JP2015105826A - Taste substance evaluation system using a domain other than taste receptor cells - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide means for precisely evaluating taste substance in a repeatable manner.SOLUTION: A taste substance evaluation system is a method for evaluating a taste substance or a taste inhibitory substance. The method includes the steps of: a) allowing a specimen to come into contact with a protein complex which includes T1R1 extracellular domain and T1R3 extracellular domain or allowing a specimen to come into contact with a protein complex which includes T1R2 extracellular domain and T1R3 extracellular domain, under the presence of calcium; and b) detecting coupling of the specimen with the protein complex.

Description

  The present invention relates to a method for evaluating a taste substance using a taste receptor extracellular domain, a reagent therefor, and a method for producing the reagent.

  Taste is a sensation that occurs when a substance binds to specific receptors present on the surface of the tongue, especially when the substance is put into the mouth. The taste is composed of five basic tastes, that is, salty taste, sour taste, sweet taste, umami taste, and bitter taste, and these basic tastes are considered to be formed by integration.

  Taste substance development has so far been effective only through extraction from food materials with strong taste and accidental discovery, and evaluation of taste substances has relied on sensory tests using human tongues until now, It was difficult to develop effective taste substances. Instead of a human sensory test, it is also possible to isolate taste cells present in the taste buds on the surface of the tongue and use them to evaluate the taste-improving effect. However, those that have isolated the taste cells present in the taste buds on the surface of the tongue cannot be used for time-consuming evaluations because they have very weak adhesion to the culture plate and die immediately. In addition, since there are no established taste cells, the evaluation method is not practical in terms of taste cell supply and test reproducibility.

  At present, saltiness and sourness are said to be sensed through several ion channel receptors expressed on the cell membrane on the proximal side of taste cells present in the taste buds on the tongue surface. On the other hand, sweet taste, umami taste, and bitter taste are thought to be detected by G protein-coupled receptor (GPCR), which is a membrane protein present in taste cells, and signal transduction via the G protein coupled thereto, Specifically, sweet taste is received by T1R2 and T1R3 heterodimers (sweet receptors) belonging to the T1R family, umami taste is received by T1R1 and T1R3 heterodimers (umami receptors) belonging to the T1R family, and bitterness is It has been shown to be received by about 30 molecules (bitter taste receptors) named the T2R family.

  As a method for objectively identifying a taste substance, an assay using a cell line expressing the receptor has been reported (Patent Document 1). However, the assay using cells has a problem in stability and reproducibility of cells, and also has a problem that adsorption to the cell membrane and response by other cell membrane proteins also occur simultaneously. In addition, it has been difficult to prepare a sample that retains the functional state of the receptor taste recognition region as well as the full length of the receptor, and the analysis at the molecular level has not been sufficiently performed so far.

JP 2005-500318 A

  An object of this invention is to provide the means for evaluating a taste substance accurately with sufficient reproducibility.

  The present inventors have made it possible to purify the extracellular domain of a taste receptor in the form of a heterodimer, which is a functional unit, by coexisting calcium ions. It was found that taste substances and taste inhibitory substances can be evaluated.

That is, the present invention includes the following inventions.
(1) A method for evaluating a taste substance or taste inhibitor,
a) contacting a test substance with a protein complex containing the extracellular domain of T1R1 and the extracellular domain of T1R3 in the presence of calcium ions, or a protein complex containing the extracellular domain of T1R2 and the extracellular domain of T1R3 A step of contacting the body with a test substance, and b) detecting a binding of the test substance to the protein complex,
Including said method.
(2) The method according to (1), wherein in step b), a structural change of the protein complex due to binding of the test substance to the protein complex is detected.
(3) The method according to (2), wherein the extracellular domain is fluorescently labeled, and the structural change of the protein complex is detected by FRET analysis.
(4) Reagent for evaluating a taste substance or taste inhibitory substance containing the following a) and b) in an aqueous solvent:
a) calcium ions,
b) A protein complex comprising the extracellular domain of T1R1 and the extracellular domain of T1R3, or a protein complex comprising the extracellular domain of T1R2 and the extracellular domain of T1R3.
(5) The reagent according to (4), wherein the calcium ion concentration in the aqueous solvent is 0.1 to 100 mM.
(6) A method for producing a reagent for evaluating a taste substance or taste inhibitor,
a) expressing in a host cell a gene encoding the extracellular domain of T1R1 and a gene encoding the extracellular domain of T1R3, or a gene encoding the extracellular domain of T1R2 and a gene encoding the extracellular domain of T1R3;
b) obtaining a fraction containing the extracellular domain of T1R1 and the extracellular domain of T1R3 or the extracellular domain of T1R2 and the extracellular domain of T1R3 from the culture; and c) adding calcium ions to the fraction, Separating and recovering the protein from the liquid fraction,
Said method.

  According to the present invention, the extracellular domain of a taste receptor can be purified in the form of a heterodimer that is a functional unit, and a taste substance or taste inhibitory substance is evaluated accurately and reproducibly using this heterodimer. It becomes possible.

FIG. 1 is a graph showing a titration curve obtained by FRET analysis.

  The present inventors have succeeded for the first time in obtaining the extracellular domain of a taste receptor belonging to the T1R family in the form of a heterodimer that is a functional unit. And, when the taste receptor was extremely unstable in the state where the taste substance as a ligand was not bound, it was found that by coexisting calcium ions, it can be stably maintained even in the absence of the taste substance, A protein sample was successfully prepared in the presence / absence of a tastant. Therefore, physicochemical taste substance binding analysis using a purified protein sample for the taste receptor has become possible for the first time.

  Furthermore, as a result of the binding analysis on the taste substance in which the receptor response was observed, it was conceived that the FRET signal change due to this taste substance binding can be used as a taste substance binding analysis system. We have succeeded in analyzing the affinity and determining the binding constants even for taste substances with weak affinity that cannot be measured in principle by the method of action analysis.

  Taste cells are one of a group of cells that are organized to form the taste buds of the tongue, and are present, for example, in the lingual papillae, such as the leaf-like, cocoon-shaped, and circumvallate papilla. Taste cells are also found in tissues such as the palate and esophagus and stomach. Sweet taste receptors and umami receptors, which are taste receptors, are membrane proteins present in taste cells and belong to the G protein-coupled receptor (GPCR) family. In these receptors, three types of proteins belonging to the T1R family (T1R1, T1R2, and T1R3) function as heterodimers, and T1R1 / T1R3 heterodimers function as umami receptors and T1R2 / T1R3. Heterodimers are responsible for sweetness acceptance.

  GPCRs usually have 7 transmembrane regions and corresponding transmembrane domains, and some have N-terminal domain, extracellular domain, cytoplasmic domain, and C-terminal domain depending on the type. These domains can be structurally identified using methods well known to those skilled in the art, such as sequence analysis programs that identify hydrophobic and hydrophilic domains.

  The extracellular domain refers to the domain of the T1R protein that protrudes from the cell membrane and is exposed outside the cell. Such a domain may include an N-terminal domain that is also exposed extracellularly. The extracellular domain also serves as a binding and recognition site for many taste substances, particularly umami and sweet substances.

  The present invention is characterized in that the extracellular domain of a taste receptor belonging to the T1R family that is a taste receptor is used in the form of a heterodimer that is a functional unit. A protein complex including the extracellular domain of T1R1 and the extracellular domain of T1R3 (hereinafter sometimes referred to as a T1R1 / T1R3 extracellular domain complex) functions as an umami receptor, for example, the extracellular domain of T1R1 It can be produced by introducing a gene encoding and a gene encoding the extracellular domain of T1R3 into a host cell, and purifying the protein complex of interest from the obtained cell culture. A protein complex containing the extracellular domain of T1R2 and the extracellular domain of T1R3 (hereinafter sometimes referred to as T1R2 / T1R3 extracellular domain complex) functions as a sweet taste receptor, for example, the extracellular domain of T1R2 It can be produced by introducing the gene to be encoded and the gene encoding the extracellular domain of T1R3 into a host cell, and purifying the protein complex of interest from the culture of the gene-introduced cell. Hereinafter, the T1R1 / T1R3 extracellular domain complex and the T1R2 / T1R3 extracellular domain complex may be collectively referred to as an extracellular domain complex or a protein complex. In addition, for a protein or protein complex whose amino acid sequence has been determined, a polypeptide known by a method known to those skilled in the art, such as chemically polymerizing each amino acid, is synthesized based on the sequence. It can also be prepared according to a method (peptide synthesis method).

  The amino acid sequences of T1R1, T1R2 and T1R3, and their extracellular domains, and the base sequences of the genes encoding them are available from public databases (GenBank, EMBL, DDBJ). Genes derived from organisms with unknown sequences can be obtained by cloning using known sequence information. A method for obtaining a desired gene by cloning is well known in the field of molecular biology. For example, if the gene sequence is known, a suitable genomic library can be created by restriction endonuclease digestion and screened using a probe complementary to the desired gene sequence. Once the sequence is isolated, the DNA can be amplified using standard amplification methods such as polymerase chain reaction (PCR) to obtain an amount of DNA suitable for transformation (gene transfer). For example, Molecular Cloning: A Laboratory Manual 3rd Edition (Sambrook & Russell, Cold Spring Harbor) Laboratory Press, 2001). Alternatively, cDNA may be obtained by reverse transcription polymerase chain reaction (RT-PCR) using RNA prepared from cells or tissues expressing the target protein as a template.

  T1R1, T1R2 and T1R3 are not particularly limited, but mammals such as primates including humans and monkeys, rodents, pigs, monkeys, dogs and cats, and non-mammals such as fish, amphibians, reptiles, birds Those derived from can be used.

  Specific examples of T1R1, T1R2 and T1R3 are shown below together with their accession numbers. Human T1R1 (amino acid sequence: DAA000012, base sequence: BK000153), human T1R2 (amino acid sequence: DAA00000019, base sequence: BK000151), human T1R3 (amino acid sequence: DAA000001, base sequence: BK000152), medaka T1R1 (amino acid sequence: BAE78481, Base sequence: AB200905), medaka T1R2a (amino acid sequence: BAE788482.2, base sequence: AB200906), medaka T1R3 (amino acid sequence: BAE784485.1, base sequence: AB200909).

  Proteins functionally equivalent to the above T1R1, T1R2 and T1R3, and their extracellular domains can also be used in the present invention. For example, T1R1 having the above-mentioned specific amino acid sequence or a protein functionally equivalent to its extracellular domain is 70% or more, preferably 80% or more, more preferably 90% or more, and more preferably the specific amino acid sequence. Consists of an amino acid sequence having a homology or identity of 95% or more, most preferably 99% or more, a protein having the activity of T1R1 or its extracellular domain, and one or several amino acids in its specific amino acid sequence Is a protein having an amino acid sequence deleted, substituted, inserted or added, and having the activity of T1R1 or its extracellular domain. The same applies to T1R2 and T1R3 and their extracellular domains.

  The amino acid substitution in the above is preferably a conservative substitution. Examples of amino acid conservative substitutions include glycine (Gly) and proline (Pro), glycine and alanine (Ala) or valine (Val), leucine (Leu) and isoleucine (Ile), glutamic acid (Glu) and glutamine (Gln) ), Aspartic acid (Asp) and asparagine (Asn), cysteine (Cys) and threonine (Thr), threonine and serine (Ser) or alanine, lysine (Lys) and arginine (Arg). Are known. When a plurality of amino acids are deleted, substituted, inserted or added, the number of amino acids to be deleted, substituted, inserted or added is usually 2 to 10, preferably 2 to 5, more preferably Two to three.

  Genes functionally equivalent to the genes encoding the T1R1, T1R2 and T1R3 and the extracellular domain thereof can also be used in the present invention. Genes include nucleic acids, which include single and double stranded DNA (including cDNA) and RNA (including mRNA). For example, as a gene functionally equivalent to the gene encoding T1R1 having the specific base sequence or its extracellular domain, the specific base sequence and 70% or more, preferably 80% or more, more preferably 90% As mentioned above, a gene encoding a protein consisting of a base sequence having homology or identity of 95% or more, most preferably 99% or more, and having activity of T1R1 or its extracellular domain can be mentioned. The same applies to the genes encoding T1R2 and T1R3 and their extracellular domains.

  Functionally equivalent genes also include conservatively modified variants (eg, synonymous codon substitutions) and complementary sequences for a particular base sequence. Specifically, synonymous codon substitution is accomplished, for example, by generating a sequence in which the third position of one or more selected codons is replaced with a mixed base and / or deoxyinosine residue. be able to. With respect to a particular base sequence, a conservatively modified variant refers to a nucleic acid that encodes the same or essentially the same amino acid sequence.

  A cell producing a T1R1 / T1R3 extracellular domain complex used in the present invention (for example, a transformant) is prepared by combining a gene encoding the extracellular domain of T1R1 and a gene encoding the extracellular domain of T1R3 together with a host cell. Or separately. That is, the gene can be introduced by preparing a vector containing the above two genes and introducing it into a host cell, or by preparing a vector containing each gene and introducing it into a host cell. The gene introduction may be performed by homologous recombination. Although two genes may be introduced into different host cells, it is preferable to introduce the two genes into the same host cell from the viewpoint of stably producing a protein complex. A method is preferably used in which the obtained cells are cultured in a medium, a fraction containing the T1R1 / T1R3 extracellular domain complex is obtained from the culture, and the fraction is purified and collected from this fraction in the presence of calcium ions. Alternatively, the T1R1 / T1R3 extracellular domain complex can be produced by expressing a gene encoding the extracellular domain of T1R1 and a gene encoding the extracellular domain of T1R3 in a cell-free protein synthesis system. The same applies to cells producing the T1R2 / T1R3 extracellular domain complex.

  The type of host into which the gene is introduced is not limited, and unicellular eukaryotes such as bacteria, fungi and various yeasts, or live cells of animals or plants can be arbitrarily selected. Specific examples of the host include bacteria belonging to the genus Escherichia such as Escherichia coli and the genus Bacillus such as Bacillus subtilis. In addition, yeasts such as Saccharomyces cerevisiae and Schizosaccharomyces pombe, animal cells such as COS cells and CHO cells, and insect cells such as S2, Sf9, and Sf21 can also be used.

  The vector for linking genes for gene introduction is not particularly limited as long as it can be used for introducing genes into host cells, and examples thereof include plasmids, phages and cosmids. Examples of the plasmid include E. coli plasmids (for example, pET21a (+), pET32a (+), pET39b (+), pET40b (+), pET43.1a (+), pET44a (+), pKK223-3, pGEX4T, pUC118, pUC119, pUC18, pUC19, etc.), plasmids derived from Bacillus subtilis (eg, pUB110, pTP5, etc.), plasmids derived from yeast (eg, YEp13, YEp24, YCp50, etc.), and plasmids for Pichia pastoris (eg, pPICZ, pPICZα, pHIL-D2, pPIC3.5, pHIL-S1, pPIC9, pPIC9K, pPIC6, pGAPZ, pPIC9K, pPIC3.5K, pAO815, pFLD, pJL-IX, pJL-SX, etc.) pAc vector etc. are mentioned. In addition, a commercially available cloning vector such as pCR4-TOPO (registered trademark) may be used for cloning and sequence confirmation.

  In gene introduction by homologous recombination, a gene obtained by synthesizing the gene sequence of the gene sequence at both ends of the gene sequence and the linear gene sequence including the introduced gene sequence by PCR or the like can be used without using a plasmid. . This linear gene has a homologous sequence with the gene on the host genome at both ends of the sequence, and is introduced into the host chromosome via this homologous sequence.

  In the above vector, in order to ensure that the inserted gene is expressed, an appropriate promoter is linked upstream of the gene. The promoter to be used may be appropriately selected by those skilled in the art depending on the host. A constitutive promoter or expression-inducible promoter may be used. A constitutive promoter refers to a promoter that allows a structural gene to be expressed at a certain level regardless of stimulation inside or outside the host cell. Examples of the constitutive promoter include promoters such as glyceraldehyde-3-phosphate dehydrogenase gene, RNA polymerase gene, DNA gyrase gene, 5s rRNA gene, 16s rRNA gene, and 23s rRNA gene, and PGK. However, it is not limited to these.

  The expression-inducible promoter means a promoter that can induce the expression of a gene linked downstream by an external factor such as a drug, strong light, UV, injury, high temperature, low temperature, salt, or the like. Examples of inducible promoters include, but are not limited to, T7 promoter, Tac promoter, Lac promoter, Trp promoter, λ-PL promoter, and the like.

  In addition to the promoter and the gene of interest, a cis element such as an enhancer, a splicing signal, a poly A addition signal, a selection marker, a ribosome binding sequence (SD sequence), and the like may be linked to the vector, if desired. Further, the gene sequence to be introduced may contain a selection marker. A selectable marker can be used to select a recombinant strain into which a target gene has been introduced into a host. Examples of selectable markers include formaldehyde resistance markers, drug resistance markers such as kanamycin, ampicillin, tetracycline, chloramphenicol, and auxotrophic markers such as leucine, histidine, lysine, methionine, arginine, tryptophan, and uracil. It is not limited to this.

  In order to link the genes, a known DNA ligase is used. Preferably, a recombinant vector can be obtained by performing a ligation reaction under defined conditions using a commercially available ligation kit such as Ligation high (Toyobo Co., Ltd.). If necessary, these vectors are purified by a boil method, an alkaline SDS method, a magnetic bead method and a commercially available kit using these principles, and further concentrated by an ethanol precipitation method, a polyethylene glycol precipitation method, or the like. It can be concentrated by means.

  The gene introduction method is not particularly limited, and examples thereof include an electric pulse method, a method using calcium ions, a protoplast method, and an electroporation method.

  In the method of inserting a target gene at an arbitrary position on the genome by homologous recombination, the target gene is inserted into a sequence homologous to the sequence on the genome together with a promoter, and this DNA fragment is introduced into the cell by electroporation. It can be carried out by causing homologous recombination. At the time of introduction into the genome, a strain in which homologous recombination has occurred can be easily selected by using a DNA fragment in which a target gene and a selection marker gene are linked. In addition, a gene linked to a drug resistance gene and a gene that becomes lethal under specific conditions is inserted into the genome by homologous recombination by the above method, and then becomes lethal under specific conditions with the drug resistance gene. The target gene can also be introduced using homologous recombination in the form of replacing the gene.

  The medium for culturing the resulting transgenic cells contains a carbon source, a nitrogen source, inorganic salts, etc., and any medium can be used as long as it can efficiently culture the transgenic cells. May be used.

  As the carbon source, for example, sugars such as glucose, polyols such as glycerin, alcohols such as ethanol, or organic acids such as pyruvic acid, succinic acid or citric acid can be used. Moreover, as a nitrogen source, for example, peptone, meat extract, yeast extract, casein hydrolyzate, soybean koji alkali extract, alkylamines such as methylamine, ammonia or a salt thereof, and the like can be used. In addition, salts such as phosphate, carbonate, sulfate, magnesium, calcium, potassium, iron, manganese, zinc, a specific amino acid, a specific vitamin, an antifoaming agent, and the like may be used as necessary. Moreover, you may add the gene expression inducer according to the introduce | transduced promoter to a culture medium as needed.

  The culture is usually performed under aerobic conditions such as shaking culture or aeration and agitation culture, preferably at 0 to 40 ° C, more preferably at 10 to 37 ° C, and particularly preferably at 15 to 37 ° C. During the culture period, the pH of the medium can be appropriately changed within the range in which the activity of the protein complex is not impaired, but is preferably in the range of about pH 3-9. The pH is adjusted using an inorganic or organic acid, an alkaline solution, or the like. During culture, an antibiotic such as ampicillin or tetracycline may be added to the medium as necessary.

  Purification of the target protein from the culture or its fractions (including culture fluids, culture supernatants, cultured cells, cell disruptions, etc.) is performed by ion exchange resin method, precipitation method, crystallization method, recrystallization method. It can be carried out by combining the concentration method and other methods. When a protein complex is produced in a cell, the cell is destroyed by a generally known method, for example, a mechanical method, an enzymatic method using lysozyme, or a chemical treatment using a surfactant. It is preferable to do. When the protein complex is produced outside the cells, the culture solution is used as it is or the cells are removed by centrifugation or the like.

  In the purification process of the protein complex from the culture, the present inventors added calcium ions to the culture, more specifically, added calcium ions to separate the solid and liquid, and recovered the protein from the liquid fraction. Thus, it was found that the T1R1 / T1R3 extracellular domain complex or the T1R2 / T1R3 extracellular domain complex can be obtained efficiently and stably.

  Calcium ions can be added, for example, as a calcium salt. The calcium salt is not particularly limited, but a water-soluble calcium salt is preferably used. Examples of the water-soluble calcium salt include inorganic acid calcium such as calcium chloride and calcium nitrate, and organic acid calcium such as calcium formate, calcium acetate, and calcium propionate. The concentration of calcium ions coexisting at the time of purification is appropriately adjusted, but it is preferably adjusted to be, for example, 5 to 200 mM, preferably 10 to 100 mM, more preferably 20 to 50 mM, and further preferably 30 to 40 mM.

  In the production of the T1R1 / T1R3 extracellular domain complex, when the T1R1 extracellular domain and the T1R3 extracellular domain are expressed in a cell into which a gene encoding them is introduced, either the T1R1 extracellular domain or the T1R3 extracellular domain is A T1R1 extracellular domain or T1R3 extracellular domain that is overexpressed and does not form a complex may inhibit the formation and purification of the T1R1 / T1R3 extracellular domain. By precipitating and removing excess T1R1 extracellular domain or T1R3 extracellular domain in the presence of calcium ions, the T1R1 / T1R3 extracellular domain complex can be effectively purified and recovered. In addition, the T1R1 / T1R3 extracellular domain complex is unstable and difficult to maintain a function-retaining state, but the T1R1 / T1R3 extracellular domain complex is stabilized by the coexistence of calcium ions. Can be maintained. In the production of the T1R2 / T1R3 extracellular domain complex, either the T1R2 extracellular domain or the T1R3 extracellular domain is overexpressed, and the T1R2 extracellular domain or the T1R3 extracellular domain that does not form a complex is a T1R2 / T1R3 cell. The formation and purification of the outer domain may be inhibited, but the T1R2 / T1R3 extracellular domain complex is effectively removed by precipitating and removing excess T1R2 extracellular domain or T1R3 extracellular domain in the presence of calcium ions. Can be purified and recovered. Similarly, by coexisting calcium ions, the T1R2 / T1R3 extracellular domain complex can be stably maintained.

  Further, in the production of the T1R1 / T1R3 extracellular domain complex, a substance (known ligand) known to bind to the umami receptor T1R1 / T1R3 heterodimer at the time of purification, for example, a known umami taste By coexisting substances, the T1R1 / T1R3 extracellular domain complex can be further stabilized and efficiently purified. Similarly, in the production of the T1R2 / T1R3 extracellular domain complex, a substance (known ligand) known to bind to the sweet receptor T1R2 / T1R3 heterodimer at the time of purification, for example, a known By allowing a sweet substance to coexist, the T1R2 / T1R3 extracellular domain complex can be further stabilized and efficiently purified. Known umami substances for human taste receptors include amino acids such as glutamic acid and aspartic acid, nucleotides such as inosinic acid, guanylic acid and xanthylic acid, organic acids such as succinic acid, and salts thereof. Known sweet substances for human taste receptors include, for example, glucose, sucrose, fructose, aspartame, acesulfame potassium, erythritol, stevia, sucralose and the like.

  When the purified T1R1 / T1R3 extracellular domain complex and T1R2 / T1R3 extracellular domain complex are used for evaluation of taste substances and taste inhibitors, the ligand is preferably removed immediately before use. Since the ligand is usually a low-molecular substance, it can be easily removed by a known method such as ultrafiltration.

  In one embodiment, the extracellular domain complex of the present invention can be purified and recovered as follows. The culture solution is centrifuged, the supernatant is collected, cooled in a low temperature chamber (0 to 10 ° C.), adjusted to pH 6.5 to 8.5, added with a known ligand and a water-soluble calcium salt, stirred, and left to stand. To precipitate the precipitate. Further, the supernatant is filtered, and the filtrate is passed through an affinity column equilibrated with a buffer containing a known ligand and a water-soluble calcium salt to bind the target protein, that is, the extracellular domain complex. After washing the column, the target protein is eluted. The elution of the target protein can be confirmed by absorption at UV 280 nm.

In one embodiment, the method for evaluating a taste substance or taste inhibitor of the present invention comprises:
a) In the presence of calcium ions, the T1R1 / T1R3 extracellular domain complex (protein complex containing the extracellular domain of T1R1 and the extracellular domain of T1R3) obtained as described above is brought into contact with the test substance. And b) detecting the binding of the test substance to the T1R1 / T1R3 extracellular domain complex,
including. When binding of the test substance to the T1R1 / T1R3 extracellular domain complex is confirmed, the test substance can be determined as an umami substance or an umami inhibitor. In the determination, it is preferable to compare with a control sample containing no umami substance or a sample containing a known umami substance.

In another embodiment, the method for evaluating a taste substance or taste inhibitor of the present invention comprises:
a) In the presence of calcium ions, the T1R2 / T1R3 extracellular domain complex (protein complex containing the extracellular domain of T1R2 and the extracellular domain of T1R3) obtained as described above is brought into contact with the test substance. And b) detecting the binding of the test substance to the T1R2 / T1R3 extracellular domain complex,
including. When the binding of the test substance to the T1R2 / T1R3 extracellular domain complex is confirmed, the test substance can be determined as a sweet substance or a sweet taste inhibiting substance. In the determination, it is preferable to compare with a control sample containing no sweet substance or a sample containing a known sweet substance.

  Binding of the analyte can be detected using, for example, changes in spectroscopic properties (eg, fluorescence, absorbance, refractive index), hydrodynamic (eg, shape) properties, or dissolution properties. For example, isothermal titration calorimetry and the like can be mentioned.

  In vivo, it is known that a gustatory substance binds to a taste receptor and conversion of a chemical stimulus into an electrical signal is initiated. The activated G protein then alters the properties of the target enzyme, channel and other effector proteins. The present inventors have found that the T1R1 / T1R3 extracellular domain complex and the T1R2 / T1R3 extracellular domain complex obtained as described above bind to ligand binding of umami receptor and sweet receptor, respectively, in the presence of calcium ions. It has been found that the presence or degree of the binding of the test substance can be evaluated at the protein level without using cells, while maintaining the function. Conventionally, since the T1R1 / T1R3 extracellular domain complex and the T1R2 / T1R3 extracellular domain complex cannot be stably maintained while maintaining the ligand binding function, the presence or absence or degree of binding of taste substances and taste inhibitors However, the present inventors have made it possible to bind a taste substance or an inhibitor to the T1R1 / T1R3 extracellular domain complex and the T1R2 / T1R3 extracellular domain complex by coexisting calcium ions. Has been found to be accurately evaluated with good reproducibility.

  Furthermore, the present inventors have found that the T1R2 / T1R3 extracellular domain complex undergoes a structural change when a sweet substance is bound. That is, in the taste receptor, it is considered that conversion from extracellular information to intracellular information is initiated by the structural change of the extracellular domain. On the other hand, although it binds to the T1R1 / T1R3 extracellular domain complex, it itself does not change the structure of the T1R1 / T1R3 extracellular domain complex, and also does not cause a structural change when a known umami substance coexists. The test substance can be evaluated as an umami-inhibiting substance and binds to the T1R2 / T1R3 extracellular domain complex, but itself does not change the structure of the T1R2 / T1R3 extracellular domain complex. In this case, a test substance that does not cause a structural change can be evaluated as a sweetness inhibiting substance.

  In other words, a tastant is an activator of a taste receptor, for example, a compound that binds to, stimulates, activates, enhances activation, and enhances or upregulates taste transmission. For example, a working substance. Taste inhibitors, for example, compounds that bind to taste receptors, partially or completely inhibit stimulation, reduce, interfere with, delay activation, inactivate or down-regulate taste transmission, e.g., antagonists It is a substance.

  Therefore, in the present invention, detection of the binding of the test substance to the T1R1 / T1R3 extracellular domain complex or the T1R2 / T1R3 extracellular domain complex is performed by detecting the T1R1 / T1R3 extracellular domain complex or the T1R2 / T1R3 extracellular domain. This is preferably carried out by detecting structural changes in the complex.

  According to the method for evaluating a taste substance of the present invention, it is possible to measure not only the presence / absence of a structural change that occurs upon binding of a test substance to a protein complex, but also the degree. By measuring the degree of structural change, the taste intensity exhibited by the test substance can be quantitatively evaluated. A test substance that produces a greater structural change at a lower concentration gives a strong taste, whereas a test substance that produces a smaller structural change at a higher concentration gives a weak taste.

  The structural change of the T1R1 / T1R3 extracellular domain complex and the T1R2 / T1R3 extracellular domain complex is preferably detected by FRET analysis. FRET (Forster resonance energy transfer) is a phenomenon that occurs when two fluorescent substance pairs (donor and acceptor) approach a distance of 10 nm or less, and a part of the excitation energy received by the donor. This is a phenomenon that is transferred to the acceptor without interfering with fluorescence.

  When the T1R2 extracellular domain and the T1R3 extracellular domain are labeled with a donor or an acceptor, respectively, FRET occurs when a structural change of the T1R2 / T1R3 extracellular domain complex occurs, and the binding of the test substance It was found that structural changes due to can be detected. The same applies to the T1R1 / T1R3 extracellular domain complex.

  When light of the excitation wavelength of the donor is irradiated, FRET occurs when the donor and the acceptor are at a short distance, and the fluorescence of the donor decreases and the fluorescence of the acceptor increases. The fluorescence of the acceptor increases and the fluorescence of the acceptor decreases. A donor in FRET is a fluorescent substance that absorbs excitation light, a part of its energy is emitted as fluorescence, and the other part moves to a nearby acceptor. An acceptor in FRET does not receive direct excitation light. A fluorescent substance that absorbs energy from a donor and emits fluorescence.

  In a method of detecting FRET by measuring fluorescence intensity, a fluorescence intensity ratio between donor fluorescence intensity and acceptor fluorescence intensity is usually used as an index of FRET. At this time, in the fluorescence channel for detecting the acceptor, in addition to acceptor fluorescence derived from FRET, leakage of donor fluorescence and acceptor fluorescence directly excited by the laser are measured. The FRET analysis can be measured using a fluorescence spectrophotometer.

  In the present invention, a fluorescent protein is preferably used as the fluorescent substance, but may be a fluorescent protein modified by a genetic engineering technique for the purpose of enhancing the fluorescence intensity. The fluorescent protein is not particularly limited, but Sirius, EBFP, SBP2, EBP2, Azurite, mKalama1, TagBFP, mBlueberry, mTurquoise, ECFP, Cullean, TagCFP, AmCian, mTP1, MCid, P , AG (Azami-Green), mAG1, ZsGreen, EmGFP (Emerald), EGFP, GP2, T-Sapphire, HyPer, and other blue fluorescent proteins; TagYFP, mAmetrine, EYFP, YFP, Venus, CitrineP turboYFP, ZsYellow, mBana, etc. Yellow fluorescent proteins of mKO1, KO (Kusabila Orange), mOrange, mOrange2, mKO2, etc. Orange fluorescent proteins; mStrawberry, TurboFP602, mRP1, JRed, KillerRed, mCherry, KeimaRed, HcRed, mRasberry, mKate2, TagFP635, mPlum, eggFP650, Neptun, P70, etc.

  A combination of fluorescent proteins preferable as a FRET pair is not particularly limited as long as the fluorescence spectrum emitted from the donor overlaps with the excitation spectrum of the acceptor. For example, CFP and YFP, AG and KO, Cerulean and Venus, etc. (The left side is a donor, and the right side is an acceptor). Which of the two extracellular domains is labeled with a donor or acceptor can be appropriately determined. For example, in the case of a T1R1 / T1R3 extracellular domain complex, the T1R1 extracellular domain may be labeled with a donor and the T1R3 extracellular domain may be labeled with an acceptor, and in the case of a T1R2 / T1R3 extracellular domain complex, the T1R2 The extracellular domain may be labeled with a donor and the T1R3 extracellular domain may be labeled with an acceptor.

  The method for labeling the T1R1 extracellular domain, the T1R2 extracellular domain, and the T1R3 extracellular domain is not particularly limited. Preferably, the extracellular domain and the fluorescent protein are linked so that their DNA frames coincide with each other and expressed. The gene can be introduced into a vector, introduced into the host with this expression vector, the resulting transgenic cell is cultured, and the desired fusion protein is collected from the culture.

  A test substance to be tested as a candidate substance for a taste substance or a taste inhibitory substance may be a small compound or a biological substance such as a protein, nucleic acid, or lipid. Although compounds that are soluble in aqueous solutions are very often tested, essentially any compound can be used as a potential analyte in the evaluation method of the present invention. Assays for screening chemical libraries can also be designed by automating the assay steps and supplying compounds from convenient sources.

  The present invention also relates to a reagent for evaluating an umami substance or an umami inhibitor comprising an calcium solvent and a protein complex containing an extracellular domain of T1R1 and an extracellular domain of T1R3 in an aqueous solvent. Furthermore, the present invention relates to a reagent for evaluating a sweetening substance or a sweetness inhibiting substance containing calcium ions and a protein complex containing the extracellular domain of T1R2 and the extracellular domain of T1R3 in an aqueous solvent. The reagent can be suitably used in the method for evaluating a taste substance or taste inhibitory substance described above. The aqueous solvent is not particularly limited, but preferably water, particularly ion-exchanged water is used. Mixtures of water and other solvents such as alcohols may be used. In the protein complex contained in the reagent, each extracellular domain may be fluorescently labeled.

  In the reagent, the calcium ion concentration in the aqueous solvent is, for example, 0.1 mM to 100 mM, preferably 0.2 to 20 mM, more preferably 0.5 to 5 mM, and further preferably 1 to 2 mM. The same calcium concentration is preferably used in the operation using the protein complex in the evaluation method of the present invention.

  The reagent may comprise instructions for use in the evaluation of taste substances or taste inhibitors, control samples free of taste substances (especially umami substances or sweet substances), and / or known taste substances (especially umami substances or A sweet substance may be provided as a kit.

  EXAMPLES Hereinafter, although an Example demonstrates this invention in detail, the scope of the present invention is not limited to the range of an Example.

  In order to analyze the binding of the tastant to the T1R taste recognition region, each protomer of the heterodimeric sample was fluorescently labeled and FRET analysis was performed. Each protomer was prepared as a different fluorescent protein fusion, and the fluorescence change accompanying the taste substance binding was observed.

(Protein expression)
Amino acid sequence of metabotropic glutamate receptor 1 extracellular domain used for crystallographic analysis (Kunishima et al., Nature, 407: 971-977 (2000); Tsuchiya et al., Proc. Natl. Acad. Sci. USA, 99: 2660-2665 (2002)), using a sequence alignment produced by Clustal W (Thompson et al., (1994) Nucleic Acids Res. 22 (22): 4673-80). The extracellular domain of the protein is identified as amino acids 1-474 in the full-length amino acid sequence (SEQ ID NO: 1) for T1R2a and identified as amino acids 1-491 in the full-length amino acid sequence (SEQ ID NO: 2) for T1R3a It was.

  In order to produce pAc-mfT1r2aL-Ce and pAc-mfT1r3aL-Ve as expression vectors for fluorescence resonance energy transfer (FRET) analysis, a gene encoding T1r2a-LBD followed by Cerulean (Ce: blue fluorescence as a donor) Protein variant) and FLAG-tag, or T1r3-LBD followed by Venus (Ve: yellow fluorescent protein variant as acceptor) and FLAG-tag, and EcoRI and pAc5.1 / V5-HisA vector (Invitrogen) Subcloning was performed between PmeI sites or between KpnI and PmeI sites, respectively (Koushik et al., Biophys J 91, L99-L101, 2006). pAc-mfT1r2aL-Ce and pAc-mfT1r3L-Ve were transfected into S2 cells by transfection using the calcium phosphate method in Schneider insect medium supplemented with 10% FBS at 27 ° C. to transiently express the protein I let you. The amino acid sequence of mfT1r2aL-Ce is shown in SEQ ID NO: 3, and the amino acid sequence of mfT1r3L-Ve is shown in SEQ ID NO: 4.

(Purification of protein complex)
The culture solution obtained by cell culture is centrifuged (500 × g, 20 minutes), the supernatant is recovered (2000 × g, 20 minutes), and phenylmethylsulfonyl fluoride (PMSF) is added at a final concentration of 0.5 mM. (PMSF was dissolved in 1 ml of DMSO and added to 1 L of the culture solution).

It was cooled in a low greenhouse (4 ° C.) and adjusted to about pH 7.8 by adding 1M Tris (pH 9.0). Alanine (final concentration 0.1 M), CaCl ₂ (40 mM) and EDTA (1 mM) as ligands were added and stirred well. When calcium was added, a white precipitate was deposited. After stirring well, the mixture was allowed to stand for 30 minutes.

  After confirming that the pH was in the range of 7.8 to 8.0, the mixture was centrifuged (4000 × g, 60 minutes), and the supernatant was filtered through a 0.2 μm filter (Pall Vac Cap 90PF).

The filtrate was added to a column to which a FLAG resin (4 ml was used for 1 L of filtrate) equilibrated with a buffer (20 mM Tris pH 8.0, 0.1 M alanine, 0.3 M NaCl, 2 mM CaCl ₂ ) was added. It was run using a peristaltic pump (3 to 4 ml / min when the filtrate was 1 L, about 15 ml / min when the filtrate was 3 L) and circulated overnight to bind the protein to the resin.

  The resin was washed with buffer. Subsequently, the FLAG peptide was dissolved in a buffer solution to a concentration of 100 μg / ml to elute the protein. The FLAG peptide solution (5CV) was pipetted several times over the column. The flow rate was adjusted to be 1 ml / min or less.

The eluate was taken in a tube by 1 CV, and absorption at UV280 nm was confirmed by nanodrop. The solution was concentrated to about 5 mg / ml and dialyzed overnight with 10 mM alanine, 20 mM Tris, 0.3 M NaCl, 2 mM CaCl ₂ , pH 8.0. The pH was adjusted at 4 ° C.

(Sample preparation)
The dialyzate was concentrated (about 3 mg / ml) and used after ultracentrifugation (154000 × g, 30 minutes). This was dissolved in buffer A (20 mM Tris, 0.3 M NaCl, 2 mM CaCl ₂ pH 8.0) so that the protein concentration was about 3 μg / ml, and then shaken in a low temperature chamber for 5 minutes. And dispensed into tubes. The tube was shaken up and down to mix well to obtain a protein solution.

  As a ligand solution, a 1M solution of alanine, which is known as a sweet substance, and a 100 mM solution of glutamine were prepared. The preparation method of the ligand solution is as follows. First, the ligand was dissolved in buffer A, cooled to 4 ° C., adjusted to pH 8.0, then made up, and filtered through a filter. For example, in the case of alanine, 100 mM, 10 mM, and 1 mM solutions were prepared from the filtrate, and a ligand solution was prepared so as to be between 5 μM and 100 mM. Specifically, 500 μl of the desired double-concentration alanine solution or glutamine solution was prepared, and the protein solution was dispensed in the same amount to finally make 1 ml.

  In this way, three or more samples having the same ligand concentration were prepared. After preparation, it was left overnight in a cold room.

(Measuring method)
The cell holder and incubator were set to 4 ° C. The Fluoromax 4 lamp was turned on 30 minutes before the measurement. Using LAMP and WATER files, it was confirmed that there was no wavelength shift. The measurement was performed at a single point.

The measurement conditions are as follows.
Measuring device: FluoroMax (HORIBA Scientific)
Excitation: 433 nm, Emission: 475 nm
Excitation: 433 nm, Emission: 526 nm
Maximum trial 5
Target std. error 2%
Slit Excitation: 4nm
Slit Emission: 5nm

(Create titration curve)
The measured value was the average of the values measured twice. Only buffer solution was measured in the same manner and subtracted as background. A scatter plot was drawn by dividing the fluorescence intensity at 526 nm by the fluorescence intensity at 475 nm (FRET index), the ligand concentration on the horizontal axis, and the FRET index on the vertical axis. The obtained titration curve is shown in FIG.

EC ₅₀ was determined by fitting with KaleidaGraph using the formula m4 + m1 * (m0 / m2) ^ m3 / (1+ (m0 / m2) ^ m3 (Hill's formula). ₅₀ was 168 ± 19 μM and the EC ₅₀ of glutamine was 12.7 ± 2.7 μM, indicating that glutamine gave a stronger sweetness compared to alanine.

  From the above, it was confirmed that the heterodimer of the extracellular domain obtained in Example 1 increased the FRET signal by the binding of the tastant. Moreover, it was confirmed that the degree of taste exhibited by the taste substance can be quantitatively evaluated based on the concentration of the test substance and the FRET signal intensity. This FRET signal change was observed in the same concentration range as the taste substance binding (measured by isothermal titration calorimetry) to the T1R extracellular domain that was not fluorescently labeled. Therefore, the heterodimer of the extracellular domain of T1R was tasted. It became clear that the structure changed with the material binding. In addition, it was possible to analyze the affinity and determine the binding constants for tasting substances with weak affinity that could not be measured in principle by other biomolecular interaction analysis methods including calorimetry.

  The taste substance binding to the T1R extracellular domain not labeled with fluorescence was measured as follows using isothermal titration calorimetry.

A heterodimeric sample of T1r2a-LBD and T1r3-LBD includes a gene encoding T1r2a-LBD followed by Factor Xa recognition site, FLAG-tag, His-tag, or T1r3-LBD followed by Factor Xa recognition site, FLAG-tag and His-tag were subcloned between the EcoRI and PmeI sites or the KpnI and PmeI sites of pAc5.1 / V5-HisA vector (Invitrogen), respectively, and similarly transfected using the calcium phosphate method After the transfection into S2 cells, a stable expression strain was selected. From the culture medium obtained by this cell culture, the protein is purified in the same manner as described above, dialyzed against 20 mM Tris (pH 8.0), 0.3 M NaCl, and 2 mM CaCl ₂ , and then iTC200 (manufactured by GE Healthcare Biosciences). ) Was used to analyze taste substance binding by isothermal titration calorimetry.

Claims (6)

A method for evaluating a taste substance or taste inhibitor,
a) contacting a test substance with a protein complex containing the extracellular domain of T1R1 and the extracellular domain of T1R3 in the presence of calcium ions, or a protein complex containing the extracellular domain of T1R2 and the extracellular domain of T1R3 A step of contacting the body with a test substance, and b) detecting a binding of the test substance to the protein complex,
Including said method.   The method according to claim 1, wherein in step b), a structural change of the protein complex due to binding of the test substance to the protein complex is detected.   The method according to claim 2, wherein the extracellular domain is fluorescently labeled, and the structural change of the protein complex is detected by FRET analysis. Reagents for evaluating taste substances or taste inhibitors containing the following a) and b) in an aqueous solvent:
a) calcium ions,
b) A protein complex comprising the extracellular domain of T1R1 and the extracellular domain of T1R3, or a protein complex comprising the extracellular domain of T1R2 and the extracellular domain of T1R3.   The reagent of Claim 4 whose calcium ion concentration in an aqueous solvent is 0.1-100 mM. A method for producing a reagent for evaluating a taste substance or a taste inhibitor,
a) expressing in a host cell a gene encoding the extracellular domain of T1R1 and a gene encoding the extracellular domain of T1R3, or a gene encoding the extracellular domain of T1R2 and a gene encoding the extracellular domain of T1R3;
b) obtaining a fraction containing the extracellular domain of T1R1 and the extracellular domain of T1R3 or the extracellular domain of T1R2 and the extracellular domain of T1R3 from the culture; and c) adding calcium ions to the fraction, Separating and recovering the protein from the liquid fraction,
Said method.

Images