodiment will be described in detail below.

[Calibration curve creation process]
First, the above-described ligand fluorescence sensor protein is brought into contact with a standard solution containing a ligand having a known concentration, and the fluorescence intensity is measured.
The ligand is not particularly limited and includes the same ligands as those exemplified in the above-mentioned “ligand fluorescence sensor protein”.
As the standard solution, a solution containing one type of ligand may be used, or a solution containing a plurality of types of ligands may be used. Among them, in order to accurately create a calibration curve, it is preferable to use a solution containing a plurality of types of ligands as the standard solution.

The ligand fluorescent sensor protein may be suspended in a solvent or immobilized on a solid support.
The solvent for suspending the ligand fluorescent sensor protein may be any solvent that does not affect the ligand binding ability and fluorescence characteristics of the ligand fluorescent sensor protein. Specific examples of the solvent include water, sodium chloride solution (for example, 0.9% (w / v) NaCl), glucose solution (for example, 5% glucose), surfactant-containing solution (for example, 0.01 % Polysorbate 20), pH buffer solution (as buffering agents, for example, HEPES-KOH, Tris-HCl, acetic acid-sodium acetate, citric acid-sodium citrate, phosphoric acid, boric acid, MES, PIPESHEPES-KOH, Tris-HCl , Acetic acid-sodium acetate, citric acid-sodium citrate, phosphoric acid, boric acid, MES, PIPES, and the like), and the like.
Examples of the solid support include those similar to those described in the above << Ligand fluorescence sensor protein >>.

Fluorescence intensity may be measured using a known stationary fluorescence measuring device. Next, a calibration curve is created from the obtained fluorescence intensity and a known ligand concentration.

[Fluorescence measurement process]
Subsequently, the fluorescence intensity is measured by bringing the ligand fluorescence sensor protein into contact with a solution containing an unknown concentration of the ligand. The fluorescence intensity may be measured using a known stationary fluorescence measuring device.
The solution containing an unknown concentration of the ligand is not particularly limited. For example, a body fluid sample collected from a human or non-human organism, a cell extract collected from a human or non-human organism, a human or non-human organism Examples include, but are not limited to, culture supernatants of cells collected from, extracts of cultured cells derived from human or non-human organisms, culture supernatants of cultured cells derived from humans or non-human organisms, and the like.

More specifically, as the body fluid sample, for example, blood, serum, plasma, urine, puffy coat, saliva, semen, chest exudate, cerebrospinal fluid, tear fluid, sputum, mucus, lymph fluid, ascites, pleural effusion, amniotic fluid , Bladder lavage fluid, bronchoalveolar lavage fluid and the like, but are not limited thereto.

[Density determination process]
Next, in the calibration curve creation step, a ligand concentration with respect to the fluorescence intensity measured in the fluorescence measurement step is determined based on the created calibration curve.
The preparation of the calibration curve and the determination of the ligand concentration relative to the fluorescence intensity may be performed using commercially available data analysis software or the like.

≪Method for detecting change in ligand concentration over time≫
By using the above-mentioned cells or the above-mentioned non-human organisms, the ratio of the fluorescence intensity in the presence of a high concentration of ligand to the fluorescence intensity in the absence of the ligand under physiological conditions is sufficiently high. Variations in the concentration of physiological and / or pathological ligands in living non-human organisms can be conveniently detected.

<First embodiment>
In one embodiment, the present invention provides a method for detecting a change in ligand concentration over time in a living cell, comprising the step of measuring fluorescence intensity over time using the above-described cells.

According to the detection method of the present embodiment, it is possible to easily detect a change with time in the concentration of a ligand in a living cell. In addition, by using cells in which multiple types of the above-described ligand fluorescent sensor proteins having different excitation wavelengths and emission wavelengths are introduced or expressed, each of them is localized in different organelles of the same cell, and in the same field of view of the fluorescence microscope. Provided is a technique for optically analyzing temporal and / or spatial changes (spatiotemporal dynamics) of various ligand concentrations accompanying detection of physiological and / or pathological changes of the cells simultaneously or almost simultaneously. be able to.

In the detection method of the present embodiment, the fluorescence intensity can be measured using a fluorescence microscope having a stationary fluorescence measurement device while the cells are alive. Furthermore, by measuring the fluorescence intensity continuously, it is possible to measure a change in fluorescence intensity over time.

<Second embodiment>
In one embodiment, the present invention provides a method for detecting a change in ligand concentration over time in a living non-human organism, comprising the step of measuring fluorescence intensity over time using the non-human organism described above.

According to the detection method of this embodiment, the ratio of the fluorescence intensity in the presence of a high concentration of ligand to the fluorescence intensity in the absence of the ligand is sufficiently high under physiological conditions, Alternatively, variations in pathological ligand concentration can be detected. In addition, by using a non-human organism containing cells expressing a plurality of types of ligand fluorescent sensor proteins having different excitation wavelengths and emission wavelengths in one or more locations in the organism, each is localized in different organelles of the same cell, Alternatively, it can be localized in different cells in the organism and detected simultaneously or nearly simultaneously in the same field of view of the fluorescence microscope, and the ligand concentration over time and / or associated with physiological and / or pathological changes in the non-human organism A technique for optically analyzing a spatial change (spatiotemporal dynamics) can be provided.

In the detection method of this embodiment, the fluorescence intensity can be measured using a fluorescence microscope or the like having a stationary fluorescence measurement device while the non-human organism is still alive. Furthermore, by measuring the fluorescence intensity continuously, it is possible to measure a change in fluorescence intensity over time.

Hereinafter, the present invention will be described in more detail with reference to Examples and Comparative Examples, but the present invention is not limited to these Examples and the like.

[Example 1] Construction of ATP-specific fluorescent sensor protein Reagents, etc. ATP, ADP, and AMP were purchased from Wako Pure Chemical Industries, Ltd., and GTP was purchased from Sigma-Aldridge. Other reagents such as sodium fluoride (NaF), oligomycin, isoproterenol and 3- (3,4-dichlorophenyl) -1,1-dimethylurea (DCMU) were purchased from Sigma-Aldridge. dATP was purchased from Thermo Scientific. All polynucleotides for primers were purchased from Sigma-Aldridge. Ligation was performed using T4 DNA ligase in each optimum buffer (Takara). PrimeSTAR HS DNA polymerase (Takara Bio Inc.) was used for PCR. The PCR reaction product or restriction enzyme digestion product was purified by electrophoresis (agarose gel) as a routine, and then gel extraction (QIAquick, Qiagen) was performed. For the isolation of plasmid DNA from E. coli, an Axy Prep ™ miniprep kit (Axygenbio, Corning Japan Co., Ltd.) was used.

2. Design of ATP Fluorescence Sensor Protein and Corresponding Negative Control Protein The ATP fluorescence sensor protein of the present invention uses the epsilon (ε) subunit of Bacillus subtilis F ₀ F ₁ -ATP synthase as the ATP binding domain, BFP (Wachter, R. et al., Biochemistry 2960, 9759-9765 (1997)), Citrine (Griesbeck, O. et al., J. Biol. Chem. 276, 29188-29194 (2001)), and mAple (Shaner, N). C. et al., Nature Methods 5: 545-551 (2008)).
BFP and mApple are described in Zhao, Y. et al. (Science. 333, 1888-1891 (2011)), amino acid substitution mutations were introduced in several places (SEQ ID NOs: 1, 2, 7, and 8). The excitation wavelengths of the fluorescent proteins BFP, Citrine, and mA Apple are 380, 490, and 550 nm, respectively, and the emission wavelength ranges are 410 to 600, 505 to 650, and 575 to 700 nm, respectively. Various polypeptide linkers were ligated to the amino terminus and carboxyl terminus of the ε subunit, and fusion proteins inserted into the fluorescent proteins BFP, Citriline, and mApple were prepared as candidate proteins for the ATP fluorescent sensor protein (MaLion B, respectively). Sometimes referred to as G and R series).

The structure of the domain and linker of the candidate protein of the ATP fluorescence sensor is as follows. First, a domain that specifically binds to ATP may be referred to as an “ATP binding domain”. The ATP binding domain is derived from the ε subunit of Bacillus subtilis F ₀ F ₁ -ATP synthase, and its amino acid sequence is listed in the sequence listing as SEQ ID NO: 15. The polypeptide linker linked to the amino terminus of the ATP binding domain may be referred to as “N-terminal linker”, and the polypeptide linker linked to the carboxyl terminus of the ATP binding domain may be referred to as “C-terminal linker”. . The amino-terminal domain and the carboxyl-terminal domain of each fluorescent protein BFP, Ciline, and mAapple cleaved by insertion are hereinafter referred to as “BFP-N domain”, “BFP-C domain”, “Citrine-N domain”, It may be referred to as “Citrine-C domain”, “mApple-N domain”, and “mApple-C domain”. The amino acid sequences of the BFP-N domain and the BFP-C domain are listed as SEQ ID NOs: 1 and 2. The amino acid sequences of the Citrine-N domain and the Citrine-C domain are listed as SEQ ID NOs: 5 and 6. The amino acid sequences of the mApple-N domain and the mApple-C domain are listed as SEQ ID NOs: 7 and 8.

Therefore, the candidate proteins of the MaLion B series ATP fluorescent sensor are [BFP-N domain]-[N-terminal linker]-[ATP-binding domain]-[C-terminal linker]-from the amino terminus to the carboxyl terminus. A polypeptide domain and a polypeptide linker are arranged in the order of [BFP-C domain].
Similarly, candidate proteins for the MaLion G series ATP fluorescent sensor are [Citrine-N domain]-[N-terminal linker]-[ATP-binding domain]-[C-terminal linker] in the direction from the amino terminus to the carboxyl terminus. -A polypeptide domain and a polypeptide linker are arranged in the order of [Citrine-C domain].
In addition, candidate proteins for the MaLion R series ATP fluorescent sensor are [mAapple-N domain]-[N-terminal linker]-[ATP-binding domain]-[C-terminal linker]-in the direction from the amino terminus to the carboxyl terminus. A polypeptide domain and a polypeptide linker are arranged in the order of [mAapple-C domain].

The procedure for constructing a candidate protein for the ATP fluorescence sensor protein is as follows. First, a polynucleotide encoding the ε subunit of Bacillus subtilis F ₀ F ₁ -ATP synthase was synthesized as an ATP binding domain (Integrated DNA Technologies, Institute of Medical Biology). Next, polynucleotides encoding N-terminal linkers and C-terminal linkers of various amino acid sequences were linked to both ends of the ATP binding domain. A fused chimeric protein in which the polynucleotide encoding the N-terminal linker, ATP-binding domain, and C-terminal linker is inserted on the carboxyl terminal side from the chromophore of each fluorescent protein so that the reading frame of the amino acid sequence is not shifted. Was prepared by PCR. In order to insert the polynucleotide encoding the candidate protein of the ATP fluorescence sensor into the pRSET _A vector (Invitrogen, Life Technologies Corporation), the MaLion G series has the XhoI / BstbI site, the MaLion B and R series have the BamHI / HindIII sites. Using. The pRSET _A vector construct containing the ATP fluorescent sensor candidate protein was transformed into E. coli JM109 (DE3), and an expression vector clone containing each ATP fluorescent sensor candidate protein was isolated. Each clone of E. coli was cultured in 2.5 mL of LB medium at 20 ° C. for 3-4 days. Thereafter, the Escherichia coli suspension was centrifuged at 15,300 g for 5 minutes, pelleted, resuspended in PBS buffer solution, and sonicated for 30 seconds (130 W, 20 kHz, intensity 30%, Vibra cell ™, (SONICS & Materials, Inc.) was applied to obtain a cell lysate. 460 μL of a buffer solution (50 mM Mops-KOH (pH 7.4), 50 mM KCl, 0.5 mM MgCl ₂ , and 0.05% triton X-100) was added to 40 μL of the supernatant of the cell lysate. . In the presence or absence of ATP at a final concentration of 10 mM, the fluorescence characteristics of the ATP fluorescence sensor candidate proteins of each clone were measured using a fluorescence spectrophotometer (Hitachi F-2700, Hitachi High-Tech Science Co., Ltd.).

3. Results of Measurement of Fluorescence Characteristics of Candidate Proteins of ATP Fluorescent Sensor FIG. 1A is a ratio of the fluorescence intensity in the presence of 19 types of ATP and the fluorescence intensity in the absence of ATP (hereinafter, referred to as ATP fluorescence sensor) 1B is a histogram of the dynamic range of 27 candidate proteins of the MaLion G series ATP fluorescence sensor, and FIG. 1C is a histogram of the 47 candidate proteins of the MaLion R series ATP fluorescence sensor. which is a histogram of the _F / F _0. Here, the ATP concentration in the presence of ATP is all 10 mM. In FIG. 1A, FIG. 1B, and FIG. 1C, the horizontal axis represents the dynamic range, the scale number represents the end of each section, and the vertical axis represents the number of candidate proteins that include the dynamic range within the dynamic range section. In FIG. 1A, FIG. 1B, and FIG. 1C, if the dynamic range is 1, it means that there is no change in fluorescence characteristics with or without ATP, and if the dynamic range exceeds 1, the fluorescence becomes strong in the presence of ATP. (Hereinafter, sometimes referred to as “turn-on type”.) When the dynamic range is less than 1, the fluorescence becomes weak in the presence of ATP (hereinafter referred to as “turn-off type”). -May be referred to as -off type)).

The candidate proteins with the largest dynamic range in the MaLion B, G, and R series were named MaLion B, G, and R, respectively. The amino acid sequences of the N-terminal linker and the C-terminal linker of MaLion B are listed in SEQ ID NOs: 16 and 17, respectively. The amino acid sequence of the N-terminal linker of MaLion G is WRG (Trp-Arg-Gly), and the amino acid sequence of the C-terminal linker of MaLion G is listed in SEQ ID NO: 18. The amino acid sequence of the N-terminal linker of MaLion R is listed in SEQ ID NO: 19. The amino acid sequence of the C-terminal linker of MaLion R is PEE (Pro-Glu-Glu). The full-length amino acid sequences of MaLion B, G, and R are listed in SEQ ID NOs: 9, 10, and 11, respectively.

In the MaLion B, G, and R series, one of the candidate proteins with a dynamic range close to 1 was named negMaLion B, G, and R, respectively. The amino acid sequences of the N-terminal linker and the C-terminal linker of negMaLion B are listed in SEQ ID NOs: 27 and 28, respectively. The amino acid sequence of the N-terminal linker of SEQ ID NO: negMaLion G is PRG (Pro-Arg-Gly), and the amino acid sequence of the C-terminal linker of negMaLion G is listed in SEQ ID NO: 29. The amino acid sequence of the N-terminal linker of negMaLion R is listed in SEQ ID NO: 30. The amino acid sequence of the C-terminal linker of negMaLion R is PEG (Pro-Glu-Gly). The full length amino acid sequences of negMaLion B, G, and R are listed in SEQ ID NOs: 24, 25, and 26, respectively.

[Example 2] Analysis of fluorescence characteristics of ATP fluorescence sensor protein, etc. Purification of ATP Fluorescence Sensor Protein etc. MaLion B, G, and R and negMaLion B, G, and R may be hereinafter referred to as “ATP fluorescence sensor protein etc.”. For the purpose of protein purification, ATP fluorescence sensor protein etc. is linked to pRSET _A vector, and fusion protein containing translation start codon, histidine hexamer polypeptide etc. is introduced into E. coli JM109 (DE3) as a construct driven by T7 promoter did. E. coli containing an expression vector such as an ATP fluorescence sensor protein was cultured in 100 mL of LB medium at 20 ° C. for 4 days. Thereafter, the suspension of Escherichia coli was centrifuged at 15,300 g for 20 minutes at 4 ° C. to remove the supernatant, and the pellet was frozen and thawed three times, and sonicated on ice for 3 minutes (30 W, 20 kHz, strength 70). %, Vibra cell (trademark)) was applied to obtain a cell lysate. The centrifuged supernatant of the cell lysate is adsorbed on a PD-10 column (GE Healthcare Japan Co., Ltd.) packed with Ni-NTA agarose (Qiagen Co., Ltd.). Fusion proteins such as sensor proteins were eluted from the column. Fusion proteins such as ATP fluorescence sensor protein were quantified using a Bradford method protein assay (Bio-Rad Protein Assay, Bio-Rad Laboratories) with a calibration curve using bovine serum albumin as a standard protein.

2. Measurement of fluorescence characteristics of purified ATP fluorescence sensor protein, etc. A purified fusion protein such as ATP fluorescence sensor protein was used in a measurement experiment with a fluorescence spectrophotometer (Hitachi F-2700). In the following experiment, a Mops buffer (50 mM Mops-KOH (pH 7.4), 50 mM KCl, 0.5 mM MgCl ₂ , and 0.05% Triton X-100) was used except for the experiment for changing the pH. In experiments examining changes in fluorescence properties with ATP concentration, various final concentrations of ATP of 0-8 mM were used. In the experiment for examining the molecular specificity, ADP, AMP, GTP, or dATP among the ATP analog compounds was used at a final concentration of 10 mM. For measuring the absorption spectrum of the purified fusion protein such as the ATP fluorescence sensor protein, the concentration of the fusion protein was adjusted to 20 μM and an ultraviolet-visible light spectrophotometer (JASCO Corporation) was used. A fluorescence spectrophotometer (RX2000, Applied photophysics Limited) equipped with a stopped-flow apparatus was used for the reaction kinetic analysis of the purified fusion protein such as the ATP fluorescence sensor protein. The protein is rapidly mixed 1: 1 with solutions of different ATP concentrations, and the change in fluorescence at a given wavelength (440 nm, 520 nm and 585 nm) is recorded, and the apparent rate constant (k _app at each ATP concentration). Was calculated by fitting an exponential curve, and then k _app at each ATP concentration was plotted to determine the association constant (k _on ) and dissociation constant (k _off ), respectively. The relationship is expressed by the following formula (1).

3. FIG. 2A is an excitation spectrum diagram of MaLion B, G, and R in the presence and absence of 10 mM ATP, and FIG. 2B is the presence and absence of 10 mM ATP. FIG. 4 is an excitation spectrum and fluorescence spectrum diagram of MaLion B, G, and R below. 2A and 2B represent the excitation wavelength and the fluorescence wavelength, respectively, and the vertical axis represents the relative value of the fluorescence intensity. The solid line and the dotted line in the graph represent the spectrum curves in the presence and absence of ATP, respectively. As is clear from FIGS. 2A and 2B, MaLion B, G, and R all increase in fluorescence in the presence of 10 mM ATP by 80%, 390%, and 350%, respectively, in the absence of ATP. did. FIG. 2C is a fluorescence spectrum diagram of negMaLion B, G, and R in the presence and absence of 10 mM ATP. From FIG. 2C, negMaLion B, G, and R all increased their fluorescence in the presence of 10 mM ATP by 104%, 95%, and 106%, respectively, compared to the absence of ATP.

FIG. 3A is a graph showing an ATP concentration-dependent change in the fluorescence intensity of MaLion B, G, and R. The results of calculating the dissociation equilibrium constant (K _D ) from the plots of different ATP concentrations and fluorescence intensity changes in FIG. 3A are shown in Table 5 below.

Although there are various theories based on conventional knowledge, the intracellular ATP concentration is known to be 5 mM or less in most cases (Rangaraju et al., Cell. 156, 825-35 (2014), Traut, T. W. Mol. Cell. Biochem. 140, 1-22 (1994)). Therefore, when considering the dissociation equilibrium constants of MaLion B, G, and R, it can be expected to function satisfactorily under physiological conditions.

FIG. 3B is a graph showing the molecular specificity of the fluorescence intensities of MaLion B, G, and R. When the relative value of the fluorescence intensity in the presence of 10 mM ATP compared to the absence of ATP is defined as a normalized dynamic range, the ATP analog compound may affect the fluorescence intensity only by 10% or less of the normalized dynamic range. It was revealed. Therefore, the present ATP fluorescent sensor is very specific for ATP. Further, from this result, even if the ATP sensor protein uses the ε subunit of the same Bacillus subtilis F ₀ F ₁ -ATP synthase as the ATP binding domain, the influence of the ATP analog compound differs for each variant, and MaLion B , And G, fluorescence decreased in the presence of ADP, AMP, and dATP, but MaLion R increased fluorescence. Therefore, it can be said that the fluorescence characteristics of the individual fluorescence sensors are determined by the sequences of the N-terminal linker and the C-terminal linker linked to both ends of the ATP binding domain.

4A, 4B, and 4C are graphs showing pH-dependent changes in fluorescence intensity and dynamic range of MaLion B, G, and R in the presence or absence of ATP, respectively. 4A, 4B, and 4C, the horizontal axis represents pH, the left vertical axis represents fluorescence intensity, and the right vertical axis represents dynamic range. From FIG. 4A, FIG. 4B, and FIG. 4C, even if MaLion B, G, and R have the same ATP concentration, the fluorescence characteristics change when the pH changes, and the influence of the pH on the fluorescence characteristics is different for each variant. Different. This is the same as the influence on the fluorescence characteristics of the ATP analog compound shown in FIG. 3B. In fact, the change pattern of the fluorescence characteristics depending on the pH of the ATP fluorescence sensor negative control proteins negMaLion B, G, and R was the same as the change pattern of the fluorescence characteristics due to the pH of MaLion B, G, and R. Therefore, in an experimental system in which pH may change at the same time as the change in ATP concentration, fluorescence measurement with MaLion B, G, and R and fluorescence measurement with negMaLion B, G, and R are performed in parallel. The ATP concentration can be determined by excluding the influence of pH change.

[Example 3] Construction of vector for intracellular expression of ATP fluorescence sensor protein, etc. Construction of Vector for Expression in Mammalian Cells For expression in mammalian cells, a polynucleotide encoding MaLion G or negMaLion G was inserted into the XhoI / HindIII site of pcDNA3.1 (−) vector. A polynucleotide encoding MaLion B and R or negMaLion B and R was inserted into the BamHI / HindIII site of pcDNA3.1 (-) vector. In order to localize ATP fluorescence sensor protein and the like to mammalian cell mitochondria, a fusion protein obtained by linking a cytochrome c oxidase subunit VIII-derived localization signal sequence (SVLTPLLLRGTGSARRLVPVPKIHSL, SEQ ID NO: 44) to the amino terminus of the ATP fluorescence sensor protein Was expressed in a mitochondria-specific expression vector. That is, a polynucleotide encoding MaLion R or negMaLion R was inserted into the BamHI / NotI site of the pEYFP-Mito vector (Clontech Laboratories, Inc.). The fusion protein in which the localization signal sequence derived from the subunit VIII of cytochrome c oxidase was linked to the amino terminus of MaLion R or negMaLion R was named “mito-MaLion R” or “mito-negMaLion R”.

2. Construction of vector for nematode cell expression To localize MaLion R or negMaLion R in nematode mitochondria, mitochondrial localization signal derived from chick triaspartate aminotransferase at the amino terminus of MaLion R or negMaLion R The fusion protein to which (ALLQSRRLLSAPPRRAAATARAS, SEQ ID NO: 45) was linked was named “Cemito-MaLion R” or “Cemito-negMaLion R”. In order to express ATP fluorescence sensor protein in the pharyngeal muscle of C. elegans, each of MaLion G, negMaLion G, Cemito-MaLion R, or Cemito-negMaLion R is encoded in a vector derived from pBueScript with the myo2p promoter introduced. The polynucleotide was inserted into the XhoI / SacI site.

3. Construction of a plant cell expression vector In order to localize MaLion R or negMaLion R in the mitochondria of plants, a fusion protein in which AR791 (AT1G52080, NM_104089.3) is linked to the amino terminus of MaLion R or negMaLion R, It was named “Plmito-MaLion R” or “Plmito-negMaLion R”. In order to express an ATP fluorescence sensor protein or the like in a plant, a pGreen — 0281 vector in which 35S promoter is double-linked is added to MaLion G, negMaLion G, Cemito-MaLion R, or a codon optimized for translation in a plant (Arabidopsis thaliana), or A polynucleotide encoding each of Chemito-negMaLion R was inserted into the XhoI / SacI site.

[Example 4] Intracellular ATP concentration measurement using ATP fluorescent sensor protein, etc. Fluorescence sensor measurement of intracellular ATP in HeLa cells HeLa cells were obtained from ATCC (American Type Culture Collection, VA, USA) and modified by Dulbecco with 10% fetal bovine serum, 100 IU / mL penicillin and 100 μg / mL streptomycin. The cells were cultured in an Eagle medium (glucose 4.5 g / L, hereinafter sometimes referred to as “growth medium”) at 37 ° C. in a 5% CO ₂ atmosphere. For fluorescence microscope measurement of ATP fluorescence sensor protein and the like expressed in HeLa cells, a 3.5 cm glass bottom dish in which HeLa cells were seeded to become 50% confluent was prepared. 10 μL of Opti-MEM medium (Life Technologies Corporation, Thermo Fisher Scientific Inc.) supplemented with 0.2 μg of expression vector containing ATP fluorescence sensor protein and 0.2 μL of FuGENE HD transfection reagent (Promega Corporation) in advance. Added to the HeLa cells on the 3.5 cm glass bottom dish. After culturing for 8 hours, the culture medium was replaced with fresh growth medium, and further cultured for 2-3 days. Immediately before the fluorescence measurement, the medium was replaced with a phenol red-free growth medium.

For the fluorescence microscope measurement, an inverted microscope (IX81, Olympus Corporation) equipped with a cooled CCD camera (Cool SNAP HQ2, Photometrics) and an oil immersion objective lens (Plan Apo 60 × 1.42 NA) was used. NaF was used for the cytoplasmic ATP production inhibition experiment.

To a dish in which HeLa cells were cultured in 1.5 mL of growth medium, 0.5 mL of growth medium containing 40 mM NaF was added to make the final concentration of NaF 10 mM. Oligomycin was used for the mitochondrial ATP production inhibition experiment. To a dish in which HeLa cells were cultured in 900 μL of growth medium, 100 μL of growth medium containing 100 μg / mL of oligomycin was added to make the final concentration of oligomycin 20 μg / mL.

Fluorescent microscope images of the cells were taken every 10 seconds. MetaFluor software (Molecular Devices, LLC) was used for camera and filter control and data recording. For MaLion G monochromatic fluorescence imaging, FF01-500 / 24 was used as an excitation filter, Di02-FF520 was used as a dichroic mirror, and FF01-542 / 27 was used as a light emission filter (all are Semrock, Optline Inc.). For MaLion R monochromatic fluorescence imaging, BP535-555HQ was used as the excitation filter, DM565HQ was used as the dichroic mirror, and BA570-625HQ was used as the emission filter (all from Olympus Corporation). For simultaneous imaging of MaLion G and R cytoplasm or mitochondrial localization fusion protein, BP460-480HQ and BP535-555HQ (Olympus Corporation) are used as excitation filters, and Di01-FF493 / 574 (Semrock, Opt Corporation) is used as a dichroic mirror. Line) and BA495-540HQ and BA570-625HQ were used as the light emission filters (Olympus Corporation). For MaLion B monochromatic fluorescence imaging, FF01-377 was used as an excitation filter, Di03-FF409 was used as a dichroic mirror, and FF02-447 was used as a light emission filter (all are Semrock, Optline Inc.). All experiments were performed using a temperature controlled circulation chamber with a CO ₂ incubator.

5A, 5B, and 5C are graphs showing changes in fluorescence after administration of NaF that inhibits glycolytic ATP production to HeLa cells in which MaLion G, R, and B are expressed, respectively. In FIGS. 5A, 5B, and 5C, the vertical axis represents normalized fluorescence intensity, and the horizontal axis represents time (minutes). Three minutes after the start of measurement, NaF was added to a final concentration of 10 mM. The light gray three waveforms in each figure show the measured values in three different dishes, and the dark gray single waveform shows the average value of these three waveforms. In the turn-on type ATP fluorescence sensors MaLion G, R, and B, the fluorescence intensity decreased until 15 minutes after the addition of NaF. This means that the intracellular ATP concentration has decreased.

FIG. 5D, FIG. 5E, and FIG. 5F are graphs showing changes in fluorescence after administration of NaF that inhibits glycolytic ATP production to HeLa cells in which negMaLion G, R, and B are expressed. FIG. 5G shows the start of fluorescence measurement of each fluorescent protein expressed in HeLa cells for MaLion G, R, and B and negMaLion G, R, and B based on the experimental results of the graphs of FIGS. 5A to 5F. It is a bar graph which shows the average value and standard deviation of the normalized fluorescence intensity 25 minutes after.

From FIG. 5D, FIG. 5E, and FIG. 5F, the fluorescence intensity after NaF administration also caused a change in fluorescence intensity even in negMaLion G, R, and B, which hardly responded to the change in ATP concentration. This may be due to changes in the cytoplasmic pH (Berg, J. et al., Nat. Methods 6, 161-166 (2009)). However, as shown in FIG. 5G, when the measurement results of MaLion G, R, and B and the measurement results of negMaLion G, R, and B were combined, the former fluorescence intensity was significantly lower than the latter fluorescence intensity. . Therefore, by combining the measurement results of MaLion G, R, and B with the measurement results of negMaLion G, R, and B, the change in ATP concentration is pH like the cytoplasm of the cell in which the glycolysis is inhibited. It was possible to measure the ATP concentration excluding the influence of the pH change even under conditions that occur simultaneously with the change of.

Next, two types of ATP fluorescence sensor proteins with different fluorescence wavelengths are localized in the cytoplasm, one in the mitochondria, and inhibits ATP production in the mitochondria, but glycolytic ATP production in the cytoplasm Changes in ATP concentration in both cytoplasm and mitochondria after administration of uninhibited oligomycin were measured simultaneously. For co-transfection of two types of expression vectors of ATP fluorescence sensor protein, 0.2 μg of expression vector containing ATP fluorescence sensor protein localized in the cytoplasm, and expression vector 0. 10 μL of Opti-MEM medium (Life Technologies Corporation, Thermo Fisher Scientific Inc.) supplemented with 2 μg and 0.2 μL of FuGENE HD transfection reagent (Promega Corporation) in advance was used. Figure 6 shows fluorescence at two wavelengths corresponding to two different ATP fluorescence sensor proteins in the same field after simultaneously transfecting HeLa cells with MaLion G expression vector and mito-MaLion R expression vector. It is the graph which showed the change of fluorescence after administering oligomycin 3 minutes after the microscope imaging start.

As is clear from FIG. 6, the ATP concentration in mitochondria decreased with inhibition of mitochondrial ATP production. Cytoplasmic ATP concentration decreased immediately after administration of oligomycin but increased over 15 minutes thereafter. The cytoplasmic and mitochondrial ATP kinetics suggest that glycolytic ATP production was enhanced when intracellular ATP began to decrease due to inhibition of mitochondrial ATP production. This example is the world's first experiment that succeeded in simultaneously observing ATP dynamics in different organelles within the same cell. In cancer cells, it is known that glycolytic ATP production is more developed than mitochondrial ATP production (Warburg effect). In the development of anticancer drugs, strategies that aim to block these two ATP production systems and lead to cell death are often adopted. However, this example specifically shows that these two ATP production systems interact. Thus, it is speculated that in the future development of drugs such as anticancer agents using the ATP production system as an action point, simultaneous observation of cytoplasmic and mitochondrial ATP dynamics using an ATP fluorescent sensor protein is used for drug evaluation.

2. Fluorescence Sensor Measurement of Intracellular ATP in Brown Adipocytes Mouse WT-1 cells of immortalized brown adipocyte (preadipocyte) line were 10% fetal bovine serum, GlutaMax ™, penicillin 100 IU / mL and streptomycin 100 μg / ML in Dulbecco's modified Eagle medium (low glucose, Thermo Fisher Scientific Inc.) at 37 ° C. in a 5% CO ₂ atmosphere. WT-1 cells are dr. Contributed by Yu-Hua Tseng (Joslin Diabetes Center, Harvard Medical School, Boston, USA). WT-1 cells differentiated into brown adipocytes on glass bottom dishes by induction of differentiation with BMP (bone morphogenetic protein) -7 (Tseng et al. (Nature, 454: 1000-1004 (2008)). After reaching a confluent state, 3.3 nM BMP-7 (354-BP, R & D Systems, Inc.), 20 nM insulin (Sigma-Aldrich Co. LLC.), And 1 nM T3 (Sigma-Aldrich Co.). Cell differentiation was initiated by pretreatment for 3 days with basal medium supplemented with LLC.) Confluent cells were induced cocktail (0.5 mM 3-isobutyl-1-methylxanthine (IBMX), 0 125 mM indomethacin, 5 μM dexamethasone, 20 n Insulin (all Sigma-Aldrich Co. LLC.)) Were treated for two days with basal medium was added.

Thereafter, the medium was changed to a basic medium supplemented with 20 nM insulin and 1 nM T3, and transfection of the expression vector of the ATP fluorescence sensor protein was performed. For experiments to measure the cytoplasmic and mitochondrial ATP concentrations of differentiated WT-1 cells, 0.4 μg of MaLion G expression vector, 0.4 μg of mito-MaLion R expression vector, and 2 μL of Lipofectamine 2000 (Invitrogen) And were used for transfection. For experiments in which the concentrations of calcium ions, cAMP and mitochondrial ATP in differentiated WT-1 cells were measured, 1.0 μg of B-Geco (Zhao, Y. et al., Science. 333, 1888-91 (2011)) Using an expression vector, 0.3 μg of Flamindo2 (Odaka, H., et al., PLoS One 9, e100252 (2014)), 0.2 μg of mito-MaLion R expression vector, and 2 μL of Lipofectamine 2000 (Invitrogen) The transfection was performed. Here, pcDNA3.1 (−) vector was used as the expression vector for these fluorescent proteins. After transfection, the cells were cultured at 37 ° C. under 5% CO ₂ atmosphere for 12 hours, after which the medium was replaced with a basic medium supplemented with 20 nM insulin and 1 nM T3, and cultured at 28 ° C. for 2 days. Prior to the fluorescence observation experiment, cells were cultured for 1 day in DMEM culture supplemented with 4.5 g / L glucose without fetal calf serum or hormones. The two-wavelength fluorescence observation using MaLion G and mito-MaLion R is described in 1. The same protocol as the fluorescence sensor measurement of intracellular ATP of HeLa cells was performed.

Three-wavelength fluorescence observation was performed with a confocal microscope (FV1000, Olympus Corporation) equipped with an oil immersion objective lens (PLAPO, 60 × 1.45NA). B-Geco, Flamindo2 and mito-MaLion R were excited with 405 nm, 488 nm and 543 nm laser lights, respectively, and fluorescence was imaged at wavelengths above 405-475 nm, 500-530 nm and 560 nm, respectively. Imaging was performed every 10 seconds. For drug stimulation experiments, 200 μL of isoproterenol and phenylephrine stock solutions were added to 1.8 mL cultures to a final concentration of 1 μM and 10 μM, respectively. All experiments were performed using a temperature controlled circulation chamber with a CO ₂ incubator.

FIG. 7A is a fluorescence microscopic image of B-Geco (Ca ²⁺ ions, blue), Flamindo 2 (cAMP, green) and mito-MaLion R (ATP, red) of differentiated WT-1 cells in the same visual field. The number on the upper left of each frame represents the time (minutes) after the observation starts. FIG. 7B is a graph showing changes in normalized fluorescence intensity of each probe over time. The arrow indicates that 1 μM isopreterenol was added 5 minutes after the start of observation (between the first and second frames in FIG. 7A). Isoproterenol is a β-adrenergic receptor agonist, B-Geco is a Ca ²⁺ ion turn-on fluorescent probe (excitation wavelength 378 nm, fluorescence wavelength 446 nm), and Flamindo 2 is a cAMP turn-off fluorescent probe (excitation). Wavelength 504 nm, fluorescence wavelength 523 nm).

Therefore, from FIG. 7A and FIG. 7B, it was found that with the activation of β-adrenergic receptor by isoproterenol, cAMP first increases and ATP decreases after a little delay. In brown adipocytes, adenylate cyclase on the cell membrane activated by receptor stimulation synthesizes cAMP, which becomes signal transduction and activates cAMP-dependent PKA. Thereafter, it is known that the fatty acid is released and acts on the uncoupling protein UCP1 on the mitochondrial membrane to eliminate the proton gradient that causes the membrane potential on the mitochondrial membrane (thermal production of brown adipocytes). The results of FIGS. 7A and 7B are consistent with this proposed mechanism. In this example, in addition to cAMP and ATP fluorescent probes, Ca ²⁺ ion fluorescent probes are also expressed in the cells at the same time, and the localization of cAMP, ATP and Ca ²⁺ ions in the same cells is quantified almost simultaneously over time. It was demonstrated that it was possible to measure. Therefore, it was speculated that the ATP fluorescent sensor protein can be used to elucidate the role of calcium ions in heat production in brown adipocytes.

3. Fluorescence sensor measurement of intracellular ATP in nematode pharyngeal muscles An adult C. elegans expressing ATP fluorescence sensor protein and the like uses a small amount of cyanoacrylate glue (Aron Alpha A, Daiichi-Sankyo). Then, it was fixed on a 3.5 cm glass bottom dish and covered with a 1.7% agar gel pad having a thickness of 0.5 cm. Fixed nematodes were immersed in M9 buffer (22 mM KH ₂ PO ₄ , 86 mM NaCl, 42 mM Na ₂ HPO ₄ and 1 mM MgSO ₄ ) and subjected to microscopic imaging. Room temperature was kept at 25 ° C. and sample preparation was completed within 30 minutes.

Nematodes are imaged with a 20x dry objective lens, a Nippon Kok disk confocal scanner (CSU-10, Yokogawa), an electron multiplying charge coupled device (EM-CCD) camera (C9100-02, Hamamatsu Photonics) An inverted epifluorescence microscope (Observer D1, Zeiss) equipped with For imaging with MaKion G and MaLion R Nipkow-disc confocal illumination, optically pumped semiconductor 488 nm laser (Sapphire 488LP, 50 mW, Coherent) and 568 nm laser (Sapphire 568LP, 50 mW, Coherent), dichroic mirror and emission filter, respectively. Used with a set (Di01-T 405/488/568/647 beam splitter and FF01-524 / 628 dual band bandpass filter, Semirock). An electromagnetically driven shutter (SSH-C4RA, Sigmakoki CO., LTD.) Is disposed in the optical path of the laser beam, and the opening and closing of the shutter is synchronized with the EM-CCD camera and is controlled by MetaMorph software (Molecular Devices, LLC). . The exposure time was 100 milliseconds. Imaging was performed every 10 seconds for 30 minutes. Nematode anesthesia was performed 5 minutes after the start of imaging by applying M9 physiological saline containing 0.5% 1-phenoxy-2-propanol.

FIG. 8A and FIG. 8B are graphs showing the results of simultaneous measurement of changes in the ATP concentration in the nematode pharyngeal muscle cytoplasm and mitochondria, and the arrow indicates 0.5% 1-phenoxy-2-propanol as an anesthetic. This shows that M9 saline containing (FIG. 8A) or not (FIG. 8B) was administered 5 minutes after the start of observation.

From FIG. 8A, the cytoplasmic ATP concentration of the nematode pharyngeal muscle rapidly decreased immediately after the administration of the anesthetic agent, and the normalized fluorescence intensity decreased to 20% at the time of the anesthetic agent administration 30 seconds after the administration of the anesthetic agent. In contrast, the mitochondrial ATP concentration decreased slowly, and the normalized fluorescence intensity decreased to 20% at the time of anesthetic administration 3 minutes after the administration of the anesthetic. From this result, it is considered that the anesthetic agent first inhibited ATP production in the mitochondria, and accordingly, cytoplasmic ATP decreased.
From FIG. 8B, the ATP concentration of the nematode pharyngeal muscle did not change significantly immediately after administration of the control M9 saline in both cytoplasm and mitochondria. The normalized fluorescence intensity decreased to 80% in the mitochondria and 60% in the cytoplasm in 5 minutes from the start of observation. This is probably because ATP production in mitochondria was reduced due to fatigue of nematodes by ultraviolet laser irradiation, and the ATP concentration in the cytoplasm was also reduced accordingly. This is the first example in the world in which ATP dynamics in the same cell in a single animal individual is observed simultaneously with different organelles, and could not be achieved without the present invention. By using a nematode that constantly expresses an ATP sensor, there is a possibility that it can be used as a drug screening tool that inhibits ATP synthesis, that is, a useful tool for anticancer agents and toxicity screening.

[Example 5] Construction of cGMP-specific fluorescent sensor protein cGMP Fluorescent Sensor Protein Design The cGMP fluorescent sensor protein uses Phosphoesterase 5α (PDE5α) as the cGMP binding domain, and Citrine (Griesbeck, O. et al., J. Biol. Chem. 276, 29188-29194 (2001)) as the fluorescent protein. Was used. The excitation wavelength of the fluorescent protein Citrine is 490 nm, and the emission wavelength region is 505 to 650 nm. Various polypeptide linkers were linked to the amino terminus and carboxyl terminus of PDE5α, and a fusion protein inserted into the fluorescent protein Citrine was prepared as a candidate protein for the cGMP fluorescent sensor protein (sometimes referred to as cGull series).

The structure of the domain and linker of the candidate protein of the cGMP fluorescence sensor is as follows. First, a domain that specifically binds to cGMP may be referred to as a “cGMP binding domain”. The cGMP binding domain is derived from PDE5α and its amino acid sequence is listed in the sequence listing as SEQ ID NO: 21. The polypeptide linker linked to the amino terminus of the cGMP binding domain may be referred to as “N-terminal linker”, and the polypeptide linker linked to the carboxyl terminus of the cGMP binding domain may be referred to as “C-terminal linker”.

Therefore, cGull series cGMP fluorescent sensor candidate proteins are [Citrine-N domain]-[N-terminal linker]-[cGMP binding domain]-[C-terminal linker]-[ The polypeptide domain and the polypeptide linker are arranged in the order of [Citrine-C domain].

As a procedure for constructing a candidate protein for the cGMP fluorescent sensor protein, “ATP” of Example 1 was used except that a polynucleotide encoding PDE5α was synthesized as a cGMP binding domain (Integrated DNA Technologies, Medical and Biological Laboratories, Inc.). This was carried out using the same method as “construction of candidate protein for fluorescent sensor protein”. In the presence or absence of cGMP at a final concentration of 100 μM, the fluorescence characteristics of the cGMP fluorescence sensor candidate proteins of each clone were measured using a fluorescence spectrophotometer (Hitachi F-2700, Hitachi High-Tech Science Co., Ltd.).

Although details are omitted, when the amino acid sequences of the N-terminal side linker and the C-terminal side linker are SEQ ID NOS: 31 and 32, respectively, the dynamic range is 6.5 times, and the cGMP fluorescence sensor has the largest fluorescence intensity in the presence of cGMP. A protein was obtained.
This candidate protein with the largest dynamic range was named cGull. The full-length amino acid sequence of cGull is shown in SEQ ID NO: 12.

FIG. 9A is a fluorescence spectrum diagram of cGull in the presence and absence of 100 μM cGMP. The horizontal axis of FIG. 9A represents the fluorescence wavelength, and the vertical axis represents the relative value of the fluorescence intensity. The solid and dotted lines in the graph represent the spectral curves in the presence and absence of cGMP.
As is clear from FIG. 9A, cGull increased its fluorescence in the presence of 100 μM cGMP by 550%, respectively, compared to the absence of cGMP.

2. Intracellular cGMP fluorescence sensor measurement of HeLa cells Using the same method as in Example 4, an expression vector containing a cGMP fluorescence sensor protein was introduced into HeLa cells. Next, the medium was replaced with a phenol red-free growth medium (containing 1 mM 8-Br-cGMP) immediately before the fluorescence measurement. For the fluorescence microscope measurement, an inverted microscope (IX81, Olympus Corporation) equipped with a cooled CCD camera (Cool SNAP HQ2, Photometrics) and an oil immersion objective lens (Plan Apo 60 × 1.42 NA) was used.
For the cytoplasmic cGMP production inhibition experiment, SNAP (S-Nitroso-N-Acetyl-D, L-Penicillamine), which is a nitric oxide donor, was used. SNAP was added to a dish in which HeLa cells were cultured in 1.5 mL of growth medium to make the final concentration of SNAP 300 μM. Fluorescence microscope images of the cells were taken every 5 minutes. MetaFluor software (Molecular Devices, LLC) was used for camera and filter control and data recording. For monochromatic fluorescence imaging, FF01-500 / 24 was used as the excitation filter, Di02-FF520 was used as the dichroic mirror, and FF01-542 / 27 was used as the emission filter (all are Semrock, Optline Co., Ltd.). All experiments were performed using a temperature controlled circulation chamber with a CO ₂ incubator.

FIG. 9B shows changes in fluorescence from the start of fluorescence microscopic imaging to 20 minutes after administration of 8-Br-cGMP (1 mM) or SNP (300 μM) of nitric oxide donor to HeLa cells expressing cGull. It is an image which shows.
FIG. 9C shows the fluorescence from the start of fluorescence microscopic imaging to 20 minutes after administration of 8-Br-cGMP (1 mM) or nitric oxide donor SNAP (300 μM) to HeLa cells expressing cGull. It is a graph which shows the change of. In FIG. 9C, the vertical axis represents normalized fluorescence intensity, and the horizontal axis represents time (minutes). The light gray 2 or 3 waveforms in each figure show the measured values in 2 or 3 different dishes, and the dark gray 1 waveform shows the average value of these 3 waveforms.

In the turn-on type cGMP fluorescence sensor cGull, the fluorescence intensity increased and maintained until 5 minutes after the addition of 8-Br-cGMP, whereas the fluorescence intensity gradually increased until 20 minutes after the addition of SNAP. This means that cGMP production in cells was partially inhibited.

[Example 6] Construction of cAMP-specific fluorescent sensor protein cAMP Fluorescence Sensor Protein Design The cAMP fluorescence sensor protein uses exchange factor direct by cAMP 1 (EPAC1) as the cAMP binding domain, and mapple (Shaner, NC et al., Nature Methods 54: 5). (2008)) was used. In addition, mA Apple is Zhao, Y. et al. (Science. 333, 1888-1891 (2011)), amino acid substitution mutations were introduced in several places (SEQ ID NOs: 7 and 8). The excitation wavelength of the fluorescent protein mA Apple is 550 nm, and the emission wavelength region is 575 to 700 nm. Various polypeptide linkers were linked to the amino terminus and carboxyl terminus of EPAC1, and a fusion protein inserted into the fluorescent protein mA Apple was prepared as a candidate protein for the cAMP fluorescent sensor protein (sometimes referred to as the “Pink Flamen series”). .

The structure of the domain and linker of the candidate protein of the cAMP fluorescence sensor is as follows. First, a domain that specifically binds to cAMP may be referred to as a “cAMP binding domain”. The cAMP binding domain is derived from EPAC1, and its amino acid sequence is listed in the sequence listing as SEQ ID NO: 20. A polypeptide linker linked to the amino terminus of the cAMP binding domain may be referred to as “N-terminal linker”, and a polypeptide linker linked to the carboxyl terminus of the cAMP binding domain may be referred to as “C-terminal linker”.

Therefore, the candidate protein for the Pink Flamindo series cAMP fluorescent sensor is [mApplee-N domain]-[N-terminal side linker]-[cAMP-binding domain]-[C-terminal side linker]- A polypeptide domain and a polypeptide linker are arranged in the order of [mAapple-C domain].

As a procedure for constructing a candidate protein for the cAMP fluorescent sensor protein, “ATP” of Example 1 was used except that a polynucleotide encoding EPAC1 was synthesized as a cAMP binding domain (Integrated DNA Technologies, Institute of Medical Biology). This was carried out using the same method as “construction of candidate protein for fluorescent sensor protein”. In the presence or absence of cAMP at a final concentration of 100 μM, the fluorescence characteristics of the cAMP fluorescence sensor candidate proteins of each clone were measured using a fluorescence spectrophotometer (Hitachi F-2700, Hitachi High-Tech Science Co., Ltd.).

Although details are omitted, when the amino acid sequences of the N-terminal side linker and the C-terminal side linker are SEQ ID NOS: 33 and 34, respectively, the dynamic range is 4.5 times, and the cAMP fluorescence sensor has the largest fluorescence intensity in the presence of cAMP. A protein was obtained.
This candidate protein with the largest dynamic range was named Pink Flamindo. The full-length amino acid sequence of Pink Flamindo is shown in SEQ ID NO: 13.

FIG. 10A is a fluorescence spectrum diagram of Pink Flamindo in the presence and absence of 100 μM cAMP. The horizontal axis of FIG. 10A represents the fluorescence wavelength, and the vertical axis represents the relative value of the fluorescence intensity. The solid and dotted lines in the graph represent the spectral curve in the presence and absence of cAMP.
As is clear from FIG. 10A, the fluorescence in the presence of 100 μM cAMP was increased by 350% in the case of Pin Flamendo as compared with the absence of cAMP.

2. Fluorescence sensor measurement of intracellular cGMP in HeLa cells Using the same method as in Example 4, an expression vector containing a cAMP fluorescence sensor protein was introduced into HeLa cells. Then, immediately before the fluorescence measurement, the medium was replaced with a phenol red-free growth medium (containing 100 μM Forskolin, an adenylate cyclase activator). For the fluorescence microscope measurement, an inverted microscope (IX81, Olympus Corporation) equipped with a cooled CCD camera (Cool SNAP HQ2, Photometrics) and an oil immersion objective lens (Plan Apo 60 × 1.42 NA) was used.
In the cytosolic cAMP production inhibition experiment, IBMX (3-isobutyl-1-methylxanthine), which is a phosphodiesterase inhibitor, was used. IBMX was added to a dish in which HeLa cells were cultured in 1.5 mL of growth medium to bring the final concentration of IBMX to 500 μM. Fluorescence microscope images of the cells were taken every 5 minutes. MetaFluor software (Molecular Devices, LLC) was used for camera and filter control and data recording. For monochromatic fluorescence imaging, BP535-555HQ was used as the excitation filter, DM565HQ was used as the dichroic mirror, and BA570-625HQ was used as the emission filter (all from Olympus Corporation). All experiments were performed using a temperature controlled circulation chamber with a CO ₂ incubator.

FIG. 10B is an image showing changes in fluorescence from the start of fluorescence microscopic image capturing to 20 minutes after administration of Forskolin (100 μM) or IBMX (500 μM) to HeLa cells in which Pink Flamindo was expressed.
FIG. 10C is a graph showing changes in fluorescence from the start of fluorescence microscopic image capturing to 20 minutes after administration of Forskolin (100 μM) or IBMX (500 μM) to HeLa cells in which Pin Flamindo was expressed. In FIG. 9C, the vertical axis represents normalized fluorescence intensity, and the horizontal axis represents time (minutes). The light gray three waveforms in each figure show the measured values in three different dishes, and the dark gray single waveform shows the average value of these three waveforms.

In the turn-on type cAMP fluorescence sensor Pink Flamindo, the fluorescence intensity rapidly increased and gradually decreased until 3 minutes after the addition of Forskolin, whereas the fluorescence intensity gradually increased until 20 minutes after the addition of IBMX. This means that cAMP production in cells was partially inhibited.

[Example 7] Construction of a fluorescence sensor protein specific for BGP Design of BGP Fluorescent Sensor Protein A BGP fluorescent sensor protein was designed to observe the intracellular localization of osteocalcin (bone Gla protein (BGP)).
As the BGP fluorescence sensor protein, an anti-BGP antibody was used as a BGP binding domain, and GFP (Griesbeck, O. et al. (J. Biol. Chem. 276, 29188-29194 (2001))) was used as a fluorescence protein. The excitation wavelength of the fluorescent protein GFP is 480 nm, and the emission wavelength region is 500 to 520 nm. Further, a domain consisting of amino acid residues from the 1st to 144th N-terminal side of GFP is referred to as “GFP-N domain”, and a domain consisting of amino acid residues from 145th to 238th from the N-terminal side of GFP is referred to as “GFP”. -C domain ". The amino acid sequence of the GFP-N domain is listed in the sequence listing as SEQ ID NO: 3, and the amino acid sequence of the GFP-C domain is listed as SEQ ID NO: 4, respectively. A fusion protein inserted between the heavy chain and the light chain of an anti-BGP antibody by linking various polypeptide linkers to the amino terminus of the GFP-C domain and the carboxyl terminus of the GFP-N domain is used as a candidate for a BGP fluorescence sensor protein. Produced as a protein (sometimes referred to as gBGP series).

The configuration of the domain and linker of the candidate protein of the BGP fluorescence sensor is as follows. First, a domain that specifically binds to BGP may be referred to as a “BGP binding domain”. The BGP binding domain is derived from an anti-BGP antibody and its amino acid sequence is listed in the sequence listing as SEQ ID NO: 22 (heavy chain) and 23 (light chain). A polypeptide linker that links to the amino terminus of the GFP-C domain is an “N-terminal linker”, a polypeptide linker that binds to the carboxyl terminus of the GFP-C domain and the amino terminus of the GFP-N domain is an “intermediate linker”, and the GFP-N domain A polypeptide linker linked to the carboxyl terminus of is sometimes referred to as a “C-terminal linker”.

Therefore, the candidate proteins for the BGP fluorescent sensor of the gBGP series are [anti-BGP antibody heavy chain]-[N-terminal linker]-[GFP-C domain]-[intermediate linker]-in the direction from the amino terminus to the carboxyl terminus. The polypeptide domain and the polypeptide linker are arranged in the order of [GFP-N domain]-[C-terminal side linker]-[light chain of anti-BGP antibody].

As a procedure for constructing a candidate protein for BGP fluorescence sensor protein, polynucleotides encoding heavy and light chains of anti-BGP antibody were respectively synthesized as BGP binding domains (Integrated DNA Technologies, Institute of Medical Biology) Except for the above, the same method as in “Construction of ATP fluorescent sensor protein candidate protein” in Example 1 was used. In the presence or absence of a final concentration of 100 μM BGP7C, the fluorescence characteristics of the candidate protein of the BGP fluorescence sensor of each clone were measured using a fluorescence spectrophotometer (Hitachi F-2700, Hitachi High-Tech Science Co., Ltd.).

Although details are omitted, when the amino acid sequences of the N-terminal linker, intermediate linker, and C-terminal linker are SEQ ID NOS: 35, 36, and 37, the dynamic range is 4.0 times, and fluorescence in the presence of BGP7C A BGP fluorescence sensor protein with the highest intensity was obtained.
This candidate protein with the largest dynamic range was named gBGP. The full-length amino acid sequence of gBGP is shown in SEQ ID NO: 14, and the base sequence is shown in SEQ ID NO: 50.

FIG. 11A is an excitation / fluorescence spectrum diagram of gBGP in the presence and absence of 100 μM BGP7C. The horizontal axis in FIG. 11A represents the excitation wavelength and the fluorescence wavelength, and the vertical axis represents the relative value of the fluorescence intensity. The solid and dotted lines in the graph represent the spectral curves in the presence and absence of BGP7C.
As is clear from FIG. 11A, gBGP increased the fluorescence in the presence of 100 μM BGP7C by 300%, respectively, compared to the absence of BGP7C.

FIG. 11B is a graph showing the ligand-specific and BGP7C concentration-dependent changes in the fluorescence intensity of gBGP. The horizontal axis of FIG. 11B represents the concentration of BGP7C or Myc as a control, and the vertical axis represents the relative value of fluorescence intensity.
As is clear from FIG. 11B, it was shown that the fluorescence intensity of gBGP changes only depending on the concentration of BGP7C.

2. Fluorescence sensor measurement of intracellular BGP in HeLa cells First, a cell membrane-localized peptide (SEQ ID NO: 42), BGP7C to which mCherry is bound (hereinafter sometimes referred to as “PMmCherry-BGP7C”), and a nuclear-localized peptide A vector expressing BGP7C to which (SEQ ID NO: 43) and mCherry were bound (hereinafter sometimes referred to as “NLSmCherry-BGP7C”) was prepared.
Next, using the same method as in Example 4, an expression vector containing a BGP fluorescent sensor protein or an expression vector containing only GFP, and an expression vector containing PMmCherry-BGP7C or NLSmCherry-BGP7C were introduced into HeLa cells. Subsequently, the medium was replaced with a phenol red-free growth medium immediately before the fluorescence measurement. A confocal laser microscope (FV1000, Olympus Corporation) was used for the measurement. The laser wavelengths were 488 nm and 543 nm and were imaged at wavelengths greater than 500-530 nm and 560 nm.

FIG. 11C is an image showing fluorescence in HeLa cells expressing gBGP or GFP, and cell membrane-localized mCherry-BGP7C or cell membrane-localized mCherry.
FIG. 11D is an image showing fluorescence in HeLa cells in which gBGP or GFP and nuclear localized mCherry-BGP7C or cell membrane localized mCherry are expressed.

When the turn-on type BGP fluorescence sensor gBGP was introduced with PMmCherry-BGP7C, localization on the cell membrane was observed, and when introduced with NLSmCherry-BGP7C, localization in the nucleus was observed.

[Example 8] Construction of HSA-specific fluorescent sensor protein Design of HSA Fluorescent Sensor Protein A human serum albumin (HSA) fluorescent sensor protein was designed.
As the HSA fluorescence sensor protein, an anti-HSA antibody was used as the HSA binding domain, and GFP (Griesbeck, O., et al. (J. Biol. Chem. 276, 29188-29194 (2001))) was used as the fluorescence protein. The excitation wavelength of the fluorescent protein GFP is 480 nm, and the emission wavelength region is 500 to 520 nm. Further, a domain consisting of amino acid residues from the 1st to 144th N-terminal side of GFP is referred to as “GFP-N domain”, and a domain consisting of amino acid residues from 145th to 238th from the N-terminal side of GFP is referred to as “GFP”. -C domain ". The amino acid sequence of the GFP-N domain is listed in the sequence listing as SEQ ID NO: 3, and the amino acid sequence of the GFP-C domain is listed as SEQ ID NO: 4, respectively. A fusion protein inserted between the heavy chain and the light chain of an anti-HSA antibody by linking various polypeptide linkers to the amino terminus of the GFP-C domain and the carboxyl terminus of the GFP-N domain is used as a candidate for an HSA fluorescence sensor protein. Produced as a protein (sometimes referred to as gHSA series).

The structure of the candidate protein domain and linker of the HSA fluorescence sensor is as follows. First, a domain that specifically binds to HSA may be referred to as an “HSA binding domain”. The HSA binding domain is derived from an anti-HSA antibody. A polypeptide linker that links to the amino terminus of the GFP-C domain is an “N-terminal linker”, a polypeptide linker that binds to the carboxyl terminus of the GFP-C domain and the amino terminus of the GFP-N domain is an “intermediate linker”, and the GFP-N domain A polypeptide linker linked to the carboxyl terminus of is sometimes referred to as a “C-terminal linker”.

Therefore, the candidate proteins of the gHSA series HSA fluorescence sensor are [anti-HSA antibody heavy chain]-[N-terminal linker]-[GFP-C domain]-[intermediate linker]-in the direction from the amino terminus to the carboxyl terminus. A polypeptide domain and a polypeptide linker are arranged in the order of [GFP-N domain]-[C-terminal side linker]-[anti-HSA antibody light chain].

As a procedure for constructing a candidate protein for the HSA fluorescence sensor protein, polynucleotides encoding the heavy chain and the light chain of the anti-HSA antibody were respectively synthesized as HSA binding domains (Integrated DNA Technologies, Institute of Medical Biology) Except for the above, the same method as in “Construction of ATP fluorescent sensor protein candidate protein” in Example 1 was used. In the presence or absence of HSA at a final concentration of 2 μM, the fluorescence characteristics of candidate proteins of the HSA fluorescence sensor of each clone were measured using a fluorescence spectrophotometer (Hitachi F-2700, Hitachi High-Tech Science Co., Ltd.).

Although details are omitted, the amino acid sequences of the N-terminal linker, intermediate linker, and C-terminal linker are “LE” (Leu-Glu), “GGTGGS” (SEQ ID NO: 53), and “TR” (Thr— Arg), the dynamic range was 1.2 times, and an HSA fluorescence sensor protein having the highest fluorescence intensity in the presence of HSA was obtained.
This candidate protein with the largest dynamic range was named gHSA. The full-length amino acid sequence of gHSA is shown in SEQ ID NO: 51, and the base sequence is shown in SEQ ID NO: 52.

FIG. 12 is a fluorescence spectrum diagram of gHSA in the presence and absence of 2 μM HSA. The horizontal axis in FIG. 12 represents the fluorescence wavelength, and the vertical axis represents the relative value of the fluorescence intensity. The solid and dotted lines in the graph represent the spectral curves in the presence and absence of HSA.
As is clear from FIG. 12, gHSA increased fluorescence by 20% in the presence of 2 μM HSA as compared to the absence of HSA.

According to the present invention, a highly sensitive ligand fluorescent sensor protein can be provided regardless of the type of ligand to be detected. According to the present invention, the ratio of the fluorescence intensity in the presence of a high concentration of ligand to the fluorescence intensity in the absence of ligand under physiological conditions is sufficiently high, and the physiological and / or disease of a cell or non-human organism. Variations in the concentration of the physical ligand can be detected.

Claims (29)

1. A ligand fluorescent sensor protein that changes its fluorescence characteristics in response to a specific ligand,
   The ligand fluorescent sensor protein includes a first fluorescent protein domain, an N-terminal linker, a ligand binding domain, a C-terminal linker, and a second fluorescent protein domain,
   The fluorescent protein used for the ligand fluorescent sensor protein has a β barrel structure,
   The first fluorescent protein domain includes a β sheet region of β1 to β3 from the N-terminus of the fluorescent protein, an α helix region following the β sheet region, and a β sheet region of β4 to β6;
   The second fluorescent protein domain comprises the same β7-β11 β-sheet region of the fluorescent protein as the first fluorescent protein domain;
   The ligand fluorescent sensor protein, wherein the N-terminal linker and the C-terminal linker are each independently a polypeptide consisting of one or several amino acids.
2. The ligand fluorescent sensor protein according to claim 1, wherein the fluorescent protein is BFP, GFP, Citriline, or mAapple.
3. The ligand fluorescent sensor protein has a first fluorescent protein domain, an N-terminal linker, a ligand binding domain, a C-terminal linker, and a second fluorescent protein domain from the N-terminus toward the C-terminus. The ligand fluorescent sensor protein according to claim 1 or 2, comprising a polypeptide formed by directly connecting peptide bonds in this order.
4. The ligand fluorescent sensor protein comprises two ligand binding domains;
   From the N-terminus toward the C-terminus, the first ligand binding domain, the N-terminal linker, the second fluorescent protein domain, the first fluorescent protein domain, the C-terminal linker, and the second ligand binding The ligand fluorescent sensor protein according to claim 1 or 2, wherein the domain includes a polypeptide directly linked by a peptide bond in this order.
5. The first fluorescent protein domain includes any of the following polypeptides (B1) to (B3):
   The second fluorescent protein domain includes any of the following polypeptides (C1) to (C3):
   The ligand fluorescent sensor protein according to claim 1 or 2, wherein the first fluorescent protein domain and the second fluorescent protein domain are derived from the same fluorescent protein.
   (B1) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 1, 3, 5, or 7,
   (B2) the amino acid sequence represented by SEQ ID NO: 1, 3, 5, or 7, comprising an amino acid sequence in which one or several amino acids are deleted, inserted, substituted or added, and the second fluorescence A polypeptide that forms a β-barrel structure with a protein domain and fluoresces,
   (B3) including an amino acid sequence having an identity of 80% or more with the amino acid sequence represented by SEQ ID NO: 1, 3, 5, or 7, and forming a β barrel structure with the second fluorescent protein domain, A fluorescent polypeptide,
   (C1) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 2, 4, 6, or 8,
   (C2) the amino acid sequence represented by SEQ ID NO: 2, 4, 6, or 8, comprising an amino acid sequence in which one or several amino acids are deleted, inserted, substituted or added, and the first fluorescence A polypeptide that forms a β-barrel structure with a protein domain and fluoresces,
   (C3) comprising an amino acid sequence having 80% or more identity with the amino acid sequence represented by SEQ ID NO: 2, 4, 6, or 8, and forming a β barrel structure with the first fluorescent protein domain, Fluorescent polypeptide
6. The ligand fluorescent sensor protein according to any one of claims 1 to 5, wherein the ligand is a nucleotide or a derivative thereof, a nucleic acid, a sugar chain, a protein, a lipid complex, or a low molecular compound.
7. The ligand fluorescent sensor protein according to claim 6, wherein the nucleotide or a derivative thereof is ATP, cAMP, or cGMP.
8. The ligand fluorescent sensor protein according to claim 6, wherein the protein is an antigen or an antibody.
9. The ligand fluorescent sensor protein according to any one of claims 1 to 8, comprising a polypeptide of any one of the following (D1) to (D3).
   (D1) a polypeptide comprising the amino acid sequence represented by any of SEQ ID NOs: 9 to 14,
   (D2) an amino acid sequence represented by any one of SEQ ID NOs: 9 to 14, including an amino acid sequence in which one or several amino acids are deleted, inserted, substituted or added, and the polypeptide (D1) Polypeptides having the same ability to bind to a ligand and fluorescent properties;
   (D3) Ligand binding ability and fluorescence, which include the amino acid sequence of 80% or more identity with the amino acid sequence represented by any of SEQ ID NOs: 9 to 14, and are identical to the polypeptide (D1) Polypeptide having properties
10. The ligand fluorescent sensor protein according to any one of claims 1 to 9, further comprising an organelle localization signal peptide.
11. The ligand fluorescent sensor protein according to claim 10, wherein the organelle localization signal peptide is a mitochondrial localization signal peptide.
12. The ligand fluorescent sensor protein according to claim 10, wherein the organelle localization signal peptide is a nuclear localization signal peptide.
13. The ligand fluorescent sensor protein according to any one of claims 1 to 12, further comprising a cell membrane-permeable peptide.
14. A polynucleotide encoding the ligand fluorescent sensor protein according to any one of claims 1 to 13.
15. An expression vector comprising the polynucleotide according to claim 14.
16. A cell comprising at least one type of ligand fluorescent sensor protein according to any one of claims 1 to 13.
17. A cell having a chromosome containing at least one polynucleotide encoding the ligand fluorescent sensor protein according to any one of claims 1 to 13.
18. A cell comprising at least one expression vector according to claim 15.
19. A non-human organism comprising the cell according to any one of claims 16 to 18.
20. The ligand fluorescent sensor protein according to any one of claims 1 to 13, the polynucleotide according to claim 14, the expression vector according to claim 15, and the cell according to any one of claims 16 to 18. And a ligand concentration measurement kit comprising at least one selected from the group consisting of non-human organisms according to claim 19.
21. A calibration curve creating step of contacting the ligand fluorescence sensor protein according to any one of claims 1 to 13 with a standard solution containing a ligand of a known concentration, measuring fluorescence intensity, and creating a calibration curve;
    A fluorescence measurement step of measuring fluorescence intensity by contacting the ligand fluorescence sensor protein with a solution containing a ligand of unknown concentration;
    In the calibration curve creating step, based on the created calibration curve, a concentration determining step for determining a ligand concentration with respect to the fluorescence intensity measured in the fluorescence measuring step;
    A method for determining a ligand concentration in a test sample.
22. A method for detecting a change in ligand concentration over time in a living cell, comprising the step of measuring fluorescence intensity over time using the cell according to any one of claims 16 to 18.
23. A method for detecting a change in a ligand concentration over time in a living non-human organism, comprising the step of measuring fluorescence intensity over time using the non-human organism according to claim 19.
24. An ATP fluorescent sensor protein whose fluorescence characteristics change in response to an ATP concentration specifically, the ATP fluorescent sensor protein comprising a first fluorescent protein domain and an N-terminal linker from the N-terminus toward the C-terminus A polypeptide in which an ATP-binding domain, a C-terminal linker, and a second fluorescent protein domain are directly linked by a peptide bond in this order,
    The first fluorescent protein domain includes a β sheet region of β1 to β3, an α helix region following this, and a β sheet region of β4 to β6 from the N terminus of the fluorescent protein BFP, Citrine or mA Apple,
    The second fluorescent protein domain comprises a β-sheet region of β7-β11 of the same fluorescent protein as the first fluorescent protein domain;
    The ATP binding domain consists of the ε subunit of F ₀ F ₁ -ATP synthase,
    Each of the N-terminal side linker and the C-terminal side linker is a polypeptide consisting of one or several amino acids, and is an ATP fluorescent sensor protein.
25. (A11) The ATP binding domain is the amino acid sequence of SEQ ID NO: 15, the first and second fluorescent protein domains are the amino acid sequences of SEQ ID NOS: 1 and 2, respectively, and the N-terminal and C-terminal linkers are MaLion B polypeptides, which are the amino acid sequences of SEQ ID NOs: 16 and 17, respectively;
    (A12) The ATP-binding domain, the first fluorescent protein domain, the second fluorescent protein domain, the N-terminal linker, and the C-terminal linker are each independently SEQ ID NOs: 15, 1, 2, 16, and 17 amino acid sequences or amino acid sequences in which one or several amino acids are deleted, substituted or added to the amino acid sequences of SEQ ID NOs: 13, 1, 2, 14, and 15, and MaLion B poly A MaLion B polypeptide having the same ATP binding ability and fluorescence properties as the peptide (A11);
    (B11) The ATP-binding domain is the amino acid sequence of SEQ ID NO: 15, the first and second fluorescent protein domains are the amino acid sequences of SEQ ID NOs: 5 and 6, respectively, and the N-terminal and C-terminal linkers are MaLion G polypeptide, which is the amino acid sequence of WRG (Trp-Arg-Gly) and SEQ ID NO: 18, respectively,
    (B12) The ATP binding domain, the first fluorescent protein domain, the second fluorescent protein domain, the N-terminal side linker and the C-terminal side linker are each independently SEQ ID NO: 15, 5, 6, WRG (Trp- Arg-Gly) and the amino acid sequence of SEQ ID NO: 18, or SEQ ID NOs: 15, 5, 6, WRG (Trp-Arg-Gly) and the amino acid sequence of SEQ ID NO: 18 have one or several amino acids deleted, A MaLion G polypeptide that is a substituted or added amino acid sequence and has the same ATP binding ability and fluorescence characteristics as the MaLion G polypeptide (B11);
    (C11) The ATP binding domain is the amino acid sequence of SEQ ID NO: 15, the first and second fluorescent protein domains are the amino acid sequences of SEQ ID NOs: 7 and 8, respectively, and the N-terminal and C-terminal linkers are MaLion R polypeptide, which is the amino acid sequence of SEQ ID NO: 19 and PEE (Pro-Glu-Glu), respectively
    (C12) The ATP binding domain, the first fluorescent protein domain, the second fluorescent protein domain, the N-terminal linker and the C-terminal linker are SEQ ID NOs: 15, 7, 8, 19, and PEE (Pro -Glu-Glu) amino acid sequence or SEQ ID NOs: 15, 7, 8, 19 and amino acid sequence of PEE (Pro-Glu-Glu) with one or several amino acids deleted, substituted or added 25. At least one polypeptide selected from the group consisting of MaLion R polypeptides that are sequences and have the same ATP binding ability and fluorescence properties as MaLion R polypeptides (C11). ATP fluorescence sensor protein.
26. (A21) a MaLion B polypeptide consisting of the amino acid sequence of SEQ ID NO: 9,
    (A22) The amino acid sequence of SEQ ID NO: 9 consists of an amino acid sequence in which one or several amino acids are deleted, substituted or added to the amino acid sequence excluding the amino acid sequences of SEQ ID NOS: 16 and 17, and MaLion B A MaLion B polypeptide having the same ATP binding ability and fluorescence properties as the polypeptide (A21);
    (B21) a MaLion G polypeptide consisting of the amino acid sequence of SEQ ID NO: 10,
    (B22) one of amino acid sequences excluding the amino acid sequence of WRG (Trp-Arg-Gly) at positions 146 to 148 of SEQ ID NO: 10 and the amino acid sequence of SEQ ID NO: 18 among the amino acid sequences of SEQ ID NO: 10 or MaLion G polypeptide consisting of an amino acid sequence in which several amino acids are deleted, substituted or added, and having the same ATP binding ability and fluorescence characteristics as MaLion G polypeptide (B21);
    (C21) a MaLion R polypeptide consisting of the amino acid sequence of SEQ ID NO: 11,
    (C22) One of the amino acid sequences of SEQ ID NO: 11 excluding the amino acid sequence of PEE (Pro-Glu-Glu) at positions 288 to 290 of SEQ ID NO: 11 and the amino acid sequence of SEQ ID NO: 19 or It is selected from the group consisting of an MaLion R polypeptide consisting of an amino acid sequence in which several amino acids are deleted, substituted or added, and having the same ATP binding ability and fluorescence characteristics as MaLion R polypeptide (C21). The ATP fluorescent sensor protein according to claim 24 or 25, comprising at least one polypeptide.
27. A fluorescent composition comprising the ATP fluorescent sensor protein according to any one of claims 24 to 26.
28. The fluorescent composition according to claim 27, wherein the ATP fluorescent sensor protein is immobilized on a solid support.
29. 29. The fluorescent composition according to claim 28, wherein the ATP fluorescent sensor protein is at least two types of ATP fluorescent sensor proteins having different pairs of first and second fluorescent protein domains.

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