Method for analyzing G-protein-coupled receptor

Abstract

The present invention provides a method capable of expressing and functioning a G protein coupled receptor (GPCR). A method for expressing a GPCR polypeptide, said method comprising a step of expressing a GPCR polypeptide in a cell, said GPCR polypeptide being derived from the expression of the GPCR polypeptide in a target GPCR (wherein, the expression of the GPCR polypeptide in the target GPCR (wherein, the expression of the GPCR polypeptide in the target GPCR; and an amino acid sequence in which at least one of amino acid residues different from the consensus amino acid sequence is changed to an amino acid residue of the consensus amino acid sequence at a position corresponding to the amino acid residue of the consensus amino acid sequence in the amino acid sequence of the olfactory receptor. The consensus amino acid sequence is an amino acid sequence derived from a comparison of an amino acid sequence of the target GPCR and an amino acid sequence of a GPCR encoded by an ortholog of the target GPCR in a vertebrate.

Description

Technical Field

[0001] The present invention relates to a method for analyzing G protein coupled receptors. Background Art

[0002] As a constituent member of an organism, cells recognize changes in the surrounding physiological conditions and change their behavior. Biological individuals use their senses to perceive changes in the external environment to adopt adaptive behaviors. The appropriate response of each life unit to endogenous information chemicals such as pheromones and neurotransmitters, or exogenous information chemicals such as odorants, is the foundation of the mechanism of the organism. Responsible for these chemical receptions are receptor proteins possessed by each tissue cell. In recent years, many biological individuals lacking specific receptor genes have been created, confirming the necessity of receptors in physiological functions. In addition, various drugs that use receptors as target molecules have been put into practical use as drugs to adjust physiological functions. On the other hand, there are still a large number of receptors whose functions are unclear.

[0003] The representative receptor possessed by organisms is the G protein-coupled receptor (GPCR). It is characterized by having a seven-transmembrane structure, which is converted into an activated structure by binding to an agonist, and transmits signals basically through interaction with G proteins in cells. It is known that there are about 800 GPCRs in humans, of which about half are not directly involved in physiological functions in the body related to sensation. Information on human GPCRs other than olfactory receptors and vomeronasal receptors can be obtained from the database (G protein-coupled receptors (IUPHAR / BPS Guide to PHARMACOLOGY, http: / / www.guidetopharmacology.org / GRAC / FamilyDisplayForward?familyId=694)). As of November 2017, 134 GPCRs are used as target molecules for drugs approved in the United States (Non-patent Document 1). In addition, it is shown that receptors that are believed to be involved only in sensation are actually expressed in tissues other than sensory tissues and participate in specific physiological functions in the body.

[0004] For example, the vomeronasal 1 receptor (VN1R) is a GPCR of the Class A family, and there are 5 genes in humans. Based on the analysis of the same gene in mice, it is predicted that it is expressed in the nasal cavity and is responsible for the recognition of pheromones (Non-Patent Document 2). Regarding VN1R1 in humans, only one case of functional analysis in cultured cells has been reported, and the report showed that it recognizes low-molecular volatile compounds (Non-Patent Document 2). In addition, in a large-scale survey in Sweden, it was studied what kind of differences in properties are caused by the genetic polymorphism of VN1R1. The results showed that different sexual characteristics in women were confirmed, suggesting the potential function of VN1R1 (Non-Patent Document 3). Regarding the other VN1R2~5, there are no reports of functional analysis such as the determination of the recognized substances.

[0005] Taste receptor TAS1R (taste 1 receptor) is a gene family discovered as a GPCR expressed in the tongue. There are three genes TAS1R1, TAS1R2, and TAS1R3 in the human genome, all of which are classified as Class C GPCRs. In the taste of the tongue, TAS1R1 and TAS1R2 are expressed in different taste cells and are co-expressed with TAS1R3. Class C GPCRs, including metabotropic glutamate receptors, are known to function by forming dimers, and TAS1R is no exception. When TAS1R3 is co-expressed with either TAS1R1 or TAS1R2, HEK293 cells respond to odorants. The TAS1R1 / TAS1R3 complex and the TAS1R2 / TAS1R3 complex receive umami and sweet substances. It is reported that by expressing the TAS1R2 / TAS1R3 complex in cultured cells and screening, it is determined that the complex functions as an allosteric regulation and enhances the sweetness of the material (non-patent literature 4). Similarly, by expressing the TAS1R1 / TAS1R3 complex, it is possible to obtain an evaluation result that the inosinic acid that enhances the umami taste improves the activity of the complex (non-patent literature 5). If these TAS1R proteins can be obtained as more stable proteins or expressed in larger quantities on the membrane of cultured cells, it is possible to efficiently determine the material that improves umami taste and sweetness.

[0006] TAS2R (taste 2receptor), which is also a taste receptor, is a gene family found as a bitter receptor expressed in the tongue, and there are 25 genes in the human genome. It is shown that it is classified as a GPCR of Class A type, identifying various bitter substances, but people TAS2R41, TAS2R42, TAS2R45, TAS2R48 and TAS2R60 have not yet successfully analyzed the function, and it is still unclear what kind of substance it identifies. In patent document 1, it is disclosed that in order to be able to use TAS2R to evaluate bitterness and determine bitterness regulating substances, a fusion protein with G protein is made in order to efficiently analyze the function of TAS2R. The expression of TAS2R is not limited to the tongue, and expression is also confirmed in the respiratory system, circulatory system, and nervous system, showing that in addition to receiving bitterness in the tongue, it is also responsible for various physiological effects (non-patent literature 6). In addition, expression is also confirmed in the tissue cells of canceration such as ovarian cancer and prostate cancer.

[0007] Trace amine associated receptor (TAAR) is a GPCR of the Class A family, and is composed of 6 genes in humans. TAAR1, which was first discovered, was shown to be responsible for recognizing neurotransmitters in the brain. As a result, the association between TAAR1 and schizophrenia, depression, poisoning, and Parkinson's disease has been studied, and efforts have been made to use its agonists for the treatment of neuropathic pain (Patent Document 2). On the other hand, TAARs other than TAAR1 are highly expressed in the olfactory epithelium, selectively and highly sensitively recognize volatile amines, and show a role in producing the sense of odor (Non-Patent Document 7).

[0008] Mas-related G-protein-coupled receptor (Mrgpr) is a GPCR of the Class A family, and there are 10 genes in humans. Its expression has been confirmed in the sensory nervous system and its related tissues such as the peripheral skin, and it perceives itching and pain by recognizing agonists (Non-patent Document 8). In addition, in mice, based on the fact that sensory nerve cells expressing MrgprB4 are involved in pleasant touch, the exploration of pleasure enhancers targeting MrgprB4 has been disclosed (Patent Document 3). In recent years, it has been taught that MrgprE and MrgprF among Mrgpr are also expressed in various tissues other than sensory nerves such as the ileum, and are responsible for a variety of physiological functions, but there are no reports of agonists (Non-patent Document 9).

[0009] In general, when analyzing the function of GPCR, the method of expressing it in cultured cells is simple and the most widely used. Regarding its analysis method, a lot of methods have been designed. Despite this, many functional analyses have failed. GPCRs that are unsuccessful in functional analysis and are unclear about which molecule is used as a ligand to identify are generally represented as GPR (G-protein-coupled receptor, G protein-coupled receptor) X (X is an arbitrary number). Regarding these GPR groups, one of the reasons for the backward determination of ligands can be cited as insufficient factors that stabilize the GPCR and express it on the cell membrane in cultured cells different from the original tissue cells, even if the target GPCR is to be expressed in cultured cells.

[0010] (Patent Document 1) Japanese Patent No. 6924590

[0011] (Patent Document 2) Japanese Patent Publication No. 2019-517524

[0012] (Patent Document 3) Japanese Patent Application Publication No. 2019-118340

[0013] (Non-patent document 1) Sriram K, Insel PA. Mol Pharmacol. 93(4): 251-258 (2018)

[0014] (Non-patent document 2) Shirokova E, et al. FASEB J. 22(5): 1416-25 (2008)

[0015] (Non-patent document 3) Henningsson, S., Hovey, D., Vass, K. et al. Transl Psychiatry 7, e1102 (2017)

[0016] (Non-patent document 4) Servant G, Tachdjian C, Tang XQ, Werner S, Zhang F, Li

[0017] (Non-patent document 5) Nelson, G., Chandrashekar, J., Hoon, M. et al. Nature 416, 199-202 (2002)

[0018] (Non-patent document 6) Tuzim K., Korolczuk AJ Transl Med 19, 440 (2021)

[0019] (Non-patent document 7) Liberles SD Curr Opin Neurobiol. 34: 1-7 (2015)

[0020] (Non-patent document 8) Liu Q, et al. Cell 139(7):1353-1365 (2009)

[0021] (Non-patent document 9) Juan M. et al. Neuroscience Letters, Volume 748, 2021 Summary of the invention

[0022] The present invention provides the following 1) to 13).

[0023] 1) A method for expressing a GPCR polypeptide, wherein the method comprises:

[0024] A step of expressing a GPCR polypeptide in a cell, wherein the GPCR polypeptide consists of an amino acid sequence in which at least one of the amino acid residues different from a consensus amino acid sequence in an amino acid sequence of a target GPCR (excluding olfactory receptors) is changed to an amino acid residue of the consensus amino acid sequence at a corresponding position,

[0025] The consensus amino acid sequence is an amino acid sequence derived from an alignment of the amino acid sequence of the target GPCR and the amino acid sequence of a GPCR encoded by an ortholog of the target GPCR in a vertebrate.

[0026] 2) A method for expressing a GPCR polypeptide, wherein the method comprises:

[0027] A step of expressing a GPCR polypeptide in a cell, wherein the GPCR polypeptide consists of an amino acid sequence in which at least one of the amino acid residues different from a consensus amino acid sequence in the amino acid sequence of the target GPCR is changed to an amino acid residue of the consensus amino acid sequence at a corresponding position,

[0028] The GPCR polypeptide consists of the following amino acid sequence, in which at least one of the amino acid residues different from the consensus amino acid sequence shown in sequence number 143 in the amino acid sequence shown in sequence number 36 of human TAAR6 is changed to an amino acid residue of the consensus amino acid sequence at the corresponding position.

[0029] 3) A method for determining the response of a target GPCR, wherein the method comprises:

[0030] A step of measuring the response of the GPCR polypeptide expressed by the method described in 1) or 2).

[0031] 4) A method for exploring a ligand of a target GPCR, wherein the method comprises:

[0032] a step of measuring the response of the GPCR polypeptide expressed by the method described in 1) or 2) in the presence of a test substance, and

[0033] A step of selecting a test substance to which the GPCR polypeptide responds.

[0034] 5) A method for evaluating and / or selecting a control agent for the recognition of a target GPCR ligand, wherein the method comprises:

[0035] a step of adding a test substance and a ligand of a target GPCR to the GPCR polypeptide expressed by the method described in 1) or 2), and

[0036] The step of determining the response of the GPCR polypeptide to the ligand.

[0037] 6) A method for evaluating taste, wherein the method comprises:

[0038] a step of adding a test substance to the GPCR polypeptide expressed by the method described in 1) or 2), and

[0039] a step of determining the response of the GPCR polypeptide to the test substance,

[0040] The GPCR polypeptide is a taste receptor polypeptide.

[0041] 7) A method for evaluating and / or selecting an inhibitor of the odor of a target GPCR ligand, wherein the method comprises:

[0042] a step of adding a test substance and a ligand of a target GPCR to the GPCR polypeptide expressed by the method described in 1) or 2), and

[0043] the step of determining the response of the GPCR polypeptide to the ligand,

[0044] The GPCR polypeptide is a trace amine-related receptor polypeptide.

[0045] 8) A method for evaluating and / or selecting an inhibitor of the odor of a target GPCR ligand, wherein the method comprises:

[0046] a step of adding a test substance to the GPCR polypeptide expressed by the method described in 1) or 2), and

[0047] a step of determining the response of the GPCR polypeptide to the test substance,

[0048] The GPCR polypeptide is a trace amine-related receptor polypeptide.

[0049] 9) An altered GPCR polypeptide, wherein the altered GPCR polypeptide consists of an amino acid sequence in which at least one of the amino acid residues different from the consensus amino acid sequence in the amino acid sequence of a target GPCR (excluding olfactory receptors) is changed to an amino acid residue of the consensus amino acid sequence at a corresponding position,

[0050] The consensus amino acid sequence is an amino acid sequence derived from an alignment of the amino acid sequence of the target GPCR and the amino acid sequence of a GPCR encoded by an ortholog of the target GPCR in a vertebrate.

[0051] 10) A modified GPCR polypeptide, wherein the modified GPCR polypeptide consists of the following amino acid sequence, in which at least one of the amino acid residues different from the consensus amino acid sequence shown in sequence number 143 in the amino acid sequence shown in sequence number 36 of human TAAR6 is changed to an amino acid residue of the consensus amino acid sequence at a corresponding position.

[0052] 11) A polynucleotide, wherein the polynucleotide encodes the altered GPCR polypeptide described in 9) or 10).

[0053] 12) A vector or a DNA fragment, wherein the vector or the DNA fragment comprises the polynucleotide described in 11).

[0054] 13) A transformed cell, wherein the transformed cell contains the vector or DNA fragment described in 12). BRIEF DESCRIPTION OF THE DRAWINGS

[0055] [ Figure 1 ]Membrane expression level of GPCR (taste receptor). DETAILED DESCRIPTION

[0056] All patent documents, non-patent documents and other publications cited in this specification are incorporated herein by reference in their entirety.

[0057] In this specification, "G-protein-coupled receptor (GPCR)" is a general term for receptors that have a seven-transmembrane structure, are converted to an activated structure by binding to a ligand, and transmit signals basically through interaction with intracellular G proteins. GPCRs include, in addition to vomeronasal receptors, taste receptors, trace amine-related receptors, Mas-related GPCRs, GPRX (X is an arbitrary number) whose ligands are not clear, etc., but are not limited to these.

[0058] In the present specification, "vomeronasal receptor" refers to VN1R (vomeronasal 1 receptor). For example, there are five types of VN1R genes in the human genome.

[0059] In this specification, "taste receptor" refers to a receptor that receives odor molecules in an organism, including umami or sweet taste receptors belonging to the TAS1R (taste 1 receptor, taste 1 receptor) family that receives umami molecules or sweet molecules and bitter taste receptors belonging to the TAS2R (taste 2 receptor, taste 2 receptor) family that receives bitter molecules. For example, there are 3 TAS1R genes in the human genome. Among these, TASR1 and TAS1R3 form a complex and function as umami receptors, and TAS1R2 and TAS1R3 form a complex and function as sweet receptors. In addition, there are 25 TAS2R genes in the human genome.

[0060] In the present invention, "trace amine associated receptor" refers to TAAR (trace amine associated receptor). For example, there are six types of TAAR genes in the human genome.

[0061] In this specification, "Mas-related G protein-coupled receptor" refers to Mrpgr (Mas-related G-protein-coupled receptor). MAS refers to a G protein-coupled receptor that binds to angiotensin. For example, there are 10 Mrpgr genes in the human genome (MAS1, MASL1, MrgprD, MrgprE, MrgprF, MrgprG, MrgprX1-4).

[0062] In this specification, "olfactory receptor" refers to Olfactory receptor or Odorant receptor. About 400 olfactory receptor genes are predicted in the human genome based on the overall sequence identity, predicted seven transmembrane regions, and whether there is a conserved partial amino acid sequence.

[0063] In the present specification, "GPCR polypeptide" refers to GPCR or a polypeptide having the equivalent function thereof. A polypeptide having the equivalent function to GPCR refers to a polypeptide having the following functions: like GPCR, it can be expressed on the cell membrane, activated by binding to a ligand, and when activated, it promotes the GDP / GTP exchange of the conjugated G protein α subunit, etc., thereby transmitting signals within the cell.

[0064] In the present specification, "functional expression" of a GPCR polypeptide in a cell means that the expressed GPCR polypeptide functions as a receptor for a corresponding ligand in the cell.

[0065] In the present specification, an "agonist" refers to a substance that binds to a receptor and activates it. On the other hand, in the present specification, an "antagonist" refers to a substance that binds to a receptor, does not activate the receptor, or inhibits the response of the receptor to an agonist.

[0066] In the present specification, "receptor agonism" means binding to a receptor to activate the receptor.

[0067] In this specification, "cross-adaptation of odor (or cross-adaptation of olfaction)" related to a target odor refers to a phenomenon in which the olfactory sensitivity to the causal substance of the target odor is reduced or changed by accustoming to the odor of a substance different from the causal substance of the target odor. The inventors of the present invention have previously discovered that "cross-adaptation of odor" is a phenomenon based on receptor agonism (International Publication No. 2016 / 194788). That is, in "cross-adaptation of odor", the receptor for the causal substance of the target odor responds to the causal substance of a different odor before responding to the causal substance of the target odor, and then, through desensitization, can only respond at a low level even if exposed to the causal substance of the target odor, and as a result, the intensity of the target odor recognized by the individual is reduced or deteriorated. The mechanism of cross-adaptation of odor caused by such receptor action is also referred to as "cross-adaptation of odor caused by receptor agonism" in this specification.

[0068] In the present specification, "inhibition of a target odor by receptor antagonism" means that the response of a receptor to a substance having a target odor is inhibited by an antagonist, and as a result, the target odor recognized by an individual is inhibited.

[0069] In the present specification, the identity of nucleotide sequences and amino acid sequences is calculated by Lipman-Pearson (Lipman-Pearson method; Science, 1985, 227: 1435-41). Specifically, it is calculated by using the homology analysis (Search homology) program of the genetic information processing software Genetyx-Win (Ver. 5.1.1; Software Development) and performing analysis with Unit size to compare (ktup) of 2.

[0070] In the present specification, "amino acid residue" refers to the 20 amino acid residues constituting proteins, alanine (Ala or A), arginine (Arg or R), asparagine (Asn or N), asparagine (Asp or D), cysteine ​​(Cys or C), glutamine (Gln or Q), glutamic acid (Glu or E), glycine (Gly or G), histidine (His or H), isoleucine (Ile or I), leucine (Leu or L), lysine (Lys or K), methionine (Met or M), phenylalanine (Phe or F), proline (Pro or P), serine (Ser or S), threonine (Thr or T), tryptophan (Trp or W), tyrosine (Tyr or Y) and valine (Val or V).

[0071] In the present specification, amino acid changes may be expressed as [original amino acid, position, changed amino acid] using the recognized IUPAC single-letter amino acid abbreviations. For example, a change of histidine at position 43 to arginine is expressed as "H43R".

[0072] In the present specification, the "equivalent position" on the amino acid sequence can be determined by aligning (aligning) the target sequence and the reference sequence (in the present invention, the amino acid sequence of the original GPCR) in a manner that gives the greatest identity. The alignment of amino acid sequences can be performed using a known algorithm, and its steps are well known to those skilled in the art. For example, the alignment can be performed using the Clustal W multiple alignment program (Thompson, JD et al, 1994, Nucleic Acids Res. 22: 4673-4680) with default settings. Alternatively, Clustal W2 or Clustal omega, which are modified versions of Clustal W, can also be used. Clustal W, Clustal W2 and Clustal omega can be used, for example, on the website of Clustal operated by University College Dublin [www.clustal.org], the website of European Bioinformatics Institute (EBI [www.ebi.ac.uk / index.html]), and the website of DNA Data Bank of Japan (DDBJ [www.ddbj.nig.ac.jp / searches-j.html]) operated by National Institute of Genetics. The position of the target sequence located at an arbitrary position of the reference sequence by the above-mentioned alignment is regarded as the "equivalent position" to the arbitrary position.

[0073] As long as one is skilled in the art, the comparison of the amino acid sequences obtained above can be optimized by making minor adjustments. Such an optimal comparison is preferably determined by considering the similarity of the amino acid sequences, the frequency of the inserted intervals, etc. Here, the similarity of the amino acid sequence refers to the ratio (%) of the number of positions where the same or similar amino acid residues exist in the sequences of both parties when two amino acid sequences are compared to the number of amino acid residues in the whole length. Similar amino acid residues refer to the 20 kinds of amino acids constituting proteins that have similar properties in terms of polarity and charge, that is, amino acid residues that undergo so-called conservative substitutions. Grouping composed of such similar amino acid residues is well known to those skilled in the art, for example, arginine and lysine or glutamine; glutamic acid and aspartic acid or glutamine; serine and threonine or alanine; glutamine and asparagine or arginine; leucine and isoleucine, etc., but are not limited thereto.

[0074] Furthermore, the alignment of the amino acid sequences obtained above can be fine-tuned and optimized based on, for example, highly conserved amino acids or amino acid motifs among GPCRs.

[0075] In this specification, "operably linked" between a control region such as a promoter and a gene means that the gene and the control region are linked in such a way that the gene can be expressed under the control of the control region. The steps of "operably linking" a gene and a control region are well known to those skilled in the art.

[0076] In this specification, "upstream" and "downstream" of a gene refer to the upstream and downstream of the transcription direction of the gene. For example, "a gene arranged downstream of a promoter" means that the gene is present on the 3' side of the promoter in the positive strand of DNA, and the upstream of a gene refers to the region on the 5' side of the gene in the positive strand of DNA.

[0077] In this specification, "homologous gene" refers to the same gene from a common ancestor. "Ortholog" is also called "ortholog", which refers to homologous genes that branched during species differentiation, exist in different biological species, and have the same or similar functions. In one example, the ortholog used in the present invention can be a GPCR gene of a species different from the biological species from which the target GPCR originates, which contains the same name as the target GPCR gene among the same genes of the target GPCR gene. When the nomenclature of the GPCR of a certain species is different from the nomenclature of the biological species from which the target GPCR originates, the ortholog is a GPCR gene with a high identity to the target GPCR gene in the specific biological species, preferably a GPCR gene with the highest identity. Alternatively, it can be a GPCR gene of a known ortholog of the target GPCR gene in the specific biological species. In another example, the ortholog used in the present invention can be a GPCR gene among the above-mentioned orthologs that is taught the possibility of branching during species differentiation by systematic numerical analysis.

[0078] In the analysis of G protein-coupled receptors (GPCRs), a method for efficiently expressing GPCRs is required.

[0079] The inventors of the present invention have conducted in-depth research on methods for efficiently expressing GPCRs. As a result, the inventors of the present invention have found that by sharing GPCRs, the membrane expression of GPCRs on cultured cells can be increased compared to the original GPCRs. There is no known example of sharing GPCRs other than olfactory receptors.

[0080] The present invention provides a method for efficiently expressing a GPCR. By using such a GPCR, it is possible to contribute to the elucidation of the function of the GPCR and the identification of a function control agent.

[0081] As shown in the Examples described below, the inventors of the present invention have found that when the amino acid sequence of a human GPCR is altered based on a common amino acid sequence derived from the amino acid sequence of the human GPCR and the amino acid sequence of a GPCR encoded by a specific ortholog of the human GPCR, and the resulting GPCR polypeptide is expressed in cells, the expression of the GPCR polypeptide in the cell membrane is increased compared to the human GPCR before the alteration (Table 7, Figure 1 ). That is, the stability of the GPCR polypeptide during expression is improved compared to the human GPCR before the modification. In this specification, the GPCR before the modification is referred to as the "original GPCR", the modification of the amino acid sequence of the GPCR based on the common amino acid sequence is referred to as "commonization", and the commonized GPCR is referred to as the "common GPCR" or "modified GPCR polypeptide".

[0082] Therefore, the above-mentioned consensus of GPCR is useful for expressing GPCR on the cell membrane of cells, especially GPCR whose functional analysis cannot be efficiently performed due to insufficient membrane expression on cultured cells. Therefore, in one embodiment, the present invention provides a method for expressing a GPCR polypeptide. The expression method of the present invention can increase the expression of GPCR (e.g., increase expression, stabilize expression), and therefore, the method is preferably a method for increasing the expression of GPCR polypeptide. The method includes the step of expressing a GPCR polypeptide in a cell, wherein the GPCR polypeptide is composed of the following amino acid sequence, in which at least one of the amino acid residues different from the consensus amino acid sequence in the amino acid sequence of the target GPCR is changed to an amino acid residue of the consensus amino acid sequence at a position corresponding thereto, and the consensus amino acid sequence is an amino acid sequence derived from the comparison of the amino acid sequence of the target GPCR and the amino acid sequence of the GPCR encoded by the ortholog of the target GPCR in vertebrates.

[0083] In the expression method of the present invention, the target GPCR is not particularly limited and can be a GPCR of any biological species, preferably a GPCR of mammals, and more preferably a human GPCR. The target GPCR can be a GPCR that can be functionally analyzed using cultured cells obtained by existing methods, or it can be an inefficient GPCR. The method of the present invention can be more appropriately applied to a GPCR that is inefficiently functionally analyzed using cultured cells obtained by existing methods.

[0084] In the expression method of the present invention, the GPCR encoded by the ortholog of the target GPCR in vertebrates is preferably a GPCR selected from the GPCRs encoded by the orthologs of the target GPCR in mammals, birds, reptiles, amphibians and fish, more preferably a GPCR selected from the GPCRs encoded by the orthologs of the target GPCR in mammals, birds, reptiles and amphibians, and further preferably a GPCR encoded by the ortholog of the target GPCR in mammals. There are no particular restrictions on the ortholog, but it is preferably an ortholog with high identity to the target GPCR gene. Here, mammals refer to species belonging to the class Mammalia of the phylum vertebrate, and about 5,500 species are known to exist. Examples of mammals include humans, chimpanzees, bonobos, gorillas, Sumatran orangutans, northern white-cheeked gibbons, drills, gelada baboons, rhesus monkeys, olive baboons, sooty mangabeys, green monkeys, ashy red colobuses, Angola colobuses, Garnett's greater galagoes, gray lemurs, Coquerel's sifakas, Philippine tarsiers, mice, rats, rabbits, cats, dogs, foxes, civet cats, ferrets, tigers, cheetahs, bears, sea lions, seals, sea otters, horses, rhinos, camels, pigs, wild boars, cattle, goats, sheep, deer, giraffes, hippopotamuses, elephants, pangolins, moles, bats, etc., but are not limited to these. Birds (Aves) refer to species belonging to the class Aves of the phylum vertebrate. Examples of birds include chickens, ducks, geese, turkeys, ostriches, pheasants, pigeons, parrots, canaries, white-rumped munias, hummingbirds, manakins, quails, and flycatchers, but are not limited to these. Reptiles (Reptilia) refer to species belonging to the class Reptilia of the phylum vertebrate. Examples of reptiles include turtles, lizards, crocodiles, iguanas, chameleons, geckos, snakes, and the like, but are not limited to these. Amphibia (Amphibia) refers to species belonging to the class Amphibia of the phylum vertebrate. Examples of amphibians include frogs, newts, salamanders, and the like, but are not limited to these. Fish (Fish) refers to a general term for species belonging to the class Hagfish, the order Lampreys, the class Chondrichthyes, and the class Osteichthyes of the phylum vertebrate. Examples of fish include hagfish, lamprey, shark, ray, tuna, bonito, salmon, trout, cod, sea bream, flounder, yellowtail, horse mackerel, and mackerel, but are not limited to these.

[0085] In a preferred example of the present embodiment, the ortholog of the target GPCR in vertebrates is a gene having the same name as the target GPCR gene among the GPCR genes possessed by the species belonging to vertebrates. Such orthologs can be selected, for example, by the following steps. The amino acid sequence of the target GPCR is set as the query sequence, and a database search is performed on a known database by BLAST of NCBI, etc. From the same gene group obtained as the result (for example, the top 500 genes, preferably the top 250 genes, more preferably the top 100 genes, and further preferably the top 50 genes), a gene having the same name as the target GPCR gene is selected. It is further preferred to encode a GPCR having a certain amino acid sequence identity with the target GPCR, for example, an amino acid sequence identity of more than 65%.

[0086] In addition, when selecting multiple genes from the same species as an ortholog, only one gene with the highest homology to the target GPCR gene can be selected. For example, when the target GPCR is a human GPCR and multiple genes of a species other than humans are selected as orthologs, the gene of the species with the highest homology to the target human GPCR gene can be selected. In addition, when the nomenclature of the GPCR of a species is different from the nomenclature in the species from which the target GPCR is derived, as an ortholog, a gene with high homology to the target GPCR gene in the species can be selected, preferably a gene with the highest homology. Alternatively, a gene known to be an ortholog of the target GPCR gene in the species can be selected.

[0087] The number of types of GPCRs encoded by orthologs of the target GPCR in vertebrates is at least 2 types, preferably at least 5 types, more preferably at least 11 types, further preferably at least 15 types, further preferably at least 30 types, further preferably 100 types, based on the number of receptors. On the other hand, the upper limit of the number of types is the total number of orthologs of the target GPCR in vertebrates, which is preferably 500 types or less, more preferably 400 types or less, further preferably 300 types or less, based on the number of receptors. The number of types of GPCRs encoded by orthologs of the target GPCR in vertebrates may be calculated as the number of receptors, for example, from 2 types to all types of orthologs of the target GPCR in vertebrates, from 5 types to all types of orthologs of the target GPCR in vertebrates, from 11 types to all types of orthologs of the target GPCR in vertebrates, 5 to 500 types, 5 to 400 types, 5 to 300 types, 11 to 500 types, 11 to 400 types, 11 to 300 types, 15 to 300 types, 30 to 300 types, or 100 to 300 types.

[0088] In the expression method of the present invention, the "consensus amino acid sequence" refers to an amino acid sequence derived from the alignment of the amino acid sequence of the target GPCR and the amino acid sequence of the GPCR encoded by the ortholog of the target GPCR in vertebrates. Specifically, the "consensus amino acid sequence" refers to an amino acid sequence composed of common residues determined according to the following criteria (i) to (iii) based on the alignment of the amino acid sequence of the target GPCR and the amino acid sequence of the GPCR encoded by the ortholog of the target GPCR in vertebrates.

[0089] (i) at each amino acid position in the alignment,

[0090] (ii) when there is an amino acid residue that is different from the amino acid residue of the target GPCR and has an occurrence frequency of 50% or more, the amino acid residue is determined as a common residue,

[0091] (i-ii) When there are two amino acid residues with an occurrence frequency of 50%, the amino acid residues of the target GPCR are determined as common residues,

[0092] (i-iii) when the amino acid residue exists in the target GPCR and the amino acid residue with an occurrence frequency of 40% or more does not exist, it is determined that there is no common residue,

[0093] (i-iv) when an amino acid residue does not exist in the target GPCR and an amino acid residue with an occurrence frequency of 60% or more exists, the amino acid residue with the highest occurrence frequency is determined as the common residue; when there are two or more amino acid residues with the highest occurrence frequency, the amino acid residue with the smallest molecular weight among the amino acid residues is determined as the common residue;

[0094] (iv) when it does not belong to any of the above cases (ii) to (i-iv), the amino acid residue of the target GPCR is determined as a consensus residue,

[0095] (ii) when the consensus residue is determined according to the criteria of (i) above, when the consensus residue at the most N-terminal side is a consensus residue at a position corresponding to the N-terminal end of the target GPCR or closer to the C-terminal end and is not a methionine residue, the consensus residue at the N-terminal end than the consensus residue consisting of the methionine residue at the position closest to the N-terminal end is changed to have no consensus residue,

[0096] (iii) When the consensus residue is determined according to the criteria of (i) above, when the consensus residue on the most N-terminal side is a consensus residue corresponding to a position closer to the N-terminus than the N-terminus of the target GPCR and is not a methionine residue, the amino acid positions closer to the N-terminus than the position of the consensus residue in the alignment are traced back one by one until a methionine residue appears, and the amino acid residue with the highest frequency of occurrence is determined as the consensus residue. When there are two or more amino acid residues with the highest frequency of occurrence, the amino acid residue with the smallest molecular weight among the amino acid residues is determined as the consensus residue.

[0097] Here, "occurrence frequency" refers to a value expressing the number of occurrences of a specific amino acid residue in each amino acid position of the amino acid sequence alignment as a percentage relative to the number of amino acid sequences provided for alignment. The alignment of amino acid sequences can be performed using a known algorithm.

[0098] (ii) of the criterion (i) is a criterion when an amino acid residue exists in a certain amino acid position of the target GPCR in the alignment of the amino acid sequence of the target GPCR and the amino acid sequence of the GPCR encoded by the ortholog of the target GPCR in vertebrates. In this case, when there is an amino acid residue that is different from the amino acid residue of the target GPCR and has an occurrence frequency of 50% or more, the amino acid residue with an occurrence frequency of 50% or more is determined as the common residue at the position. On the other hand, when the occurrence frequencies of amino acid residues other than the amino acid residue of the target GPCR are all lower than 50%, the amino acid residue of the target GPCR is determined as the common residue at the position according to the criterion (iv).

[0099] Criteria (i) (i-ii) are criteria when an amino acid residue exists in the target GPCR at a certain amino acid position in the alignment of the amino acid sequence of the target GPCR and the amino acid sequence of the GPCR encoded by the ortholog of the target GPCR in vertebrates. In this case, when there are two types of amino acid residues with an occurrence frequency of 50%, one of the two amino acid residues must be the amino acid residue of the target GPCR, and the amino acid residue of the target GPCR is determined as the common residue at the position.

[0100] (i-iii) of the criterion (i) is a criterion when an amino acid residue exists in a certain amino acid position of the target GPCR in the alignment of the amino acid sequence of the target GPCR and the amino acid sequence of the GPCR encoded by the ortholog of the target GPCR in vertebrates. At this time, if there is no amino acid residue with an occurrence frequency of 40% or more, it is determined that there is no common residue at that position. On the other hand, when there is no amino acid residue with an occurrence frequency of less than 40%, if it belongs to the above-mentioned criterion (ii), the common residue at that position is determined according to the criterion, and if it does not belong to the above-mentioned criterion (ii), the amino acid residue of the target GPCR is determined as the common residue at that position according to the criterion (iv).

[0101] (i-iv) of the criterion (i) is a criterion when an amino acid residue does not exist in a certain amino acid position of the target GPCR in the alignment of the amino acid sequence of the target GPCR and the amino acid sequence of the GPCR encoded by the ortholog of the target GPCR in vertebrates. In this case, when there is an amino acid residue with an appearance frequency of 60% or more, the amino acid residue with the highest appearance frequency is determined as the common residue at the position, and when there are two or more amino acids with the highest appearance frequency, the amino acid residue with the smallest molecular weight among the amino acids with the highest appearance frequency is determined as the common residue. For example, when there is one amino acid residue with the highest appearance frequency, the one amino acid residue is determined as the common residue at the position, and when there are two or more amino acid residues with the highest appearance frequency, the amino acid residue with the smallest molecular weight among them is determined as the common residue at the position. In addition, when the full length of the common amino acid sequence is longer than the full length of the amino acid sequence of the target GPCR on the N-terminal side by 10% or more due to the change, from the viewpoint of maintaining the structure of the GPCR, the common residue at the position corresponding to the N-terminal of the target GPCR in the common amino acid sequence is set to a methionine residue, and the common residue on the N-terminal side of the methionine residue is changed to be absent. That is, the N-terminal structure of the target GPCR can be maintained as it is. On the other hand, when the frequency of occurrence of an amino acid residue is less than 60%, the amino acid residue of the target GPCR is determined to be a common residue at that position according to criterion (iv). That is, it is determined that there is no amino acid residue at that position.

[0102] When a certain amino acid position in the alignment of the amino acid sequence of the target GPCR and the amino acid sequence of the GPCR encoded by the ortholog of the target GPCR in a vertebrate does not belong to any of the cases (ii) to (i-iv) of (i) above, the amino acid residue of the target GPCR is determined as the consensus residue at the position. In this case, when the target GPCR does not have an amino acid residue, it is sufficient to determine that there is no amino acid residue at the position in the consensus amino acid sequence.

[0103] Criterion (ii) is a criterion when determining the common residue at each amino acid position of the amino acid sequence of the target GPCR and the amino acid sequence of the GPCR encoded by the ortholog of the target GPCR in vertebrates in accordance with the above-mentioned criterion (i), the common residue located at the most N-terminal side among the common residues is a common residue at a position corresponding to the N-terminal of the target GPCR or closer to the C-terminal side thereof and is not a methionine residue. In this case, the common residue at the N-terminal is a methionine residue, in other words, the common residue at the N-terminal side of the common residue composed of the methionine residue at the position closest to the N-terminal among the common residues is changed to have no common residue in such a manner that the translation start amino acid of the GPCR polypeptide becomes a methionine residue. For example, the common residue is confirmed residue by residue from the N-terminal, and if it is not a methionine residue, it is determined that there is no common residue at that position, and this operation is repeated until the methionine residue appears for the first time. In addition, when the total length of the consensus amino acid sequence is shortened by 10% or more than the total length of the amino acid sequence of the target GPCR due to the change, the consensus residue at the most N-terminal side among the consensus residues before the change can be changed to a consensus residue composed of a methionine residue from the viewpoint of maintaining the helical structure of the GPCR. In this case, when the consensus residue at the most N-terminal side among the consensus residues before the change is asparagine, serine or threonine related to sugar chain modification and / or membrane translocation, from the viewpoint of maintaining the structure of the GPCR, the consensus residue at the most N-terminal side among the consensus residues before the change can be left unchanged, and the amino acid residue of the target GPCR on the N-terminal side of the position corresponding to the consensus residue can be used as the consensus residue. That is, the N-terminal structure of the target GPCR can be maintained as it is.

[0104] Criterion (iii) is a criterion when the consensus residue is determined at each amino acid position in the alignment of the amino acid sequence of the target GPCR and the amino acid sequence of the GPCR encoded by the ortholog of the target GPCR in vertebrates in accordance with the above criterion (i), the consensus residue at the most N-terminal side is a consensus residue corresponding to a position closer to the N-terminal side than the N-terminal side of the target GPCR and is not a methionine residue. In this case, the alignment is checked by tracing back the amino acid positions one by one from the position closer to the N-terminal side than the most N-terminal consensus residue so that the consensus residue at the N-terminal side becomes a methionine residue, in other words, so that the initiation amino acid of the translation of the GPCR polypeptide becomes a methionine residue, until a methionine residue appears, and the amino acid residue with the highest frequency of appearance is determined as the consensus residue. When there are two or more amino acid residues with the highest frequency of appearance, the amino acid residue with the smallest molecular weight among the amino acid residues is determined as the consensus residue.

[0105] The amino acid sequence composed of the consensus residues determined in this way is the consensus amino acid sequence. The GPCR polypeptide used in the present invention is a modified GPCR polypeptide composed of an amino acid sequence in which at least one of the amino acid residues different from the consensus amino acid sequence is changed to an amino acid residue of the consensus amino acid sequence at a position corresponding to it in the amino acid sequence of the target GPCR. Here, "change" refers to any concept including substitution, deletion, addition, and insertion. The difference between the amino acid sequence of the target GPCR and the consensus amino acid sequence can be found by comparing the two amino acid sequences through the alignment of the amino acid sequences using a known algorithm. For example, when the amino acid sequence of the target GPCR is compared with the consensus amino acid sequence, when the amino acid residue at a certain amino acid position of the amino acid sequence of the target GPCR is different from the amino acid residue at the position corresponding to it in the consensus amino acid sequence, the amino acid residue of the target GPCR can be replaced with the amino acid residue of the consensus amino acid sequence. Alternatively, when the amino acid sequence of the target GPCR is compared with the consensus amino acid sequence, when there is no amino acid residue at a position corresponding to a certain amino acid position of the amino acid sequence of the target GPCR in the consensus amino acid sequence, the amino acid residue at the amino acid position of the target GPCR can be deleted. Alternatively, the amino acid sequence of the target GPCR is compared with the consensus amino acid sequence, and when an amino acid residue exists in the consensus amino acid sequence at a position corresponding to a position where an amino acid residue does not exist in the amino acid sequence of the target GPCR, the amino acid residue of the consensus amino acid sequence can be inserted into the amino acid position of the target GPCR. In addition, when the amino acid sequence of the target GPCR is compared with the consensus amino acid sequence, when the N-terminus of the consensus amino acid sequence is longer than that of the amino acid sequence of the target GPCR, the N-terminus portion that only exists in the consensus amino acid sequence can be added to the N-terminus of the amino acid sequence of the target GPCR. Alternatively, when the amino acid sequence of the target GPCR is compared with the consensus amino acid sequence, when the C-terminus of the consensus amino acid sequence is longer than that of the amino acid sequence of the target GPCR, the C-terminus portion that only exists in the consensus amino acid sequence can be added to the C-terminus of the amino acid sequence of the target GPCR.

[0106] The number of acid residues changed in the amino acid sequence of the target GPCR is at least 1, preferably at least 5, more preferably at least 10, and even more preferably, all the amino acid residues in the amino acid sequence of the target GPCR that are different from those in the consensus amino acid sequence are changed to amino acid residues in the consensus amino acid sequence at corresponding positions (i.e., the amino acid sequence of the target GPCR is changed to the consensus amino acid sequence).

[0107] Alternatively, the number of amino acid residues altered in the amino acid sequence of the target GPCR is preferably at least 10% of the number of amino acid residues in the consensus amino acid sequence, more preferably at least 30%, further preferably at least 50%, further preferably at least 70%, further preferably at least 90%, further preferably 100% (i.e., the amino acid sequence of the target GPCR is altered to the consensus amino acid sequence).

[0108] In addition, when the N-terminus of the consensus amino acid sequence is longer than the amino acid sequence of the target GPCR, regardless of the number of amino acid residues, it is preferred to modify the N-terminus of the amino acid sequence of the target GPCR by adding only the N-terminus portion present in the consensus amino acid sequence as a whole. When the N-terminus of the consensus amino acid sequence is shorter than the amino acid sequence of the target GPCR, regardless of the number of amino acid residues, it is preferred to modify the N-terminus portion present in the amino acid sequence of the target GPCR by deleting only the N-terminus portion present in the amino acid sequence of the target GPCR as a whole. When the C-terminus of the consensus amino acid sequence is longer than the amino acid sequence of the target GPCR, regardless of the number of amino acid residues, it is preferred to modify the C-terminus portion present in the consensus amino acid sequence as a whole. When the C-terminus of the consensus amino acid sequence is shorter than the amino acid sequence of the target GPCR, regardless of the number of amino acid residues, it is preferred to modify the C-terminus portion present in the amino acid sequence of the target GPCR by deleting only the C-terminus portion present in the amino acid sequence of the target GPCR as a whole.

[0109] As the GPCR polypeptide used in the present invention, as long as its function is not impaired, polypeptides containing substitutions, deletions, additions or insertions of 1 to several (e.g., 1 to 10, 1 to 5, 1 to 3) amino acid residues at amino acid positions other than the target amino acid positions for changes based on the above-mentioned consensus amino acid sequence are also included in the present invention.

[0110] In a preferred example of the present embodiment, the GPCR polypeptide is composed of the following amino acid sequence, wherein at least one of the amino acid residues different from the consensus amino acid sequence represented by the sequence number (3) in the amino acid sequence represented by the sequence number (2) of the GPCR in (1) of Tables 1-1 and 1-2 below is changed to an amino acid residue of the consensus amino acid sequence at a corresponding position. More preferably, the GPCR polypeptide is a GPCR polypeptide composed of the amino acid sequence represented by any one of the sequence numbers 108 to 214 and 275 to 312 in (3) of Tables 1-1 and 1-2 below, or a combination of a GPCR polypeptide composed of the amino acid sequence represented by the sequence number 112 or 113 in (3) of Table 1-1 below and a GPCR polypeptide composed of the amino acid sequence represented by the sequence number 114. In addition, the GPCR polypeptide consisting of the amino acid sequence represented by any one of SEQ ID NOs: 108 to 112, 114 to 120, 138 to 145, 167, 183, 187, 192 and 209 has significantly improved membrane expression compared to the original GPCR. In Tables 1-1 and 1-2, the GPCR of No. 1 to 145 (1) is a human GPCR, and the accession number (Accession No.) indicates the accession number in the GenBank.

[0111] [Table 1-1]

[0112]

[0113] [Table 1-2]

[0114]

[0115] A GPCR polypeptide consisting of an amino acid sequence in which at least one of the amino acid residues different from the consensus amino acid sequence shown in the sequence number (3) in the amino acid sequence shown in the sequence number (1) of the GPCR of No. 1 to 4 in the above Table 1-1 is replaced with an amino acid residue of the consensus amino acid sequence at a position corresponding thereto is a vomeronasal receptor polypeptide. In the above Table 1-1, the vomeronasal receptor polypeptide consisting of the amino acid sequence shown in any one of the sequence numbers 108 to 111 in (3) is a consensus vomeronasal receptor consisting of a consensus amino acid sequence derived from the amino acid sequence shown in any one of the sequence numbers 1 to 4 in (2) of the vomeronasal receptor of No. 1 to 4 (1) and the amino acid sequence of a vomeronasal receptor encoded by an ortholog of the vomeronasal receptor in a vertebrate.

[0116] The GPCR polypeptide consisting of an amino acid sequence in which at least one of the amino acid residues different from the consensus amino acid sequence shown by the sequence number (3) in the amino acid sequence shown by the sequence number (1) of the GPCR of No. 5 to 30 in the above Table 1-1 is replaced with an amino acid residue of the consensus amino acid sequence at a corresponding position is a taste receptor polypeptide. Among them, the GPCR polypeptide consisting of an amino acid sequence in which at least one of the amino acid residues different from the consensus amino acid sequence shown by the sequence number (3) in the amino acid sequence shown by the sequence number (1) of the GPCR of No. 5 is replaced with an amino acid residue of the consensus amino acid sequence at a corresponding position and the GPCR polypeptide consisting of an amino acid sequence in which at least one of the amino acid residues different from the consensus amino acid sequence shown by the sequence number (3) in the amino acid sequence shown by the sequence number (1) of the GPCR of No. 7 is replaced with an amino acid residue of the consensus amino acid sequence at a corresponding position form an umami taste receptor complex. In addition, a GPCR polypeptide consisting of an amino acid sequence in which at least one of the amino acid residues different from the consensus amino acid sequence shown in the sequence number (3) in the amino acid sequence shown in the sequence number (1) of the GPCR of No. 6 is replaced with an amino acid residue of the consensus amino acid sequence at a position corresponding thereto, and a GPCR polypeptide consisting of an amino acid sequence in which at least one of the amino acid residues different from the consensus amino acid sequence shown in the sequence number (3) in the amino acid sequence shown in the sequence number (1) of the GPCR of No. 7 is replaced with an amino acid residue of the consensus amino acid sequence at a position corresponding thereto form a sweet taste receptor complex. On the other hand, a GPCR polypeptide consisting of an amino acid sequence in which at least one of the amino acid residues different from the consensus amino acid sequence shown in the sequence number (3) in the amino acid sequence shown in the sequence number (1) of the GPCR of Nos. 8 to 30 is replaced with an amino acid residue of the consensus amino acid sequence at a position corresponding thereto is a bitter taste receptor polypeptide. In the above Table 1-1, the taste receptor polypeptide composed of the amino acid sequence shown in any one of the sequence numbers 112 to 137 of (3) is a common taste receptor composed of a common amino acid sequence derived from the amino acid sequence shown in any one of the sequence numbers 5 to 30 of the taste receptor (2) of No. 5 to 30 (1) and the amino acid sequence of the taste receptor encoded by the ortholog of the taste receptor in vertebrates.

[0117] A GPCR polypeptide consisting of an amino acid sequence in which at least one of the amino acid residues different from the consensus amino acid sequence shown in the sequence number (3) in the amino acid sequence shown in the sequence number (2) of the GPCR in No. 31 to 36 of the above Table 1-1 is replaced with an amino acid residue of the consensus amino acid sequence at a position corresponding thereto is a trace amine-related receptor polypeptide. In the above Table 1-1, the trace amine-related receptor polypeptide consisting of the amino acid sequence shown in any one of the sequence numbers 138 to 143 of (3) is a consensus trace amine-related receptor polypeptide consisting of a consensus amino acid sequence derived from the amino acid sequence shown in any one of the sequence numbers 31 to 36 of the trace amine-related receptor polypeptide in (1) of No. 31 to 36 and the amino acid sequence of a trace amine-related receptor polypeptide encoded by a direct homolog of the trace amine-related receptor polypeptide in a vertebrate. Among them, the trace amine-associated receptor polypeptide composed of the amino acid sequence shown in SEQ ID NO: 143 is a common trace amine-associated receptor composed of a common amino acid sequence obtained by maintaining the N-terminal structure of the target trace amine-associated receptor polypeptide as is in the above-mentioned criterion (ii).

[0118] A GPCR polypeptide consisting of an amino acid sequence in which at least one of the amino acid residues different from the consensus amino acid sequence shown in the sequence number (3) in the amino acid sequence shown in the sequence number (2) of the GPCR of No. 37 to 46 in the above Table 1-1 is replaced with an amino acid residue of the consensus amino acid sequence at a position corresponding thereto is a Mas-related G protein coupled receptor polypeptide. In the above Table 1-1, a Mas-related G protein coupled receptor polypeptide consisting of an amino acid sequence shown in any one of the sequence numbers 144 to 153 in (3) is a consensus Mas-related G protein coupled receptor consisting of a consensus amino acid sequence derived from an amino acid sequence shown in any one of the sequence numbers 37 to 46 in (2) of the GPCR of No. 37 to 46 (1) and an amino acid sequence of a Mas-related G protein coupled receptor encoded by an ortholog of the Mas-related G protein coupled receptor in a vertebrate.

[0119] A GPCR polypeptide consisting of an amino acid sequence in which at least one of the amino acid residues different from the consensus amino acid sequence shown in the sequence number (3) in the amino acid sequence shown in the sequence number (1) of the GPCR of Nos. 47 to 108 in the above Tables 1-1 and 1-2 is replaced with an amino acid residue of the consensus amino acid sequence at a corresponding position is a GPR polypeptide. In the above Tables 1-1 and 1-2, a GPR polypeptide consisting of an amino acid sequence shown in any one of the sequence numbers 154 to 214 and 275 in (3) is a consensus GPR consisting of a consensus amino acid sequence derived from an amino acid sequence shown in any one of the sequence numbers 47 to 107 and 237 in (2) of the GPR of Nos. 47 to 108 (1) and an amino acid sequence of a GPR encoded by an ortholog of the GPR in a vertebrate.

[0120] The GPCR polypeptide consisting of an amino acid sequence in which at least one of the amino acid residues different from the consensus amino acid sequence shown in the sequence number of (3) in the amino acid sequence shown in the sequence number of (1) of the GPCR of No. 109 to 145 in the above Table 1-2 is replaced with an amino acid residue of the consensus amino acid sequence at a position equivalent thereto is a GPCR polypeptide other than a vomeronasal receptor polypeptide, a taste receptor polypeptide, a trace amine-related receptor polypeptide, a Mas-related G protein-coupled receptor polypeptide, a GPR polypeptide, and an olfactory receptor polypeptide. In the above Table 1-2, the GPCR polypeptide consisting of an amino acid sequence shown in any one of the sequence numbers 276 to 312 in (3) is a consensus GPCR consisting of a consensus amino acid sequence derived from the amino acid sequence shown in any one of the sequence numbers 238 to 274 in (2) of the GPCR of No. 109 to 145 (1) and an amino acid sequence of a GPCR encoded by an ortholog of the GPCR in a vertebrate.

[0121] The "expression" of the GPCR polypeptide of the present invention refers to the production of a translation product from a polynucleotide encoding the polypeptide, and the translation product is locally present in the cell membrane of its action site in a functional state. The GPCR polypeptide of the present invention can be expressed in cells by methods known in the art. For example, the GPCR polypeptide can be expressed in the cell membrane of the cell by introducing a vector containing a polynucleotide encoding the polypeptide into a cell that becomes a host, or by introducing a DNA fragment containing a polynucleotide encoding the polypeptide into the genome of a cell that becomes a host. Preferably, the GPCR polypeptide is transformed into a host cell using a vector containing a polynucleotide encoding the polypeptide so that it is expressed in the cell membrane of the host cell. The host cell can be any cell that can functionally express an external GPCR. As specific examples of cells, human embryonic kidney cells (HEK293 cells), Chinese hamster ovary cells (CHO cells), monkey cells (COS cells), isolated olfactory neurons, African clawed frog oocytes, insect cells, yeast or bacteria can be cited, but are not limited to these.

[0122] A polynucleotide encoding a GPCR polypeptide can be obtained using various mutation introduction techniques known in the art. For example, a polynucleotide encoding a GPCR polypeptide can be obtained by changing a nucleotide sequence encoding an amino acid residue to be changed in a polynucleotide encoding an amino acid sequence of a target GPCR to a nucleotide sequence encoding the changed amino acid residue.

[0123] The introduction of target mutations into the polynucleotide encoding the amino acid sequence of the target GPCR can be performed using various site-specific mutation introduction methods known to those skilled in the art. The site-specific mutation introduction method can be performed by any method such as the inverse PCR method, the annealing method, etc. It is also possible to use a commercially available site-specific mutation introduction kit (e.g., Stratagene's QuickChange II Site-Directed Mutagenesis Kit, QuickChange Multi Site-Directed Mutagenesis Kit, etc.).

[0124] Alternatively, a polynucleotide encoding a GPCR polypeptide can also be obtained by genome editing using an artificial DNA cleavage enzyme (artificial DNA nucleases or programmable nuclease), DNA synthesis based on its nucleotide sequence, and the like.

[0125] The polynucleotide encoding the GPCR polypeptide may include single-stranded or double-stranded DNA, cDNA, RNA or its artificial nucleic acid. The DNA, cDNA and RNA may be chemically synthesized. In addition, the polynucleotide may include the nucleotide sequence of the open reading frame (ORF) and the untranslated region (UTR). In addition, the polynucleotide may be codon optimized according to the type of cell used for expression of the GPCR. Information on the codons used by various organisms can be obtained from the Codon Usage Database ([www.kazusa.or.jp / codon / ]).

[0126] In a preferred example, the polynucleotide encoding the GPCR polypeptide consists of a base sequence represented by any one of SEQ ID NOs: 215 to 234 and 313 to 317. The polynucleotide encodes a GPCR polypeptide consisting of an amino acid sequence represented by SEQ ID NOs: 108 to 112, 114 to 120, 138 to 145, 167, 183, 187, 192, and 209, respectively.

[0127] The obtained polynucleotide encoding the GPCR polypeptide can be inserted into a vector or a DNA fragment. Preferably, the vector is an expression vector. In addition, preferably, the vector is an expression vector capable of introducing the GPCR polynucleotide into a host cell and expressing the polynucleotide in the host cell. Preferably, the vector can be a plasmid or other vector capable of self-propagation and replication outside the chromosome, or can be a vector inserted into the chromosome. As an example of a specific vector, pME18S can be cited, but it is not limited thereto.

[0128] The vector preferably comprises a polynucleotide encoding a GPCR polypeptide and a control region that can be operably linked thereto. The control region is a sequence for expressing the introduced polynucleotide encoding a GPCR polypeptide in a host cell into which the vector is introduced, and for example, expression regulatory regions such as promoters and terminators, transcription start points, etc. can be listed. The type of the control region can be appropriately selected according to the type of vector. As needed, the vector or DNA fragment may also have a drug resistance gene such as ampicillin as a selection marker. The control region and the selection marker gene may use the control region and the selection marker gene originally contained in the vector, or may be inserted into the vector together with or separately from the polynucleotide encoding the GPCR polypeptide.

[0129] Examples of DNA fragments containing polynucleotides encoding GPCR polypeptides include PCR amplified DNA fragments and restriction enzyme-cut DNA fragments. Preferably, the DNA fragment is an expression cassette containing the polynucleotide and a control region that can be operably linked thereto. Examples of control regions that can be used are the same as those in the case of vectors.

[0130] Preferably, in order to promote the cell membrane expression of the GPCR polypeptide, a polynucleotide encoding RTP (receptor transporting protein) (also referred to as RTP gene in this specification) is introduced into the cell together with the polynucleotide encoding the GPCR polypeptide. Alternatively, preferably, in order to promote the cell membrane expression of the GPCR polypeptide, a polynucleotide encoding REEP (receptor expression enhancing protein) (also referred to as REEP gene in this specification) is introduced into the cell together with the polynucleotide encoding the GPCR polypeptide. For example, a vector containing the RTP gene or REEP gene and the polynucleotide encoding the GPCR polypeptide can be constructed and introduced into the host cell, or a vector containing the RTP gene or REEP gene and a vector encoding the polynucleotide encoding the GPCR polypeptide can be introduced into the host cell separately. Examples of RTP include RTP1S, RTP3 and RTP4, and examples of RTP1S include human RTP1S. Human RTP1S is registered in GenBank as AY562235, and is a polypeptide consisting of the amino acid sequence of sequence number 236 and encoded by a gene having the nucleotide sequence of sequence number 235. Examples of REEP include REEP1 and REEP2. An example of REEP1 includes human REEP1, and an example of REEP2 includes human REEP2.

[0131] Regarding the introduction of vectors or DNA fragments into host cells, for mammalian cells, conventional transformation methods such as electroporation, lipofection, gene gun method, etc. can be used. Transformed cells into which the target vector or DNA fragment has been introduced can be selected using a selection marker. Alternatively, the introduction of the target vector or DNA fragment can also be confirmed by studying the sequence of the cell's DNA.

[0132] The GPCR polypeptide of the present invention is produced from the polynucleotide encoding the GPCR polypeptide contained in the vector or DNA fragment introduced into the cell by the above steps and inserted into the cell membrane. Therefore, the GPCR polypeptide expressed by the expression method of the present invention is expressed on the cell membrane of the genetically manipulated transformed cell in the manner of expressing the GPCR polypeptide.

[0133] The cell membrane expression (amount) of the GPCR polypeptide of the present invention can be measured by, for example, pre-fusion of a tag such as a FLAG tag to the GPCR polypeptide and using an antibody that specifically recognizes the tag by a known method such as flow cytometry.

[0134] The GPCR polypeptide expressed by the expression method of the present invention can be efficiently expressed on the cultured cell membrane compared to the original GPCR. In addition, it can be considered that the response of the GPCR polypeptide reflects the response of the original GPCR. Therefore, in another embodiment, the present invention provides a method for measuring the response of the target GPCR. The method includes the step of measuring the response of the GPCR polypeptide expressed by the expression method of the GPCR polypeptide of the present invention. According to the response measurement method of the present invention, the efficiency of the response measurement of the target GPCR can be improved, and the response of the target GPCR whose function could not be analyzed in the past due to insufficient expression on the cell membrane of the cultured cell can be measured.

[0135] The GPCR polypeptide used in the response determination method of the present invention can be any GPCR polypeptide expressed by the expression method of the present invention. The GPCR polypeptide is as described above. As a specific example of the GPCR polypeptide, a GPCR polypeptide consisting of an amino acid sequence shown in any one of sequence numbers 108 to 214 and 275 to 312, or a combination of a GPCR polypeptide consisting of an amino acid sequence shown in sequence number 112 or 113 and a GPCR polypeptide consisting of an amino acid sequence shown in sequence number 114 is preferably cited. The GPCR polypeptide can be used in any form as long as it does not lose its responsiveness to the ligand. For example, the GPCR polypeptide can be used in the form of a transformed cell expressing the GPCR polypeptide or a culture thereof, a membrane of the transformed cell having the GPCR polypeptide, an artificial lipid bilayer membrane having the GPCR polypeptide, and the like. As the GPCR polypeptide, it is preferred to use a transformed cell expressing the GPCR polypeptide or a culture thereof.

[0136] In the response determination method of the present invention, the determination of the response of the GPCR polypeptide can be carried out by any method known in the art as a method for determining the response of the GPCR. For example, it can be carried out by a method for directly or indirectly determining the amount of intracellular cAMP. For example, it is known that when the G protein-coupled receptor is activated by a ligand, adenylate cyclase is activated or inhibited by conjugating with a G protein α subunit classified as the Gs family or a G protein α subunit classified as the Gi family in the cell, so that the amount of intracellular cAMP changes. On the other hand, when the G protein-coupled receptor is activated by a ligand, it is also conjugated with proteins belonging to the Gq family such as Gαq, Gα16, and Gα15 in the cell, and the amount of calcium ions can also be increased in the cell. Therefore, by using the amount of intracellular calcium ions or the action of downstream molecules activated by it as an indicator, the response of the GPCR polypeptide can also be determined. As a method for determining the amount of cAMP, ELISA method, reporter gene detection, etc. can be listed. Methods for determining calcium ion concentration can include calcium imaging and TGFα shedding detection. TGFα shedding detection is effective for measuring the signal of GPCR when it is conjugated with Gα12 and Gα13 in cells. In addition, as an example of a method that uses the action of downstream molecules activated by the amount of cAMP as an indicator, the two-electrode membrane potential fixation method is also effective: it measures the potential changes inside and outside the cell membrane of the cystic fibrosis transmembrane conductance regulator CFTR activated by cAMP signals in African clawed frog oocytes. By using a G protein α subunit that is easily conjugated to the target GPCR and a chimeric protein with any other G protein α subunit, any of the above-mentioned response measurement methods can be used. For example, taste receptors can be efficiently conjugated with subunits belonging to the Gi family among various G protein α subunits. In summary, by using Gα15 (Gα15 / i3, Gα15 / gust) with 5 amino acids on the C-terminal side of Gαi3 and Gαgust, the signal from the activated taste receptor can be detected as an increase in the intracellular calcium ion concentration without reducing the original intracellular cAMP amount. In addition, by purifying the GPCR polypeptide according to the present invention from cells expressing the GPCR polypeptide in the cells, the binding property to the ligand can also be evaluated. The method for evaluating the binding property of the purified GPCR polypeptide to the ligand can be carried out by any method known in the art. For example, a method for measuring the heat generated by the GPCR polypeptide under the condition of ligand addition and a method for evaluating the change of the thermal stability of the GPCR polypeptide can be cited.

[0137] As described above, the GPCR polypeptide expressed by the expression method of the present invention can be efficiently expressed on the cultured cell membrane compared to the original GPCR, and it can be considered that the response of the GPCR polypeptide reflects the response of the original GPCR, and therefore, the GPCR polypeptide can be used to explore the ligand of the original GPCR. Therefore, in another embodiment, the present invention provides a method for exploring the ligand of the target GPCR. The method includes: a step of determining the response of the GPCR polypeptide expressed by the expression method of the present invention in the presence of a test substance, and a step of selecting the test substance to which the GPCR polypeptide responds. The ligand exploration method of the present invention can improve the ligand exploration efficiency of the target GPCR, and can determine the response of the target GPCR whose function could not be analyzed in the past due to insufficient expression on the cell membrane of the cultured cell.

[0138] The test substance used in the ligand search method of the present invention is not particularly limited as long as it is a substance for which it is desired to confirm whether it is a ligand of the target GPCR. The test substance may be a naturally occurring substance, or a substance artificially synthesized by chemical or biological methods, or may be a compound, or may be a composition or a mixture.

[0139] The GPCR polypeptide and its form used in the ligand search method of the present invention are the same as the GPCR polypeptide and its form used in the response measurement method of the present invention.

[0140] In the ligand search method of the present invention, a test substance is applied to the GPCR polypeptide expressed by the expression method of the present invention. Examples of methods for applying a test substance to a GPCR polypeptide include, but are not particularly limited to, adding a test substance to a culture medium for culturing cells expressing the GPCR polypeptide.

[0141] In the ligand search method of the present invention, after adding a test substance to a GPCR polypeptide, the response of the GPCR polypeptide to the test substance is measured. As a method for measuring the response, the same method as the response measurement method of the present invention can be used.

[0142] Next, based on the measured response of the GPCR polypeptide, the test substance is evaluated. The test substance that causes the response of the GPCR polypeptide can be determined as the ligand of the GPCR polypeptide, that is, the ligand of the original GPCR. Therefore, in the ligand search method of the present invention, the test substance to which the GPCR polypeptide responds is selected as the ligand of the original GPCR.

[0143] Preferably, the response of the GPCR polypeptide to the test substance is evaluated by comparing the response of the GPCR polypeptide to which the test substance is added (test group) with the response of the GPCR polypeptide in the control group. As the control group, the GPCR polypeptide to which the test substance is not added, the GPCR polypeptide to which the control substance is added, the GPCR polypeptide to which the test substance is added at a lower concentration, the GPCR polypeptide before the addition of the test substance, etc. can be listed. When the response in the test group is higher than that in the control group, it is evaluated that the GPCR polypeptide responds to the test substance, and the test substance is selected as the original GPCR ligand.

[0144] Therefore, in one embodiment of the ligand search method of the present invention, the response of the GPCR polypeptide in the presence and absence of the test substance is measured, and then it is determined whether the response in the presence of the test substance is increased compared to the response in the absence of the test substance. When the response in the presence of the test substance is higher, the test substance is selected as a ligand. In a preferred embodiment, when the response intensity of the GPCR polypeptide in the presence of the test substance is preferably 120% or more, more preferably 150% or more, and further preferably 200% or more compared to the absence of the test substance, the test substance is selected as the ligand of the original GPCR. In another preferred embodiment, when the response intensity of the GPCR polypeptide in the presence of the test substance is statistically significantly increased compared to the absence of the test substance, the test substance is selected as the ligand of the original GPCR.

[0145] In addition, as described above, the GPCR polypeptide expressed by the expression method of the present invention can be efficiently expressed on the culture cell membrane compared with the original GPCR, and it can be considered that the response of the GPCR polypeptide reflects the response of the original GPCR, and therefore, the GPCR polypeptide can be used to evaluate and / or select substances that control the recognition of ligands in the original GPCR. Therefore, in another embodiment, the present invention provides a method for evaluating and / or selecting a control agent for the recognition of a ligand of a target GPCR. The method includes: a step of adding a test substance and a target ligand to the GPCR polypeptide expressed by the expression method of the present invention, and a step of measuring the response of the GPCR polypeptide to the ligand. In one example, the vomeronasal receptor is considered to function as a pheromone receptor, and therefore, when the vomeronasal receptor polypeptide is used as the GPCR polypeptide, a pheromone substance is preferably listed as a ligand. In addition, in another example, when a taste receptor polypeptide is used as the GPCR polypeptide, an odor substance (bitter substance, umami substance, or sweet substance) is preferably listed as a ligand. As the ligand of the target GPCR, a ligand selected by the ligand search method of the present invention is preferred. By using the method for evaluating and / or selecting a controlling agent of the present invention, it is possible to efficiently evaluate or select a substance that can selectively inhibit or enhance recognition of a target ligand.

[0146] The evaluation and / or selection of the controlling agent of the present invention is a method performed in vitro or ex vivo.

[0147] The test substance used in the method for evaluating and / or selecting a control agent of the present invention is not particularly limited as long as it is a substance used as a control agent for recognition of a target ligand. The test substance may be a naturally occurring substance, or a substance artificially synthesized by chemical or biological methods, or may be a compound, or may be a composition or a mixture.

[0148] The GPCR polypeptide and its form used in the method for evaluating and / or selecting a controlling agent of the present invention are the same as the GPCR polypeptide and its form used in the method for measuring a response of the present invention.

[0149] In the method for evaluating and / or selecting a controlling agent of the present invention, a test substance and a target ligand are applied to the GPCR polypeptide expressed by the expression method of the present invention. As a method for applying a test substance and a target ligand to a GPCR polypeptide, a method of adding a test substance and a target ligand to a culture medium of cells expressing the GPCR polypeptide can be cited, but it is not particularly limited.

[0150] In the method for evaluating and / or selecting a controlling agent of the present invention, after adding a test substance and a target ligand to a GPCR polypeptide, the response of the GPCR polypeptide to the ligand is then measured. As a method for measuring the response, the same method as the response measuring method of the present invention can be used.

[0151] Next, based on the measured response, a test substance that inhibits the response of the GPCR polypeptide to the ligand is detected. The detected test substance is selected as a controller for the recognition of the ligand. Alternatively, based on the measured response, a test substance that enhances the response of the GPCR polypeptide to the ligand is detected. The detected test substance is selected as an enhancer for the recognition of the ligand.

[0152] The test substance that inhibits the response of the GPCR polypeptide to the ligand is selected as an inhibitor of the recognition of the ligand. Alternatively, the test substance that enhances the response of the GPCR polypeptide to the ligand is selected as an enhancer of the recognition of the ligand. The effect of the test substance on the response of the GPCR polypeptide to the ligand can be evaluated by comparing the response of the GPCR polypeptide (test group) to the ligand to the ligand with the response of the control group to the ligand. As an example of a control group, the GPCR polypeptide without the addition of a test substance, the GPCR polypeptide with the addition of a control substance, the GPCR polypeptide with the addition of a lower concentration of a test substance, the GPCR polypeptide before the addition of a test substance, and cells that do not express the GPCR polypeptide can be listed. Preferably, the evaluation and / or selection method of the inhibitor of the present invention includes measuring the activity of the GPCR polypeptide to the ligand in the presence and absence of a test substance.

[0153] For example, when the response in the test group is suppressed compared to the control group, the test substance can be determined as a substance that suppresses the response of the GPCR polypeptide to the target ligand, that is, a substance that suppresses the response of the original GPCR to the ligand. For example, when the response of the GPCR polypeptide in the test group is preferably suppressed to 60% or less, more preferably suppressed to 50% or less, and further preferably suppressed to 25% or less compared to the control group, the test substance can be determined as a substance that suppresses the response of the GPCR polypeptide to the target ligand, that is, a substance that suppresses the response of the original GPCR to the ligand. Alternatively, when the response of the GPCR polypeptide in the test group is statistically significantly suppressed compared to the control group, the test substance can be determined as a substance that suppresses the response of the GPCR polypeptide to the target ligand, that is, a substance that suppresses the response of the original GPCR to the ligand.

[0154] Alternatively, for example, when the response in the test group is enhanced compared to the control group, the test substance can be determined as a substance that enhances the response of the GPCR polypeptide to the target ligand, that is, a substance that enhances the response of the original GPCR to the ligand. For example, when the response of the GPCR polypeptide in the test group is preferably enhanced by 120% or more, more preferably enhanced by 150% or more, and further preferably enhanced by 200% or more compared to the control group, the test substance can be determined as a substance that enhances the response of the GPCR polypeptide to the target ligand, that is, a substance that enhances the response of the original GPCR to the ligand. Alternatively, when the response of the GPCR polypeptide in the test group is statistically significantly enhanced compared to the control group, the test substance can be determined as a substance that enhances the response of the GPCR polypeptide to the target ligand, that is, a substance that enhances the response of the original GPCR to the ligand.

[0155] In one embodiment of the method for evaluating and / or selecting a control agent of the present invention, the GPCR polypeptide is a vomeronasal receptor polypeptide expressed by the expression method of the present invention, and the ligand of the target GPCR is a pheromone substance, and the pheromone inhibitor or pheromone enhancer is evaluated and / or selected as a control agent for ligand recognition. In another embodiment of the method for evaluating and / or selecting a control agent of the present invention, the GPCR polypeptide is an umami receptor polypeptide (a combination of a common TAS1R1 and a common TAS1R3) expressed by the expression method of the present invention, the ligand of the target GPCR is an umami substance, and the umami inhibitor or umami enhancer is evaluated and / or selected as a control agent for ligand recognition. In another embodiment of the method for evaluating and / or selecting a control agent of the present invention, the GPCR polypeptide is a sweet receptor polypeptide (a combination of a common TAS1R2 and a common TAS1R3) expressed by the expression method of the present invention, the ligand of the target GPCR is a sweet substance, and the sweet inhibitor or sweet enhancer is evaluated and / or selected as a control agent for ligand recognition. In another embodiment of the method for evaluating and / or selecting a controlling agent of the present invention, the GPCR polypeptide is a bitter receptor polypeptide expressed by the expression method of the present invention, the ligand of the target GPCR is a bitter substance, and the bitter inhibitor or bitter enhancer is evaluated and / or selected as a controlling agent for ligand recognition.

[0156] The test substance determined by the above steps is a substance that can control the recognition of the ligand of the target by controlling the response of the GPCR to the ligand of the target. Therefore, the test substance determined by the above steps can be selected as a control agent for the recognition of the ligand. The substance selected as a control agent for the recognition of the ligand of the target by the evaluation and / or selection method of the control agent of the present invention can control the recognition of the ligand by controlling the response of the GPCR to the ligand.

[0157] Therefore, in one embodiment, the substance selected by the evaluation and / or selection method of the controller of the recognition of the ligand of the present invention is the active ingredient of the controller of the recognition of the ligand. Alternatively, the substance selected by the evaluation and / or selection method of the controller of the recognition of the ligand of the present invention can be contained in the compound or composition used to control the recognition of the ligand as an active ingredient for controlling the recognition of the ligand. In addition, or, the substance selected by the evaluation and / or selection method of the controller of the recognition of the ligand of the present invention can be used for the manufacture of the controller of the recognition of the ligand, or for the manufacture of the compound or composition used to control the recognition of the ligand. Using this substance, the recognition of the ligand of the target can be inhibited or enhanced.

[0158] In addition, as described above, the GPCR polypeptide expressed by the expression method of the present invention can be efficiently expressed on the cultured cell membrane compared to the original GPCR, and it can be considered that the response of the GPCR polypeptide reflects the response of the original GPCR, and therefore, the taste receptor polypeptide can be used as the GPCR polypeptide to evaluate the taste of the test substance. Therefore, in another embodiment, the present invention provides a method for evaluating taste. The method includes: a step of adding a test substance to the GPCR polypeptide expressed by the expression method of the present invention, and a step of measuring the response of the GPCR polypeptide to the test substance, and the GPCR polypeptide is a taste receptor polypeptide. The taste evaluation method of the present invention can efficiently evaluate the taste of the test substance.

[0159] The test substance used in the taste evaluation method of the present invention is not particularly limited as long as it is a substance for which the presence or degree of taste (e.g., umami, sweetness, or bitterness) is desired to be confirmed. The test substance may be a naturally occurring substance, or a substance artificially synthesized by chemical or biological methods, or may be a compound, or may be a composition or a mixture.

[0160] The GPCR polypeptide and its form used in the taste evaluation method of the present invention may be a taste receptor polypeptide, which is the same as the GPCR polypeptide and its form used in the response measurement method of the present invention.

[0161] In the taste evaluation method of the present invention, a test substance is applied to the GPCR polypeptide expressed by the expression method of the present invention. Examples of methods for applying a test substance to a GPCR polypeptide include, but are not particularly limited to, adding a test substance to a culture medium for culturing cells expressing the GPCR polypeptide.

[0162] In the taste evaluation method of the present invention, after adding a test substance to a GPCR polypeptide, the response of the GPCR polypeptide to the test substance is measured. As a method for measuring the response, the same method as the response measuring method of the present invention can be used.

[0163] Next, based on the measured response of the GPCR polypeptide, the test substance is evaluated. The test substance that causes the response of the GPCR polypeptide can be determined as a substance that presents a taste (e.g., umami, sweetness, or bitterness). The greater the intensity of the response caused by the test substance, the greater the degree of the taste of the test substance is evaluated to be (e.g., stronger umami, stronger sweetness, or stronger bitterness).

[0164] Preferably, the response of the GPCR polypeptide to the test substance is evaluated by comparing the response of the GPCR polypeptide to which the test substance is added (test group) with the response of the GPCR polypeptide in the control group. As the control group, the GPCR polypeptide to which the test substance is not added, the GPCR polypeptide to which the control substance is added, the GPCR polypeptide to which the test substance is added at a lower concentration, the GPCR polypeptide before the test substance is added, etc. can be listed. When the response in the test group is higher than that in the control group, the test substance is evaluated as a substance that presents a taste.

[0165] Therefore, in one embodiment of the taste evaluation method of the present invention, the response of the GPCR polypeptide in the presence and absence of the test substance is measured, and then it is determined whether the response in the presence of the test substance is increased compared to the response in the absence of the test substance. When the response in the presence of the test substance is higher, the test substance is evaluated as a substance that presents a taste. In a preferred embodiment, when the intensity of the response of the GPCR polypeptide in the presence of the test substance is preferably 120% or more, more preferably 150% or more, and further preferably 200% or more compared to the absence of the test substance, the test substance is evaluated as a substance that presents a taste. In another preferred embodiment, when the intensity of the response of the GPCR polypeptide in the presence of the test substance is statistically significantly increased compared to the absence of the test substance, the test substance is evaluated as a substance that presents a taste.

[0166] In one embodiment of the taste evaluation method of the present invention, the GPCR polypeptide is an umami receptor polypeptide (a combination of shared TAS1R1 and shared TAS1R3) expressed by the expression method of the present invention, and the taste of the evaluation object is umami. In another embodiment of the taste evaluation method of the present invention, the GPCR polypeptide is a sweet receptor polypeptide (a combination of shared TAS1R2 and shared TAS1R3) expressed by the expression method of the present invention, and the taste of the evaluation object is sweet. In another embodiment of the taste evaluation method of the present invention, the GPCR polypeptide is a bitter receptor polypeptide expressed by the expression method of the present invention, and the taste of the evaluation object is bitter.

[0167] In addition, as described above, the GPCR polypeptide expressed by the expression method of the present invention can be efficiently expressed on the cultured cell membrane compared to the original GPCR, and it can be considered that the response of the GPCR polypeptide reflects the response of the original GPCR. Therefore, as the GPCR polypeptide, a substance that inhibits the odor recognition of the ligand (odor substance, specifically amine) in the original GPCR can be evaluated and / or selected using a trace amine-related receptor polypeptide. The substance that inhibits the response of the GPCR polypeptide changes the ligand response of the GPCR polypeptide, even if the ligand response of the original GPCR changes, and as a result, the odor of the ligand can be selectively inhibited based on receptor antagonism. On the other hand, the substance that enhances the response of the GPCR polypeptide changes the ligand response of the GPCR polypeptide, even if the ligand response of the original GPCR changes, and as a result, the odor of the ligand can be selectively inhibited based on the cross-adaptation of the odor caused by the receptor agonism.

[0168] Therefore, in another embodiment, the present invention provides a method for evaluating and / or selecting an inhibitor of the odor of a ligand of a target GPCR. The method includes the step of measuring the response of the GPCR polypeptide after the addition of a test substance, wherein the GPCR polypeptide is a trace amine-related receptor polypeptide. Based on the measured response, a test substance that inhibits or enhances the response of the GPCR polypeptide is detected. The detected test substance is selected as an inhibitor of the odor of the target ligand. That is, the test substance that inhibits the response of the GPCR polypeptide is selected as an inhibitor of the odor of the ligand based on receptor antagonism, and the test substance that enhances the response of the GPCR polypeptide is selected as an inhibitor of the odor of the ligand based on cross-adaptation of the odor caused by receptor agonism. As the ligand of the target GPCR, it is preferably a ligand selected by the ligand exploration method of the present invention. By using the method for evaluating and / or selecting the odor inhibitor of the present invention, it is possible to efficiently evaluate or select substances that can selectively inhibit the odor of the target ligand. Even when the target ligand is a ligand of a GPCR that cannot be efficiently functionally analyzed due to insufficient expression on the cell membrane of cultured cells, it is possible to evaluate or select substances that can selectively inhibit the odor of the target ligand.

[0169] The evaluation and / or selection of the odor suppressant of the present invention is a method performed in vitro or ex vivo.

[0170] The test substance used in the method for evaluating and / or selecting an odor inhibitor of the present invention is not particularly limited as long as it is a substance that is intended to be an inhibitor of the odor of the target ligand. The test substance may be a naturally occurring substance, or a substance artificially synthesized by a chemical or biological method, or may be a compound, or may be a composition or a mixture.

[0171] The GPCR polypeptide and its form used in the method for evaluating and / or selecting the odor inhibitor of the present invention are the same as the GPCR polypeptide and its form used in the method for measuring the response of the present invention, as long as the GPCR polypeptide is a trace amine-associated receptor polypeptide.

[0172] In the method for evaluating and / or selecting an odor inhibitor of the present invention, a test substance is applied to the GPCR polypeptide expressed by the expression method of the present invention. As a method for applying a test substance to a GPCR polypeptide, a method of adding a test substance to a culture medium of cells expressing the GPCR polypeptide can be cited, but it is not particularly limited.

[0173] In the method for evaluating and / or selecting an odor inhibitor of the present invention, after adding a test substance to a GPCR polypeptide, the response of the GPCR polypeptide to the test substance is measured. As a method for measuring the response, the same method as the response measuring method of the present invention can be used.

[0174] In the first embodiment, the method for evaluating and / or selecting an odor inhibitor of the present invention includes the steps of adding a test substance and a target ligand to the GPCR polypeptide expressed by the expression method of the present invention, and measuring the response of the GPCR polypeptide to the ligand. As a method for applying a ligand to a GPCR polypeptide, the same method as the method for applying a test substance can be used. Then, based on the measured response, a test substance that inhibits the response of the GPCR polypeptide to the ligand is detected. As the detected test substance, an inhibitor of the odor of the ligand is selected.

[0175] In the first embodiment, the test substance that inhibits the response of the GPCR polypeptide to the ligand is selected as an inhibitor of the odor of the ligand based on receptor antagonism. The effect of the test substance on the response of the GPCR polypeptide to the ligand is evaluated, for example, by comparing the response of the GPCR polypeptide (test group) to which the test substance is added with the response of the GPCR polypeptide in the control group. As examples of control groups, the GPCR polypeptide to which no test substance is added, the GPCR polypeptide to which a control substance is added, the GPCR polypeptide to which a lower concentration of the test substance is added, the GPCR polypeptide before the addition of the test substance, cells that do not express the GPCR polypeptide, etc. can be listed. Preferably, the evaluation and / or selection method of the odor inhibitor of the present invention in the first embodiment includes measuring the activity of the olfactory GPCR polypeptide to the ligand in the presence and absence of the test substance.

[0176] For example, when the response in the test group is suppressed compared to the control group, the test substance can be determined as a substance that suppresses the response of the GPCR polypeptide to the target ligand, that is, a substance that suppresses the response of the original GPCR to the ligand. For example, when the response of the GPCR polypeptide in the test group is preferably suppressed to 60% or less, more preferably suppressed to 50% or less, and further preferably suppressed to 25% or less compared to the control group, the test substance can be determined as a substance that suppresses the response of the GPCR polypeptide to the ligand, that is, a substance that suppresses the response of the original GPCR to the ligand. Alternatively, when the response of the GPCR polypeptide in the test group is statistically significantly suppressed compared to the control group, the test substance can be determined as a substance that suppresses the response of the GPCR polypeptide to the ligand, that is, a substance that suppresses the response of the original GPCR to the ligand.

[0177] In a second embodiment, the method for evaluating and / or selecting an odor inhibitor of the present invention comprises the steps of adding a test substance to the GPCR polypeptide expressed by the expression method of the present invention, and measuring the response of the GPCR polypeptide to the test substance. Then, based on the measured response, a test substance that enhances the response of the GPCR polypeptide to the target ligand is detected. The detected test substance is selected as an inhibitor of the odor of the ligand.

[0178] The test substance that enhances the response of the GPCR polypeptide can reduce the response of the GPCR when it is subsequently exposed to the target ligand by enhancing the response of the GPCR in advance. As a result, the recognition of the odor of the ligand by the individual can be inhibited based on odor cross-adaptation. Therefore, in the second embodiment, an inhibitor of the odor of the ligand based on the cross-adaptation of the odor caused by receptor agonism is selected.

[0179] The effect of the test substance on the GPCR polypeptide can be evaluated, for example, by comparing the response of the GPCR polypeptide to which the test substance is added (test group) with the response in the control group. As examples of control groups, the above examples can be cited. Preferably, the evaluation and / or selection method of the odor inhibitor of the present invention in the second embodiment includes measuring the activity of the GPCR polypeptide in the presence and absence of the test substance. In addition, it is preferred that the evaluation and / or selection method of the odor inhibitor of the present invention in the second embodiment includes measuring the response of cells expressing the GPCR polypeptide and non-expressing cells to the ligand in the presence of the test substance.

[0180] For example, when the response in the test group is enhanced compared to the control group, the test substance can be determined as a substance that inhibits the response of the GPCR polypeptide to the target ligand, that is, a substance that inhibits the response of the original GPCR to the ligand. For example, when the response of the GPCR polypeptide in the test group is preferably enhanced by 120% or more, more preferably enhanced by 150% or more, and further preferably enhanced by 200% or more compared to the control group, the test substance can be determined as a substance that inhibits the response of the GPCR polypeptide to the ligand, that is, a substance that inhibits the response of the original GPCR to the ligand. Alternatively, when the response of the GPCR polypeptide in the test group is statistically significantly enhanced compared to the control group, the test substance can be determined as a substance that inhibits the response of the GPCR polypeptide to the ligand, that is, a substance that inhibits the response of the original GPCR to the ligand.

[0181] The test substance determined by the above steps is a substance that can inhibit the recognition of the odor of the target ligand by an individual by inhibiting the response of the GPCR to the target ligand. Therefore, the test substance determined by the above steps can be selected as an inhibitor of the odor of the ligand. The substance selected as an inhibitor of the odor of the target ligand by the evaluation and / or selection method of the odor inhibitor of the present invention can inhibit the odor of the ligand by inhibiting the response of the GPCR to the ligand.

[0182] Therefore, in one embodiment, the substance selected by the evaluation and / or selection method of the odor inhibitor of the ligand of the present invention can be an effective ingredient of the inhibitor of the odor of the ligand. Alternatively, the substance selected by the evaluation and / or selection method of the odor inhibitor of the ligand of the present invention can be contained in the compound or composition used to suppress the odor of the ligand as an effective ingredient for suppressing the odor of the ligand. In addition, or, the substance selected by the evaluation and / or selection method of the odor inhibitor of the ligand of the present invention can be used for the manufacture of the inhibitor of the odor of the ligand, or for the manufacture of the compound or composition for suppressing the odor of the ligand. According to this substance, the odor of the target ligand can be deodorized without causing the problem of unpleasantness caused by the strong odor of the fragrance, etc., which is generated in the deodorization method using the existing deodorant or fragrance, and causing other odors to be suppressed.

[0183] As exemplary embodiments of the present invention, the following compositions, production methods, uses, or methods are disclosed in this specification, but the present invention is not limited to these embodiments.

[0184] [1] A method for expressing a GPCR polypeptide, wherein the method comprises:

[0185] A step of expressing a GPCR polypeptide in a cell, wherein the GPCR polypeptide consists of an amino acid sequence in which at least one of the amino acid residues different from a consensus amino acid sequence in an amino acid sequence of a target GPCR (excluding olfactory receptors) is changed to an amino acid residue of the consensus amino acid sequence at a corresponding position,

[0186] The consensus amino acid sequence is an amino acid sequence derived from an alignment of the amino acid sequence of the target GPCR and the amino acid sequence of a GPCR encoded by an ortholog of the target GPCR in a vertebrate.

[0187] [2] The method described in [1], wherein the method is a method for increasing the expression of a GPCR polypeptide.

[0188] [3] A method for functionalizing a target GPCR, wherein the method comprises:

[0189] A step of expressing a GPCR polypeptide in a cell, wherein the GPCR polypeptide consists of an amino acid sequence in which at least one of the amino acid residues different from a consensus amino acid sequence in an amino acid sequence of a target GPCR (excluding olfactory receptors) is changed to an amino acid residue of the consensus amino acid sequence at a corresponding position,

[0190] The consensus amino acid sequence is an amino acid sequence derived from an alignment of the amino acid sequence of the target GPCR and the amino acid sequence of a GPCR encoded by an ortholog of the target GPCR in a vertebrate.

[0191] [4] A method as described in any one of [1] to [3], wherein the above-mentioned ortholog is an ortholog selected from orthologs in mammals, birds, reptiles, amphibians and fish, preferably an ortholog selected from orthologs in mammals, birds, reptiles and amphibians, and more preferably an ortholog in mammals.

[0192] [5] The method according to any one of [1] to [4], wherein the consensus amino acid sequence is an amino acid sequence consisting of consensus residues determined based on the alignment and in accordance with the following criteria (i) to (iii):

[0193] (i) at each amino acid position in the alignment,

[0194] (ii) when there is an amino acid residue that is different from the amino acid residue of the target GPCR and has an occurrence frequency of 50% or more, the amino acid residue is determined as a common residue,

[0195] (i-ii) When there are two amino acid residues with an occurrence frequency of 50%, the amino acid residues of the target GPCR are determined as common residues,

[0196] (i-iii) when the amino acid residue exists in the target GPCR and the amino acid residue with an occurrence frequency of 40% or more does not exist, it is determined that there is no common residue,

[0197] (i-iv) when an amino acid residue does not exist in the target GPCR and an amino acid residue with an occurrence frequency of 60% or more exists, the amino acid residue with the highest occurrence frequency is determined as the common residue; when there are two or more amino acid residues with the highest occurrence frequency, the amino acid residue with the smallest molecular weight among the amino acid residues is determined as the common residue;

[0198] (iv) when it does not belong to any of the above cases (ii) to (i-iv), the amino acid residue of the target GPCR is determined as a consensus residue,

[0199] (ii) when the consensus residue is determined according to the criteria of (i) above, when the consensus residue at the most N-terminal side is a consensus residue at a position corresponding to the N-terminal end of the target GPCR or closer to the C-terminal end and is not a methionine residue, the consensus residue at the N-terminal end than the consensus residue consisting of the methionine residue at the position closest to the N-terminal end is changed to have no consensus residue,

[0200] (iii) When the consensus residue is determined according to the criteria of (i) above, when the consensus residue on the most N-terminal side is a consensus residue corresponding to a position closer to the N-terminus than the N-terminus of the target GPCR and is not a methionine residue, the amino acid positions closer to the N-terminus than the position of the consensus residue in the alignment are traced back one by one until a methionine residue appears, and the amino acid residue with the highest frequency of occurrence is determined as the consensus residue. When there are two or more amino acid residues with the highest frequency of occurrence, the amino acid residue with the smallest molecular weight among the amino acid residues is determined as the consensus residue.

[0201] [6] The method as described in any one of [1] to [5], wherein the above-mentioned comparison is a comparison of the amino acid sequence of the above-mentioned target GPCR with the amino acid sequences of at least 2, preferably at least 5, more preferably at least 11, further preferably at least 15, further preferably at least 30, and further preferably at least 100 GPCRs encoded by the orthologs of the target GPCR in the above-mentioned vertebrate.

[0202] [7] The method according to any one of [1] to [6], wherein the target GPCR is a human GPCR.

[0203] [8] The method according to any one of [1] to [7], wherein the target GPCR is a vomeronasal receptor, a taste receptor, a trace amine-related receptor, a Mas-related G protein-coupled receptor, or a GPR.

[0204] [9] A method as described in any one of [1] to [8], wherein the GPCR polypeptide is preferably composed of the following amino acid sequence, in which at least one of the amino acid residues different from the consensus amino acid sequence shown by the sequence number (3) in the amino acid sequence shown by the sequence number (2) of the GPCR in (1) of the following Tables 2-1 and 2-2 is changed to an amino acid residue of the consensus amino acid sequence at the corresponding position.

[0205] [Table 2-1]

[0206]

[0207] [Table 2-2]

[0208]

[0209]

[10] The method according to [9], wherein the GPCR polypeptide preferably consists of an amino acid sequence represented by any one of SEQ ID NOs: 108 to 142, 144 to 214, and 275 to 312.

[0210]

[11] A method for expressing a GPCR polypeptide, wherein the method comprises:

[0211] A step of expressing a GPCR polypeptide in a cell, wherein the GPCR polypeptide consists of an amino acid sequence in which at least one of the amino acid residues different from a consensus amino acid sequence in the amino acid sequence of the target GPCR is changed to an amino acid residue of the consensus amino acid sequence at a corresponding position,

[0212] The GPCR polypeptide is composed of the following amino acid sequence, in which at least one of the amino acid residues different from the consensus amino acid sequence shown in sequence number 143 in the amino acid sequence shown in sequence number 36 of human TAAR6 is changed to an amino acid residue of the consensus amino acid sequence at a corresponding position, and preferably consists of the amino acid sequence shown in sequence number 143.

[0213]

[12] The method described in

[11] , wherein the method is a method for increasing the expression of a GPCR polypeptide.

[0214]

[13] A method for functionalizing a target GPCR, wherein the method comprises:

[0215] A step of expressing a GPCR polypeptide in a cell, wherein the GPCR polypeptide consists of an amino acid sequence in which at least one of the amino acid residues different from a consensus amino acid sequence in the amino acid sequence of the target GPCR is changed to an amino acid residue of the consensus amino acid sequence at a corresponding position,

[0216] The GPCR polypeptide is composed of the following amino acid sequence, in which at least one of the amino acid residues different from the consensus amino acid sequence shown in sequence number 143 in the amino acid sequence shown in sequence number 36 of human TAAR6 is changed to an amino acid residue of the consensus amino acid sequence at a corresponding position, and preferably consists of the amino acid sequence shown in sequence number 143.

[0217]

[14] The method according to any one of [1] to

[13] , wherein the GPCR polypeptide is expressed on the cell membrane of the cell.

[0218]

[15] The method according to any one of [1] to

[14] , wherein the method preferably further comprises the step of expressing RTP1S in the cell.

[0219]

[16] The method according to any one of [1] to

[15] , wherein the cells are HEK293 cells.

[0220]

[17] A method for measuring the response of a target GPCR, wherein the method comprises:

[0221] A step of measuring the response of the GPCR polypeptide expressed by the method of any one of [1] to

[16] .

[0222]

[18] A method for exploring a ligand of a target GPCR, wherein the method comprises:

[0223] A step of measuring the response of the GPCR polypeptide expressed by the method of any one of [1] to

[16] in the presence of a test substance, and

[0224] A step of selecting a test substance to which the GPCR polypeptide responds.

[0225]

[19] The method as described in

[18] , wherein the method preferably further comprises the step of measuring the response of the GPCR polypeptide in the absence of a test substance.

[0226]

[20] The method according to

[19] , wherein the test substance is preferably selected so as to increase the response of the GPCR polypeptide in the presence of the test substance to 120% or more of the response in the absence of the test substance.

[0227]

[21] The method according to

[19] , wherein the test substance is preferably selected so that the response of the GPCR polypeptide in the presence of the test substance is statistically significantly increased compared to the response in the absence of the test substance.

[0228]

[22] A method for evaluating and / or selecting a control agent for the recognition of a target GPCR ligand, wherein the method comprises:

[0229] a step of adding a test substance and a ligand of a target GPCR to the GPCR polypeptide expressed by the method of any one of [1] to

[16] , and

[0230] The step of determining the response of the GPCR polypeptide to the ligand.

[0231]

[23] The method according to

[22] , wherein the ligand is a ligand selected by the method described in any one of

[18] to

[21] .

[0232]

[24] A method as described in any one of

[22] or

[23] , wherein the method further comprises: based on the measured response, determining a test substance that controls the response of the GPCR polypeptide, preferably a test substance that inhibits or enhances the response of the GPCR polypeptide.

[0233]

[25] The method as described in any one of

[22] to

[24] , wherein the method further comprises the step of measuring the response of the GPCR polypeptide to the ligand without the addition of the test substance.

[0234]

[26] The method as described in

[25] , wherein the method further includes: when the response of the GPCR polypeptide to the ligand added with the test substance is suppressed compared to the response of the GPCR polypeptide to the ligand without the addition of the test substance, the test substance is determined as a substance that inhibits the response of the GPCR polypeptide to the ligand; or, when the response of the GPCR polypeptide to the ligand added with the test substance is enhanced compared to the response of the GPCR polypeptide to the ligand without the addition of the test substance, the test substance is determined as a substance that enhances the response of the GPCR polypeptide to the ligand.

[0235]

[27] A method for evaluating taste, wherein the method comprises:

[0236] a step of adding a test substance to the GPCR polypeptide expressed by the method of any one of [1] to

[16] , and

[0237] a step of determining the response of the GPCR polypeptide to the test substance,

[0238] The GPCR polypeptide is a taste receptor polypeptide.

[0239]

[28] The method as described in

[27] , wherein the method preferably further comprises the step of measuring the response of the GPCR polypeptide to which the test substance is not added.

[0240]

[29] The method as described in

[28] , wherein the test substance that increases the response of the GPCR polypeptide to which the test substance is added to 120% or more of the response of the GPCR polypeptide to which the test substance is not added is preferably evaluated as a substance that presents a taste.

[0241]

[30] The method as described in

[28] , wherein the test substance that statistically significantly increases the response of the GPCR polypeptide to which the test substance is added compared to the response of the GPCR polypeptide to which the test substance is not added is preferably evaluated as a substance that presents a taste.

[0242]

[31] The method according to any one of

[27] to

[30] , wherein the taste is preferably umami, sweetness, or bitterness.

[0243]

[32] A method for evaluating and / or selecting an inhibitor of an odor of a target GPCR ligand, wherein the method comprises:

[0244] a step of adding a test substance and a ligand of a target GPCR to the GPCR polypeptide expressed by the method of any one of [1] to

[16] , and

[0245] the step of determining the response of the GPCR polypeptide to the ligand,

[0246] The GPCR polypeptide is a trace amine-related receptor polypeptide.

[0247]

[33] The method according to

[32] , wherein the ligand is a ligand selected by the method described in any one of

[18] to

[21] .

[0248]

[34] The method as described in

[32] or

[33] , wherein the method further comprises: based on the measured response, determining a test substance that inhibits the response of the above-mentioned GPCR polypeptide.

[0249]

[35] The method as described in any one of

[32] to

[34] , wherein the method further comprises the step of measuring the response of the GPCR polypeptide to the ligand without the addition of the test substance.

[0250]

[36] The method as described in

[35] , wherein the method further includes: when the response of the GPCR polypeptide to the ligand to which the test substance is added is inhibited compared to the response of the GPCR polypeptide to the ligand to which the test substance is not added, the step of determining the test substance as a substance that inhibits the response of the GPCR polypeptide to the ligand.

[0251]

[37] A method for evaluating and / or selecting an inhibitor of an odor of a target GPCR ligand, wherein the method comprises:

[0252] a step of adding a test substance to the GPCR polypeptide expressed by the method of any one of [1] to

[16] , and

[0253] a step of determining the response of the GPCR polypeptide to the test substance,

[0254] The GPCR polypeptide is a trace amine-related receptor polypeptide.

[0255]

[38] The method according to

[37] , wherein the ligand is a ligand selected by the method described in any one of

[18] to

[21] .

[0256]

[39] The method as described in

[37] or

[38] , wherein the method further comprises: a step of determining a test substance that enhances the response of the GPCR polypeptide based on the measured response.

[0257]

[40] The method as described in any one of

[37] to

[39] , wherein the method further comprises the step of measuring the response of the GPCR polypeptide to which the test substance is not added.

[0258]

[41] The method as described in

[40] , wherein the method further includes: when the response of the GPCR polypeptide to which the test substance is added is enhanced compared to the response of the GPCR polypeptide to which the test substance is not added, the step of determining the test substance as a substance that inhibits the response of the GPCR polypeptide to the ligand.

[0259]

[42] A method as described in any one of

[17] to

[41] , wherein the response of the above-mentioned GPCR polypeptide is preferably measured by the following methods: measuring the amount of intracellular cAMP using ELISA or reporter gene detection, measuring the amount of calcium ions using calcium imaging or TGFα shedding detection, or measuring the potential changes inside and outside the cell membrane using a two-electrode membrane potential fixation method using African clawed frog oocytes.

[0260]

[43] An altered GPCR polypeptide, wherein the altered GPCR polypeptide consists of the following amino acid sequence, in which at least one of the amino acid residues different from the consensus amino acid sequence in the amino acid sequence of a target GPCR (excluding olfactory receptors) is changed to an amino acid residue in the consensus amino acid sequence at a corresponding position,

[0261] The consensus amino acid sequence is an amino acid sequence derived from an alignment of the amino acid sequence of the target GPCR and the amino acid sequence of a GPCR encoded by an ortholog of the target GPCR in a vertebrate.

[0262]

[44] An altered GPCR polypeptide as described in

[43] , wherein the above-mentioned ortholog is an ortholog selected from orthologs in mammals, birds, reptiles, amphibians and fish, preferably an ortholog selected from orthologs in mammals, birds, reptiles and amphibians, and more preferably an ortholog in mammals.

[0263]

[45] The altered GPCR polypeptide according to

[43] or

[44] , wherein

[0264] The consensus amino acid sequence is an amino acid sequence consisting of consensus residues determined based on the alignment and in accordance with the following criteria (i) to (iii):

[0265] (i) at each amino acid position in the alignment,

[0266] (ii) when there is an amino acid residue that is different from the amino acid residue of the target GPCR and has an occurrence frequency of 50% or more, the amino acid residue is determined as a common residue,

[0267] (i-ii) When there are two amino acid residues with an occurrence frequency of 50%, the amino acid residues of the target GPCR are determined as common residues,

[0268] (i-iii) when the amino acid residue exists in the target GPCR and the amino acid residue with an occurrence frequency of 40% or more does not exist, it is determined that there is no common residue,

[0269] (i-iv) when an amino acid residue does not exist in the target GPCR and an amino acid residue with an occurrence frequency of 60% or more exists, the amino acid residue with the highest occurrence frequency is determined as the common residue; when there are two or more amino acid residues with the highest occurrence frequency, the amino acid residue with the smallest molecular weight among the amino acid residues is determined as the common residue;

[0270] (iv) when it does not belong to any of the above cases (ii) to (i-iv), the amino acid residue of the target GPCR is determined as a consensus residue,

[0271] (ii) when the consensus residue is determined according to the criteria of (i) above, when the consensus residue at the most N-terminal side is a consensus residue at a position corresponding to the N-terminal end of the target GPCR or closer to the C-terminal end and is not a methionine residue, the consensus residue at the N-terminal end than the consensus residue consisting of the methionine residue at the position closest to the N-terminal end is changed to have no consensus residue,

[0272] (iii) When the consensus residue is determined according to the criteria of (i) above, when the consensus residue on the most N-terminal side is a consensus residue corresponding to a position closer to the N-terminus than the N-terminus of the target GPCR and is not a methionine residue, the amino acid positions closer to the N-terminus than the position of the consensus residue in the alignment are traced back one by one until a methionine residue appears, and the amino acid residue with the highest frequency of occurrence is determined as the consensus residue. When there are two or more amino acid residues with the highest frequency of occurrence, the amino acid residue with the smallest molecular weight among the amino acid residues is determined as the consensus residue.

[0273]

[46] An altered GPCR polypeptide as described in any one of

[43] to

[45] , wherein the above-mentioned comparison is a comparison of the amino acid sequence of the above-mentioned target GPCR with the amino acid sequences of at least 2, preferably at least 5, more preferably at least 11, further preferably at least 15, further preferably at least 30, and further preferably at least 100 GPCRs encoded by the orthologs of the target GPCR in the above-mentioned vertebrate.

[0274]

[47] The modified GPCR polypeptide as described in any one of

[43] to

[46] , wherein the target GPCR is a human GPCR.

[0275]

[48] ​​The modified GPCR polypeptide as described in any one of

[43] to

[47] , wherein the target GPCR is a vomeronasal receptor, a taste receptor, a trace amine-related receptor, a Mas-related G protein-coupled receptor, or a GPR.

[0276]

[49] An altered GPCR polypeptide as described in any one of

[43] to

[48] , wherein the altered GPCR polypeptide is preferably composed of the following amino acid sequence, in which at least one of the amino acid residues different from the consensus amino acid sequence shown by the sequence number (3) in the amino acid sequence shown by the sequence number (2) of the GPCR in (1) of Tables 2-1 and 2-2 above is changed to an amino acid residue of the consensus amino acid sequence at the corresponding position.

[0277]

[50] The altered GPCR polypeptide as described in

[49] , wherein the altered GPCR polypeptide is preferably composed of an amino acid sequence represented by any one of SEQ ID NOs: 108 to 142, 144 to 214, and 275 to 312.

[0278]

[51] A modified GPCR polypeptide, wherein the modified GPCR polypeptide consists of the following amino acid sequence, in which at least one of the amino acid residues different from the consensus amino acid sequence shown in sequence number 143 in the amino acid sequence shown in sequence number 36 of human TAAR6 is changed to an amino acid residue of the consensus amino acid sequence at a corresponding position, preferably consisting of the amino acid sequence shown in sequence number 143.

[0279]

[52] A polynucleotide, wherein the polynucleotide encodes the altered GPCR polypeptide described in any one of

[43] to

[51] .

[0280]

[53] The polynucleotide according to

[52] , wherein the polynucleotide consists of a base sequence represented by any one of SEQ ID NOs: 215 to 234 and 313 to 317.

[0281]

[54] A vector or a DNA fragment, wherein the vector or the DNA fragment comprises the polynucleotide described in

[52] or

[53] .

[0282]

[55] A transformed cell, wherein the transformed cell contains the vector or DNA fragment described in

[54] .

[0283]

[56] The transformed cell according to

[55] , wherein the transformed cell preferably further contains: a vector or a DNA fragment containing a polynucleotide encoding RTP1S.

[0284]

[57] The transformed cell according to

[55] or

[56] , wherein the cell is a HEK293 cell.

[0285] Example

[0286] Hereinafter, the present invention will be described in more detail with reference to examples.

[0287] Example 1 Preparation and analysis of shared receptors

[0288] 1) Production of receptor genes

[0289] When designing a common olfactory receptor, a search for candidate genes identical to the target olfactory receptor gene is performed using NCBI BLAST, and orthologous gene groups are identified for the obtained gene groups.

[0290] Specifically, when a human receptor (GPCR) is used as a target receptor, in the identification of orthologs, genes having the same name as the target receptor are selected as orthologs from genes with similarity to each other obtained by BLAST search.

[0291] For example, in the case of TAAR1 of the human trace amine-related receptor, the amino acid sequence of human TAAR1 (NP_612200.1) was set as the query sequence, and 249 genes containing TAAR1 in the name were determined as orthologs from the 250 genes with the same homology obtained by BLAST search. The amino acid sequences of these 249 genes plus human TAAR1, a total of 250 genes (Tables 3-1 to 4), were aligned and analyzed and the common amino acids were determined as follows. For human TAAR2, TAAR5, TAAR6, TAAR8 and TAAR9, the ortholog group was also determined in the same way. When human TAAR6, TAAR8 or TAAR9 is used as the target receptor, the orthologs determined are the orthologs of primates and the orthologs of mammals other than primates. When TAAR2 or TAAR5 is used as the target receptor, the orthologs determined are the orthologs of primates, the orthologs of mammals other than primates, and the orthologs of birds. When TAAR1 is used as the target receptor, the orthologs determined are the orthologs of primates, the orthologs of mammals other than primates, the orthologs of birds, the orthologs of reptiles, and the orthologs of amphibians.

[0292] For example, in the case of VN1R1 of the human vomeronasal receptor, the amino acid sequence of human VN1R1 (NP_065684.1) was set as the query sequence, and a BLAST search was performed to determine 22 genes whose names contained vomeronasal type-1 receptor 1 among the 250 genes with the highest homology. VN1R is a receptor gene family with great diversity between identical genes, and 7 genes among these identical genes that have more than 65% amino acid identity with human VN1R1 were determined as orthologs. The amino acid sequences of these 7 genes plus human VN1R1, a total of 8 genes, were compared and analyzed and the common amino acids were determined as follows. Ortholog groups were also determined for human VN1R2, VN1R4, and VN1R5. When VN1R1, VN1R2, VN1R4 or VN1R5 is used as the target receptor, the orthologs determined are the orthologs in mammals (primates).

[0293] For example, in the case of TAS1R1 of the human taste receptor (umami receptor), the amino acid sequence of human TAS1R1 (NP_619642.2) is set as the query sequence, and from the 250 genes with the same homology obtained by BLAST search, 249 genes whose names contain TAS1R1 are determined as orthologs. The amino acid sequences of these 249 genes plus human TAS1R1, a total of 250 genes, are compared and analyzed and the common amino acids are determined as described below. Ortholog groups are also determined for human TAS1R2 and TAS1R3. When TAS1R1, TAS1R2 or TAS1R3 is used as the target receptor, the orthologs determined are orthologs of primates, orthologs of mammals other than primates, orthologs of birds, orthologs of reptiles, orthologs of amphibians, and orthologs of fish.

[0294] For example, in the case of TAS2R8 of a human taste receptor (bitter receptor), the amino acid sequence of human TAS2R8 (NP_076407.1) was set as a query sequence, and 90 genes containing TAS2R8 in the name were determined as orthologs from among the 250 genes with the same identity obtained by BLAST search. The amino acid sequences of these 90 genes plus human TAS2R8, a total of 91 genes, were aligned and analyzed and the common amino acids were determined as follows. Ortholog groups were similarly determined for human TAS2R16, TAS2R38, TAS2R42, TAS2R45, TAS2R46, TAS2R31, TAS2R1, TAS2R3, TAS2R4, TAS2R5, TAS2R7, TAS2R9, TAS2R10, TAS2R13, TAS2R14, TAS2R20, TAS2R30, TAS2R39, TAS2R40, TAS2R43, TAS2R50, and TAS2R60. When human TAS2R45 is used as the target receptor, the orthologs determined are the orthologs of primates, and when human TAS2R8, TAS2R16, TAS2R38, TAS2R42 or TAS2R46 is used as the target receptor, the orthologs determined are the orthologs of primates and the orthologs of mammals other than primates.

[0295] For example, in the case of MrgprE of the human Mas-related G protein-coupled receptor, the amino acid sequence of human MrgprE (NP_001034254.2) is set as the query sequence, and 72 genes whose names contain MrgprE are identified as orthologs from the 250 genes with the highest homology obtained by BLAST search. The amino acid sequences of these 72 genes plus human MrgprE, a total of 73 genes, are aligned and analyzed and the common amino acids are determined as described below. Ortholog groups are also determined for human MrgprF, MAS1, MAS1L, MRGPRD, MRGPRG, MRGPRX1, MRGPRX2, MRGPRX3 and MRGPRX4. When human MrgprE or MrgprF is used as the target receptor, the orthologs determined are orthologs of primates and orthologs of target mammals other than primates.

[0296] For example, in the case of GPR3 ​​of human GPR, the amino acid sequence of human GPR3 ​​(NP_005272.1) is set as the query sequence, and 234 genes whose names contain GPR3 ​​are determined as orthologs from the 250 genes with the highest homology obtained by BLAST search. The amino acid sequences of these 234 genes plus human GPR3, a total of 235 genes, are aligned and analyzed and the common amino acids are determined as follows. Ortholog groups are also determined for human GPRs other than GPR3, GPR17, GPR31, GPR50, GPR65, GPR68, and GPR42 described in Table 4. In addition, in the case of human GPR17, genes whose names contain "uracil nucleotide / cysteinyl leukotriene receptor" known as orthologs are determined as orthologs. Similarly, in the case of human GPR31, genes whose names include "12-(S)-hydroxy-5,8,10,14-eicosatetraenoic acid receptor" were identified as orthologs, in the case of human GPR50, genes whose names include "melatonin-related receptor" were identified as orthologs, in the case of human GPR65, genes whose names include "psychosine receptor" were identified as orthologs, and in the case of human GPR68, genes whose names include "ovarian cancer G-protein coupled receptor 1" were identified as orthologs. In the case of human GPR42, genes containing "GPR42" in their names and genes containing "free fatty acid receptor3" in their names (GPCR genes with high identity to the GPR42 gene) were identified as orthologs.

[0297] Among the human GPCRs other than those mentioned above, in the case of ADGRB1, the amino acid sequence of human ADGRB1 (NP_001693.2) was set as the query sequence, and 234 genes whose names contained ADGRB1 were determined as orthologs from the 250 genes with the highest homology obtained by BLAST search. The amino acid sequences of these 234 genes plus human ADGRB1, a total of 235 genes, were compared and analyzed and the common amino acids were determined as described below. Ortholog groups were also determined for human ADGRB2, ADGRB3, and ADGRD1. In addition, in the case of human ADGRA1, genes containing "ADGRA1" in the name and genes containing "GPR123" in the name (GPCR genes with high homology to the ADGRA1 gene) were determined as orthologs. Similarly, in the case of human ADGRA2, genes containing "ADGRA2" and genes containing "GPR124" were identified as orthologs, and in the case of human ADGRA3, genes containing "ADGRA3" and genes containing "GPR125" were identified as orthologs. In addition, in the case of human ADGRC1, genes containing "EGF LAG seven-pass G-type receptor 1" (GPCR genes with high homology to the ADGRC1 gene) were identified as orthologs. Similarly, in the case of human ADGRC2, genes whose names include "cadherin EGF LAG seven-pass G-type receptor 2" are determined as orthologs, in the case of human ADGRC3, genes whose names include "cadherin EGF LAG seven-pass G-type receptor 3" are determined as orthologs, in the case of human ADGRD2, genes whose names include "adhesion G protein-coupled receptor D2" or "G-protein coupled receptor 144" are determined as orthologs, and in the case of human ADGRE1, genes whose names include "adhesion G protein-coupled receptor E1" or "adhesion G protein-coupled receptor E1 isoform" are determined. 2precursor (adhesion G protein-coupled receptor E1 isotype 2 precursor)" gene as a direct homolog, in the case of human ADGRE2,The genes whose names contain “adhesion G protein-coupled receptor E2” are determined as orthologs. In the case of human ADGRE3, the genes whose names contain “adhesion G protein-coupled receptor E3” are determined as orthologs. In the case of human ADGRE5, the genes whose names contain “adhesion G protein-coupled receptor E5” or “CD97 antigen” are determined as orthologs. In the case of human ADGRF1, the genes whose names contain “adhesion G-protein coupled receptor F1” or “adhesion G-protein coupled receptor F1 isoform 1 precursor” are determined as orthologs. In the case of human ADGRF3, the genes whose names contain “adhesion G-protein coupled receptor F3 (adhesion G protein coupled receptor F3)" or "G-protein coupled receptor 113 (G protein coupled receptor 113)" is determined as an ortholog. In the case of human ADGRF4, the gene whose name contains "adhesion G-protein coupled receptor F4 (adhesion G protein coupled receptor F4)" or "G-protein coupled receptor 115 (G protein coupled receptor 115)" is determined as an ortholog. In the case of human ADGRF5, the gene whose name contains "adhesion G-protein coupled receptor F5 (adhesion G protein coupled receptor F5)" or "G-protein coupled receptor 116 (G protein coupled receptor 116)" is determined as an ortholog. In the case of human ADGRG1, the gene whose name contains "adhesion G-protein coupled receptor G1 (adhesion G protein coupled receptor G1)" or "adhesion G-protein coupled receptor G1isoform b" is determined. The gene of "adhesion G protein-coupled receptor G1 isotype b precursor" is a direct homolog, and in human ADGRG2,The genes whose names contain “adhesion G-protein coupled receptor G2(Adhesion G protein coupled receptor G2)” or “G-protein coupled receptor 64(G protein coupled receptor 64)” are determined as orthologs. In the case of human ADGRG4, the genes whose names contain “adhesion G-protein coupled receptor G4(Adhesion G protein coupled receptor G4)” or “G-protein coupled receptor 112(G protein coupled receptor 112)” are determined as orthologs. In the case of human ADGRG5, the genes whose names contain “adhesion G-protein coupledreceptor G5(Adhesion G protein coupled receptor G5)” or “G-protein coupled receptor 114(G protein coupled receptor 114)” are determined as orthologs. In the case of human ADGRG6, the genes whose names contain “adhesion G-protein coupled receptor G6(Adhesion G protein coupled receptor G6)” or “G-protein coupledreceptor 126(G protein coupled receptor 126)" was identified as an ortholog; in the case of human ADGRG7, genes whose names contained "adhesion G-protein coupled receptor G7(Adhesion G protein coupled receptor G7)" or "adhesionG-protein coupled receptor G7 isoform 2precursor(Adhesion G protein coupled receptor G7 isoform 2 precursor)" were identified as orthologs; in the case of human ADGRL1, genes whose names contained "adhesion G protein-coupled receptor L1(Adhesion G protein coupled receptor L1)" or "adhesion G protein-coupledreceptor L1 isoform 1precursor(Adhesion G protein coupled receptor L1 isoform 1 precursor)" were identified as orthologs; in the case of human ADGRL2, genes whose names contained "adhesion G-protein coupled receptor L2(Adhesion G protein coupled receptor L2)" or "latrophilin and seven transmembrane domain-containing protein (containing spider toxin and seven transmembrane domains)" gene as a direct homolog, in the case of human ADGRL3,Genes whose names contain "adhesion G-protein coupled receptor L3" or "latrophilin-3" are determined as orthologs. In the case of human ADGRL4, genes whose names contain "adhesion G-protein coupled receptor L4" or "latrophilin-2isoform" are determined as orthologs. In the case of human Chrm-4 / M4R, genes whose names contain "M4R" are determined as orthologs. In the case of human F2RL1, genes whose names contain "proteinase-activated receptor 2" are determined as orthologs. In the case of human GRM5, genes whose names contain "metabotropic glutamate receptor 5" are determined as orthologs. In the case of human APLNR, genes whose names contain "apelin receptor" as an ortholog, in the case of human CALCRL, a gene whose name includes "calcitonin gene-related peptide type 1" as an ortholog, in the case of human GLP2R, a gene whose name includes "glucagon-like peptide 2 receptor" as an ortholog, in the case of human MC4R, a gene whose name includes "MC4R", "Melanocortin receptor 4", "Melanocortin-4 receptor" or "Melanocortin 4 receptor" as an ortholog, and in the case of human CCR6, a gene whose name includes "CC chemokine receptor type 6" as an ortholog.

[0298] In Table 4, for each human GPCR, the total number of the gene of the human GPCR and the determined orthologs is shown as the reference gene number.

[0299] [Table 3-1]

[0300] Reference genes 1 trace amine-associated receptor 1 (Homo sapiens) 2 Trace amine-related receptor 1 [Bonobo (Pan paniscus)] 3 Trace amine receptor 1 [Chimpanzee (Pan troglodytes)] 4 Trace amine-associated receptor 1 [Gorilla gorilla gorilla] 5 Trace amine receptor 1 [Hylobates moloch] 6 Trace amine-related receptor 1 [Macaca mulatta] 7 Prediction: Trace amine-related receptor 1 [White-naped mangabey (Cercocebus atys)] 8 Trace amine receptor 1 [Macaca nemestrina] 9 Trace amine receptor 1 [Sumatran orangutan (Pongo abelii)] 10 Trace amine-related receptor 1 [Green monkey (Chlorocebus sabaeus)] 11 Prediction: Trace amine-related receptor 1 [Drillus leucophaeus] 12 Trace amine-associated receptor 1 [Gelada baboons (Theropithecus gelada)] 13 Prediction: Trace amine-related receptor 1 [Colobus angolensis palliates] 14 Trace amine-related receptor 1 [East African baboon (Papio anubis)] 15 Trace amine receptor 1 [White-cheeked gibbon (Nomascus leucogenys)] 16 Trace amine receptor 1 [Trachypithecus francoisi] 17 Trace amine-associated receptor 1 [Peruvian night monkey (Aotus nancymaae)] 18 Trace amine-related receptor 1 [Golden monkey (Rhinopithecus roxellana)] 19 Low-quality protein: trace amine-associated receptor 1-like [Ugandan red colobus (Piliocolobus tephrosceles)] 20 Prediction: Trace amine-related receptor 1 [Rhinopithecus bieti] 21 Trace amine receptor 1 [Sapajus apella] 22 Trace amine receptor 1 [Panamanian capuchin monkey (Cebus imitator)] 23 Trace amine-related receptor 1 [Common marmoset (Callithrix jacchus)] 24 Trace amine-associated receptor 1 [Bolivian squirrel monkey (Saimiri boliviensis boliviensis)] 25 Trace amine-related receptor 1 [Carlito syrichta] 26 Trace amine-associated receptor 1 [Blue whale (Balaenoptera musculus)] 27 Trace amine-related receptor 1 [Balaenoptera acutorostrata scammoni] 28 Trace amine receptor 1 [African elephant (Loxodonta africana)] 29 Trace amine-related receptor 1 [Sperm whale (Physeter catodon)] 30 Trace amine-related receptor 1 [Killer whale (Orcinus orca)] 31 Prediction: Trace amine-related receptor 1 [Lipotes vexillifer] 32 Trace amine receptor 1 isoform X2 (isoform X2) [Phocoena sinus] 33 Trace amine-related receptor 1 [Long-finned pilot whale (Globicephala melas)] 34 Trace amine receptor 1 [Bottlenose dolphin (Tursiops truncatus)] 35 Trace amine receptor 1 [Monodon monoceros] 36 Prediction: Trace amine-related receptor 1 [Hipposideros armiger] 37 Trace amine receptor 1 [Rhinolophus ferrumequinum] 38 Trace amine-related receptor 1 homotype X2 [Yangtze River porpoise (Neophocaena asiaeorientalis asiaeorientalis)] 39 Trace amine receptor 1 [Pacific white-necked dolphin (Lagenorhynchus obliquidens)] 40 Trace amine-related receptor 1 isotype X1 [Delphinapterus leucas] 41 Trace amine-related receptor 1 [Greater rat-eared bat (Myotis myotis)] 42 Trace amine-related receptor 1 [Gray mouse lemur (Microcebus murinus)] 43 Trace amine-related receptor 1 [Molossus molossus] 44 Prediction: Trace amine-related receptor 1 [Propithecus coquereli] 45 Trace amine receptor 1 [Pteropus vampyrus] 46 Trace amine receptor 1 [Angora shorthair bat (Pteropus alecto)] 47 Trace amine receptor 1 [Otolemur garnettii] 48 Low-quality protein: trace amine-related receptor 1 [(Sus scrofa)] 49 Trace amine-associated receptor 1 [Desmodus rotundus] 50 Trace amine-associated receptor 1 [Phyllostomus discolor] 51 Trace amine-related receptor 1 [Big brown bat (Eptesicus fuscus)] 52 Trace amine receptor 1 [North American otter (Lontra canadensis)] 53 Trace amine-related receptor 1 [Tupaia chinensis] 54 Trace amine-associated receptor 1 [Grizzly bear (Ursus arctos horribilis)] 55 Trace amine-associated receptor 1 [Nine-banded armadillo (Dasypus novemcinctus)] 56 Trace amine-related receptor 1 [Giant panda (Ailuropoda melanoleuca)] 57 Trace amine receptor 1 [Enhydra lutris kenyoni] 58 Trace amine-related receptor 1 [West Indian manatee (Trichechus manatus latirostris)] 59 Trace amine receptor 1 [Little brown bat (Myotis lucifugus)] 60 Prediction: Trace amine-related receptor 1 [Brown's myotis brandtii] 61 Prediction: Trace amine-associated receptor 1 [Natal long-fingered bat (Miniopterus natalensis)] 62 Trace amine receptor 1 [Mustela erminea] 63 Trace amine-related receptor 1 [Alpaca (Vicugna pacos)] 64 Trace amine-associated receptor 1 [California sea lion (Zalophus californianus)] 65 Prediction: Trace amine-related receptor 1 [Mustela putorius furo]

[0301] [Table 3-2]

[0302] Reference genes 66 Trace amine-associated receptor 1 [Jamaican fruit bat (Artibeus jamaicensis)] 67 Trace amine-related receptor 1 [Chinese pangolin (Manis pentadactyla)] 68 Prediction: Trace amine-related receptor 1 [Equus przewalskii] 69 Prediction: Trace amine-related receptor 1 [Odobenus rosmarus divergens] 70 Trace amine-associated receptor 1 [elephant seal (Mirounga leonina)] 71 Trace amine-associated receptor 1 [Pipistrellus kuhlii] 72 Trace amine-associated receptor 1 [Egyptian fruit bat (Rousettus aegyptiacus)] 73 Trace amine-associated receptor 1 [Weddell seal (Leptonychotes weddellii)] 74 Trace amine-related receptor 1 [Malayan pangolin (Manis javanica)] 75 Trace amine-associated receptor 1 [Two-toed sloth (Choloepus didactylus)] 76 Trace amine receptor 1 [Callorhinus ursinus] 77 Trace amine-associated receptor 1 [Hawaiian monk seal (Neomonachus schauinslandi)] 78 Prediction: Trace amine-associated receptor 1 [Chrysochloris asiatica] 79 Trace amine receptor 1 [Echinops telfairi] 80 Trace amine-associated receptor 1 [Harbor seal (Phoca vitulina)] 81 Trace amine receptor 1 [Little brown bat (Myotis lucifugus)] 82 Trace amine-associated receptor 1 [Spanish mole (Talpa occidentalis)] 83 Low-quality protein: trace amine-related receptor 1 [Indian flying fox (Pteropus giganteus)] 84 Trace amine-associated receptor 1 [Grey seal (Halichoerus grypus)] 85 Trace amine receptor 1 [Camelus dromedarius] 86 Prediction: Trace amine-related receptor 1 [Ceratotherium simum simum] 87 Trace amine receptor 1 [Oryx dammah] 88 Prediction: Low-quality protein: trace amine-related receptor 1-like [Galeopterus variegatus] 89 Trace amine receptor 1 [Suricata suricatta] 90 Trace amine-related receptor 1 [sheep (Ovis aries)] 91 Trace amine receptor 1 [Hyaena hyaena] 92 Trace amine receptor 1 [Persian cat (Felis catus)] 93 Trace amine receptor 1 [Camelus ferus] 94 Trace amine receptor 1 [Acinonyx jubatus] 95 Trace amine receptor 1 [Puma yagouaroundi] 96 Trace amine-associated receptor 1 [Puma concolor] 97 Prediction: Trace amine-related receptor 1 [David's myotis bat (Myotis davidii)] 98 Prediction: Trace amine-related receptor 1 [Marmota marmota marmota] 99 Trace amine-associated receptor 1 [Marmota flaviventris] 100 Prediction: Trace amine-related receptor 1 [Panthera tigris altaica] 101 Trace amine-related receptor 1 [Thirteen-striped ground squirrel (Lctidomys tridecemlineatus)] 102 Trace amine-related receptor 1 [Buffalo (Bubalus bubalis)] 103 Prediction: Trace amine-related receptor 1 [Orvctolagus cuniculus] 104 Trace amine-associated receptor 1 [Arctic ground squirrel (Urocitellus parryii)] 105 Prediction: Trace amine-related receptor 1 [goat (Capra hircus)] 106 Prediction: Trace amine-related receptor 1 [Wild yak (Bos mutus)] 107 Trace amine receptor 1 [cattle (Bos taurus)] 108 Prediction: Trace amine-related receptor 1 [Chinchilla lanigera] 109 Trace amine-related receptor 1 homotype X1 [Yangtze River porpoise (Neophocaena asiaeorientalis asiaeorientalis)] 110 Trace amine-related receptor 1 homotype X1 [Phocoena sinus] 111 Trace amine-related receptor 1-like [naked mole rat (Heterocephalus glaber)] 112 Trace amine-related receptor 1-like [naked mole rat (Heterocephalus glaber)] 113 Trace amine receptor 1 [Odocoileus virginianus texanus] 114 Prediction: Trace amine-related receptor 1 [Condylura cristata] 115 Prediction: Trace amine-related receptor 1 [Western European hedgehog (Erinaceus europaeus)] 116 Trace amine receptor 1 [Fukomys damarensis] 117 Trace amine-associated receptor 1 [Israeli mole rat (Nannospalax galili)] 118 Low-quality protein: trace amine-associated receptor 1 [American beaver (Castor canadensis)] 119 Prediction: Trace amine-related receptor 1 [Shrew (Sorex araneus)] 120 Trace amine receptor 1 [Sturnira hondurensis] 121 Trace amine-associated receptor 1 [Chilean degus (Octodon degus)] 122 Prediction: Trace amine-associated receptor 1 [Jaculus jaculus] 123 Prediction: Trace amine-related receptor 1 [Dipodomys ordii] 124 Trace amine-related receptor 1 [Tasmanian devil (Sarcophilus harrisii)] 125 Trace amine-related receptor 1 [Dipodomys spectabilis] 126 Trace amine-related receptor 1 [Guinea pig (Cavia porcellus)] 127 Prediction: trace amine related receptor 1 [(Monodelphis domestica)] 128 Trace amine-related receptor 1-like [Trichosurus vulpecula] 129 Trace amine receptor 1 [White-footed mouse (Peromyscus leucopus)] 130 Trace amine receptor 1 [Microtus ochrogaster]

[0303] [Table 3-3]

[0304]

[0305] [Table 3-4]

[0306]

[0307] [Table 4]

[0308]

[0309] Regarding the GPCRs No. 1 to 145 in Table 4, the alignment analysis of the determined gene group was performed using ClustalW. Based on the alignment results, Jalview was used to design a common receptor. In this alignment, when there is an amino acid residue that is different from the amino acid residue of the reference amino acid sequence and has an occurrence frequency of 50% or more at each amino acid position of the original receptor amino acid sequence of the target receptor that becomes the benchmark, the amino acid residue of the reference amino acid sequence is changed to the amino acid residue. In addition, when there is an amino acid residue that is different from the amino acid residue of the reference amino acid sequence and has an occurrence frequency of 50% at each amino acid position of the original receptor amino acid sequence of the target receptor that becomes the benchmark, the amino acid residue of the reference amino acid sequence is not changed when the occurrence frequency of the amino acid residue of the reference amino acid sequence is also 50%. In this alignment, when there is a deletion with an occurrence frequency of 40% or more at each amino acid position of the original receptor amino acid sequence of the target receptor that becomes the benchmark, the amino acid residue of the reference amino acid sequence is changed to a deletion. For example, there is a deletion with a frequency of 40% or more at the position of the amino acid position corresponding to the C-terminus, such as TAAR1, and therefore, the amino acid residues of the reference amino acid sequence are changed to deletion. In addition, for example, deletions are confirmed at a frequency of 40% or more at the position corresponding to the starting methionine position in the human sequence, such as TAAR1, TAAR2, MAS1L, GPR135, and ADGRG4, and therefore, the amino acid residues of the reference amino acid sequence are changed to deletions, and the initial methionine is selected as the starting methionine in the amino acid sequence after the change through the above steps, and the amino acid sequence before it is deleted. Among them, in this method, when the common residue on the N-terminal side of the common residue composed of the methionine residue at the position closest to the N-terminus is changed to a non-common residue in TAAR6, the total length of the amino acid sequence of the common receptor generated as a result becomes shorter than the total length of the amino acid sequence of the original receptor by more than 10%, and therefore, this method is not followed, but the N-terminal structure of the original receptor is maintained as it is instead. Specifically, the common residue asparagine at the most N-terminal side is an important amino acid residue for sugar chain modification and membrane migration, so the common residue is not changed, and the N-terminal structure of the original receptor on the N-terminal side is maintained as it is compared to the position corresponding to the common residue. On the other hand, in this alignment, when there is an amino acid with an occurrence frequency of 60% or more at the position corresponding to the deletion position of the original receptor amino acid sequence of the target receptor as the benchmark, the amino acid with the highest conservation is inserted into the deletion position of the benchmark amino acid sequence for modification. When there are two or more amino acids with the highest conservation, the amino acid with the smallest molecular weight is inserted for modification.For example, as in TAS2R1, an amino acid with an occurrence frequency of 60% or more is present on the N-terminal side of the amino acid position corresponding to the N-terminus, and therefore, an amino acid residue is added on the N-terminal side of the reference amino acid sequence.

[0310] The topology of the receptor under design was confirmed using TMHMM (Transmembrane Hidden Markov Model). Each receptor to be designed was required to have a seven-transmembrane structure.

[0311] Regarding the GPCRs No. 1 to 5, 7 to 13, 31 to 38, 60, 76, 80, 85 and 102 in Table 4, the DNA sequences encoding the designed receptor polypeptides were obtained by DNA synthesis based on the optimization of the base sequence codons corresponding to the amino acid sequence for expression in human cultured cells. EcoRI and XhoI sites were added to both ends of the base sequence, and the prepared EcoRI and XhoI sites were inserted downstream of the Flag-Rho tag sequence on the pME18S vector. The gene encoding human RTP1S that transfers the GPCR protein produced in the cultured cells to the cell membrane was inserted into the EcoRI and XhoI sites of another pME18S vector to prepare the pME18S-RTP1S vector.

[0312] 2) Preparation of cultured cells transformed with receptor genes 1

[0313] For flow cytometry, 3.3 × 10 HEK293 cells suspended in DMEM (Nacalai) were seeded in each well of a 6-well plate. 5 After 24 hours, the reaction solution of the composition shown in Table 5 was prepared, and after standing in a clean bench for 20 minutes, it was added to each well of a 6-well plate. Here, the receptor gene refers to the gene of the GPCR No. 1 to 4, 8 to 13, 31 to 38, 60, 76, 80, 85 and 102 in Table 4. The cells were cultured in an incubator maintained at 37°C and 5% CO2 for 24 hours. As a control, cells (mock) that did not express the receptor were prepared.

[0314] [Table 5]

[0315]

[0316] 3) Preparation of cultured cells transformed with receptor genes 2

[0317] For flow cytometry, 3.3 × 10 HEK293 cells suspended in DMEM (Nacalai) were seeded in each well of a 6-well plate. 5After 24 hours, a reaction solution with the composition shown in Table 6 was prepared for the cells, and allowed to stand in a clean bench for 20 minutes before being added to each well of a 6-well plate. The cells were cultured for 24 hours in an incubator maintained at 37°C and 5% CO2. As a control, cells not expressing the receptor (mock) were prepared.

[0318] [Table 6]

[0319]

[0320] *“c” indicates mutualization.

[0321] 4) Determination of the amount of receptor protein on the cell membrane (flow cytometry)

[0322] The cells recovered using Cellstripper were treated with an anti-FLAG antibody (COSMO BIO) as a primary antibody on ice for 1 hour. After washing the cells, PE (phycoerythrin)-conjugated anti-mouse IgG (ABCAM Co., Ltd.) as a secondary antibody was treated on ice for 30 minutes. After washing, 0.25 μg of 7-AAD (7-aminoactinomycin D, 7-Aminoactinomycin D, Fujifilm Wako Pure Chemical Industries, Ltd.) was added, and the average value of the PE signal of the 7-AAD-negative cell cluster was measured by a cell flow cytometry system (BD) as an indicator of the amount of receptors on the cell membrane. In each experiment, cells expressing FLAG-M2 acetylcholine receptors were used as positive controls (PC), and cells not expressing receptors were used as negative controls (NC), and the average value of the PE signal was calculated in the same way as above. The PE signal of NC was set to 0% and the PE signal of PC was set to 100% for standardization, and the PE signal (%) of each receptor was calculated as the membrane expression level (cell surface expression).

[0323] 5) Results

[0324] The expression level of each receptor on HEK293 cells was analyzed using flow cytometry (2) and 4) above. As shown in Table 7, the average values ​​of the three experiments were compared, and the increase in membrane expression obtained by the communal method was confirmed for all receptors analyzed for membrane expression, specifically VN1R1, VN1R2, VN1R4, VN1R5, TAS2R8, TAS2R16, TAS2R38, TAS2R42, TAS2R45, TAS2R46, TAAR1, TAAR2, TAAR5, TAAR6, TAAR8, TAAR9, MrgprE, MrgprF, GPR31, GPR65, GPR82, GPR88 and GPR171.

[0325] [Table 7]

[0326]

[0327] Using flow cytometry (3) and 4) above), the expression levels of the original receptor of TAS1R1 and the original receptor of TAS1R3, the shared receptor of TAS1R1 and the shared receptor of TAS1R3, the original receptor of TAS1R1 and the shared receptor of TAS1R3, or the combination of the shared receptor of TAS1R1 and the original receptor of TAS1R3 on HEK293 cells were analyzed. Figure 1 As shown, it was found that the membrane expression level of TAS1R1 increased when either of the two original receptors TAS1R1 and TAS1R3 was expressed in HEK293 cells compared to when the two original receptors TAS1R1 and TAS1R3 were co-expressed. In particular, the highest amount of expression was observed when both TAS1R1 and TAS1R3 were co-expressed.

[0328] The accession numbers and amino acid sequences of the original GPCRs of the above examples and the amino acid sequences of the common GPCRs are shown in the following Tables 8-1 and 8-2. In addition, the DNA sequences encoding the common GPCRs consisting of the amino acid sequences shown in SEQ ID NOs. 108 to 112, 114 to 120, 138 to 145, 167, 183, 187, 192 and 209 are shown in SEQ ID NOs. 215 to 234 and 313 to 317.

[0329] [Table 8-1]

[0330]

[0331] [Table 8-2]

[0332]

Claims

1. A method for expressing a G protein coupled receptor (GPCR) polypeptide, wherein: The method comprises: A step of expressing a GPCR polypeptide in a cell, wherein the GPCR polypeptide consists of an amino acid sequence in which at least one of the amino acid residues different from a consensus amino acid sequence in the amino acid sequence of a target GPCR is changed to an amino acid residue of the consensus amino acid sequence at a corresponding position, wherein the target GPCR does not include an olfactory receptor, The consensus amino acid sequence is an amino acid sequence derived from an alignment of the amino acid sequence of the target GPCR and the amino acid sequence of a GPCR encoded by an ortholog of the target GPCR in a vertebrate.

2. The method of claim 1, wherein: The consensus amino acid sequence is an amino acid sequence consisting of consensus residues determined based on the alignment and in accordance with the following criteria (i) to (iii): (i) at each amino acid position in the alignment, (ii) when there is an amino acid residue that is different from the amino acid residue of the target GPCR and has an occurrence frequency of 50% or more, the amino acid residue is determined as a common residue, (i-ii) When there are two amino acid residues with an occurrence frequency of 50%, the amino acid residues of the target GPCR are determined as common residues, (i-iii) when the amino acid residue exists in the target GPCR and the amino acid residue with an occurrence frequency of 40% or more does not exist, it is determined that there is no common residue, (i-iv) when an amino acid residue does not exist in the target GPCR and an amino acid residue with an occurrence frequency of 60% or more exists, the amino acid residue with the highest occurrence frequency is determined as the common residue; when there are two or more amino acid residues with the highest occurrence frequency, the amino acid residue with the smallest molecular weight among the amino acid residues is determined as the common residue; (iv) when it does not belong to any of the above cases (ii) to (i-iv), the amino acid residue of the target GPCR is determined as a consensus residue, (ii) when the consensus residue is determined according to the criteria of (i) above, when the consensus residue at the most N-terminal side is a consensus residue at a position corresponding to the N-terminal end of the target GPCR or closer to the C-terminal end and is not a methionine residue, the consensus residue at the N-terminal end than the consensus residue consisting of the methionine residue at the position closest to the N-terminal end is changed to have no consensus residue, (iii) When the consensus residue is determined according to the criteria of (i) above, when the consensus residue on the most N-terminal side is a consensus residue corresponding to a position closer to the N-terminus than the N-terminus of the target GPCR and is not a methionine residue, the amino acid positions closer to the N-terminus than the position of the consensus residue in the alignment are traced back one by one until a methionine residue appears, and the amino acid residue with the highest frequency of occurrence is determined as the consensus residue. When there are two or more amino acid residues with the highest frequency of occurrence, the amino acid residue with the smallest molecular weight among the amino acid residues is determined as the consensus residue.

3. The method according to claim 1 or 2, wherein: The target GPCR is a human GPCR.

4. The method according to any one of claims 1 to 3, wherein: The GPCR polypeptide is composed of the following amino acid sequence, in which at least one of the amino acid residues different from the consensus amino acid sequence shown by the sequence number (3) in the amino acid sequence shown by the sequence number (2) of the GPCR in (1) of Tables 1 and 2 below is changed to an amino acid residue of the consensus amino acid sequence at the corresponding position. [Table 1] [Table 2] 5. The method of claim 4, wherein: The GPCR polypeptide consists of an amino acid sequence represented by any one of SEQ ID NOs: 108-142, 144-214, and 275-312.

6. A method for expressing a GPCR polypeptide, wherein: The method comprises: A step of expressing a GPCR polypeptide in a cell, wherein the GPCR polypeptide consists of an amino acid sequence in which at least one of the amino acid residues different from a consensus amino acid sequence in the amino acid sequence of the target GPCR is changed to an amino acid residue of the consensus amino acid sequence at a corresponding position, The GPCR polypeptide consists of the following amino acid sequence, in which at least one of the amino acid residues different from the consensus amino acid sequence shown in sequence number 143 in the amino acid sequence shown in sequence number 36 of human TAAR6 is changed to an amino acid residue of the consensus amino acid sequence at the corresponding position.

7. The method of claim 6, wherein: The GPCR polypeptide consists of the amino acid sequence shown in SEQ ID NO:

143.

8. A method for measuring the response of a target GPCR, wherein: The method comprises: A step of measuring the response of the GPCR polypeptide expressed by the method according to any one of claims 1 to 7.

9. A method for exploring a ligand of a target GPCR, wherein: The method comprises: a step of measuring the response of the GPCR polypeptide expressed by the method of any one of claims 1 to 7 in the presence of a test substance, and A step of selecting a test substance to which the GPCR polypeptide responds.

10. A method for evaluating and / or selecting a control agent for the recognition of a target GPCR ligand, wherein: The method comprises: a step of adding a test substance and a ligand of a target GPCR to the GPCR polypeptide expressed by the method of any one of claims 1 to 7, and The step of determining the response of the GPCR polypeptide to the ligand.

11. A method for evaluating taste, wherein: The method comprises: a step of adding a test substance to the GPCR polypeptide expressed by the method of any one of claims 1 to 7, and a step of determining the response of the GPCR polypeptide to the test substance, The GPCR polypeptide is a taste receptor polypeptide.

12. A method for evaluating and / or selecting an inhibitor of an odor of a target GPCR ligand, wherein: The method comprises: a step of adding a test substance and a ligand of a target GPCR to the GPCR polypeptide expressed by the method of any one of claims 1 to 7, and the step of determining the response of the GPCR polypeptide to the ligand, The GPCR polypeptide is a trace amine-related receptor polypeptide.

13. A method for evaluating and / or selecting an inhibitor of an odor of a target GPCR ligand, wherein: The method comprises: a step of adding a test substance to the GPCR polypeptide expressed by the method of any one of claims 1 to 7, and a step of determining the response of the GPCR polypeptide to the test substance, The GPCR polypeptide is a trace amine-related receptor polypeptide.

14. The method according to any one of claims 8 to 13, wherein: The response of the GPCR polypeptide is measured by the following methods: measuring the amount of intracellular cAMP by ELISA or reporter gene detection, measuring the amount of calcium ions by calcium imaging or TGFα shedding detection, or measuring the potential change inside and outside the cell membrane by the two-electrode membrane potential fixation method using Xenopus oocytes.

15. An altered GPCR polypeptide, wherein: The altered GPCR polypeptide is composed of the following amino acid sequence, in which at least one of the amino acid residues different from the consensus amino acid sequence in the amino acid sequence of the target GPCR is changed to an amino acid residue of the consensus amino acid sequence at a corresponding position, wherein the target GPCR does not include an olfactory receptor, The consensus amino acid sequence is an amino acid sequence derived from an alignment of the amino acid sequence of the target GPCR and the amino acid sequence of a GPCR encoded by an ortholog of the target GPCR in a vertebrate.

16. The altered GPCR polypeptide of claim 15, wherein The consensus amino acid sequence is an amino acid sequence consisting of consensus residues determined based on the alignment and in accordance with the following criteria (i) to (iii): (i) at each amino acid position in the alignment, (ii) when there is an amino acid residue that is different from the amino acid residue of the target GPCR and has an occurrence frequency of 50% or more, the amino acid residue is determined as a common residue, (i-ii) When there are two amino acid residues with an occurrence frequency of 50%, the amino acid residues of the target GPCR are determined as common residues, (i-iii) when the amino acid residue exists in the target GPCR and the amino acid residue with an occurrence frequency of 40% or more does not exist, it is determined that there is no common residue, (i-iv) when an amino acid residue does not exist in the target GPCR and an amino acid residue with an occurrence frequency of 60% or more exists, the amino acid residue with the highest occurrence frequency is determined as the common residue; when there are two or more amino acid residues with the highest occurrence frequency, the amino acid residue with the smallest molecular weight among the amino acid residues is determined as the common residue; (iv) when it does not belong to any of the above cases (ii) to (i-iv), the amino acid residue of the target GPCR is determined as a consensus residue, (ii) when the consensus residue is determined according to the criteria of (i) above, when the consensus residue at the most N-terminal side is a consensus residue at a position corresponding to the N-terminal end of the target GPCR or closer to the C-terminal end and is not a methionine residue, the consensus residue at the N-terminal end than the consensus residue consisting of the methionine residue at the position closest to the N-terminal end is changed to have no consensus residue, (iii) When the consensus residue is determined according to the criteria of (i) above, when the consensus residue on the most N-terminal side is a consensus residue corresponding to a position closer to the N-terminus than the N-terminus of the target GPCR and is not a methionine residue, the amino acid positions closer to the N-terminus than the position of the consensus residue in the alignment are traced back one by one until a methionine residue appears, and the amino acid residue with the highest frequency of occurrence is determined as the consensus residue. When there are two or more amino acid residues with the highest frequency of occurrence, the amino acid residue with the smallest molecular weight among the amino acid residues is determined as the consensus residue.

17. The altered GPCR polypeptide of claim 15 or 16, wherein The target GPCR is a human GPCR.

18. The altered GPCR polypeptide according to any one of claims 15 to 17, wherein The GPCR polypeptide is composed of the following amino acid sequence, in which at least one of the amino acid residues different from the consensus amino acid sequence shown by the sequence number (3) in the amino acid sequence shown by the sequence number (2) of the GPCR in (1) of Tables 1 and 2 above is changed to an amino acid residue of the consensus amino acid sequence at the corresponding position.

19. The altered GPCR polypeptide of claim 18, wherein The altered GPCR polypeptide consists of an amino acid sequence represented by any one of SEQ ID NOs: 108-142, 144-214, and 275-312.

20. An altered GPCR polypeptide, wherein: The altered GPCR polypeptide is composed of the following amino acid sequence, in which at least one of the amino acid residues different from the consensus amino acid sequence shown in sequence number 143 in the amino acid sequence shown in sequence number 36 of human TAAR6 is changed to an amino acid residue of the consensus amino acid sequence at a corresponding position.

21. The altered GPCR polypeptide of claim 20, wherein The altered GPCR polypeptide consists of the amino acid sequence shown in SEQ ID NO:

143.

22. A polynucleotide, wherein The polynucleotide encodes the altered GPCR polypeptide according to any one of claims 15 to 21.

23. A vector or DNA fragment, wherein: The vector or DNA fragment comprises the polynucleotide of claim 22.

24. A transformed cell, wherein: Containing the vector or DNA fragment according to claim 23.

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