CN108329393A - The taste receptors of T1R oligomerics and the cell line for expressing the receptor and its purposes in identification of taste compounds - Google Patents

The taste receptors of T1R oligomerics and the cell line for expressing the receptor and its purposes in identification of taste compounds Download PDF

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CN108329393A
CN108329393A CN201710563289.1A CN201710563289A CN108329393A CN 108329393 A CN108329393 A CN 108329393A CN 201710563289 A CN201710563289 A CN 201710563289A CN 108329393 A CN108329393 A CN 108329393A
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receptor
taste
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protein
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M·T·佐勒
李晓东
L·斯塔谢夫斯基
S·奥康奈尔
S·佐祖利亚
J·E·阿德勒
徐红
F·埃切韦里
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Femenisher Co
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Senomyx Inc
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Priority claimed from US09/897,427 external-priority patent/US6955887B2/en
Priority claimed from US10/035,045 external-priority patent/US7241880B2/en
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Priority claimed from CNB028128303A external-priority patent/CN100489102C/en
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    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
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    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/72—Assays involving receptors, cell surface antigens or cell surface determinants for hormones
    • G01N2333/726—G protein coupled receptor, e.g. TSHR-thyrotropin-receptor, LH/hCG receptor, FSH

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Abstract

The present invention relates to the discoveries that T1R receptors are assembled to form functional taste receptor.Specifically, present invention discover that coexpression T1R1 and T1R3 can generate the taste receptors of response delicate flavour stimulant (including monosodium glutamate).It has also been found that coexpression T1R2 and T1R3 receptors can generate the taste receptors of response sweet stimulus object (including naturally occurring and artificial sweetener).The invention further relates to purposes of the different oligomerization taste receptors in analysis comprising T1R1/T1R3 and T1R2/T1R3, for identifying the compound for responding delicate flavour stimulant and sweet stimulus object respectively.The invention additionally relates to structures to stablize under the conditions of composing type or induction type or instantaneously co-express the cell line that T1R1 and T1R3 or T1R2 and T1R3 is combined.The present invention also provides purposes of these cell lines in the high flux screening analysis of analysis, particularly utilization fluorescence imaging detection receptor active based on cell, for identifying delicate flavour and Sweet flavor modifier compound.Finally, the present invention relates to following discovery, i.e., certain compounds, such as Lactitol (lactisole), inhibit hT1R2/T1R3 and T1R1/T1R3 receptor actives, to inhibit sweet taste and delicate flavour to feel that it may be unique sweet taste and umami receptor to prompt these receptors.

Description

It the taste receptors of T1R oligomerics and expresses the cell line of the receptor and its is reflecting Determine the purposes in taste compounds
The application is No. 200910137899.0 " the TIR oligomerics of application for a patent for invention submitted on June 26th, 2002 The divisional application of taste receptors and the cell line for expressing the receptor and its purposes in identification of taste compounds ".
The cross reference of related application
This application claims to following priority application, i.e. the U.S. Provisional Application 60/300 that on June 26th, 2001 proposes, 434, the U.S. Provisional Application of the U.S. utility application proposition on July 13rd, 09/897,427,2001 proposed on July 3rd, 2001 60/304,749, the U.S. for the U.S. Provisional Application proposition on November 21st, 60/310,493,2001 that August in 2001 proposes on the 8th The U.S. Provisional Application of provisional application proposition on December 14th, 60/331,771,2001 is carried on January 3rd, 60/339,472,2002 60/372,090, the 2002 year April 22 of U.S. Provisional Application proposed U. S. application on the April 15th, 10/035,045,2002 gone out The U.S. Provisional Application 60/374,143 that day proposes, introduces for reference in full.
Background of invention
Invention field
Part of the present invention is related to being assembled to form the discovery of functional taste receptor in relation to T1R receptors.Specifically, of the invention It was found that coexpression T1R1 and T1R3 can generate response delicate flavour (umami taste) stimulant (including monosodium glutamate) the sense of taste by Body.It has also been found that coexpression T1R2 and T1R3 receptors can generate response sweet stimulus object (including naturally occurring and artificial sweetener) Taste receptors.
The invention further relates to purposes of the different oligomerization taste receptors in analysis comprising T1R1/T1R3 and T1R2/T1R3, use Respond the compound of delicate flavour stimulant and sweet stimulus object respectively in identification.
The invention additionally relates to structures stablize under the conditions of composing type or induction type or instantaneous coexpression T1R1 and T1R3 or The cell line of T1R2 and T1R3 combinations.
The present invention also provides these cell lines based on cell analysis, particularly using fluorescence imaging detection receptor live Property high flux screening analysis in purposes, for identifying delicate flavour and sweet tea taste modulatory compound.
Background technology
Gustatory system provides the sensory information about extraneous chemical constituent.It is believed that mammal is at least five kinds of Basic taste sensation pattern：Sweet tea, hardship, acid, salty and fresh (umami).For example, see Kawamura et al., Introduction to Umami:A Basic Taste(1987)；Kinnamon et al., Ann.Rev.Physiol, 54:715-31(1992)； Lindemann,Physiol.Rev.,76:718-66(1996)；Stewart et al., Am.J.Physiol, 272:1-26 (1997).It is believed that one or more different albumen that each taste modalities is expressed by the taste receptor cells of tongue surface Matter receptor-mediated (Lindemann, Physol.Rev.76:718-716(1996)).Identification hardship, the sense of taste of sweet tea and delicate flavour stimulant Receptor belongs to G protein coupled receptor (GPCR) superfamily (Hoon et al., Cell 96:451(1999)；Adler et al., Cell 100:693(2000)).(it is believed that other taste modalities are by Ion channel mediation.)
G protein coupled receptor mediates many other physiological functions, such as endocrine function, exocrine function, heart rate, fat Solution acts on and glycometabolism.The biochemical analysis and molecular cloning of this many receptoroid have been discovered that about these receptor work( Many basic principles of energy.For example, United States Patent (USP) 5,691,188 describes after ligand is combined with GPCR, how this receptor passes through By conformational change, the GDP combined by promoting G α subunits surface GTP to substitute, subsequent G α subunits are detached with G β and G γ subunits, from And lead to heterologous trimerization G-protein activation.Free G α subunits and G β γ compounds can activate the downstream of multi-signal pathway Element.
The present invention relates to the ternary T1R types of sense of taste specificity GPCR.Previously assumed that T1R receptors exercised sweet receptor function (Hoon et al., Cell 96:541-51(1999)；Kitagawa et al., Biochem Biophys Res.Commun.283: 236-42(2001)；Max et al., Nat.Genet.28:58-63(2001)；Montmayeur et al., Nat.Neurosci.4: 412-8(2001)；Sainz et al., J.Neurochem.77:896-903 (2001)), nearest Nelson et al. (2001) is proved Rat T1R2 and T1R3 synergy in identifying sweet stimulus object.The present invention relates to two discoveries.First, with rat T1R2/ T1R3 is identical, hT1R2 and the T1R3 synergy in identifying sweet stimulus object.Secondly, hT1R1 and T1R3 are in identification delicate flavour thorn Swash synergy in object.Therefore, in mammals, T1R2/T1R3 may play sweet receptor function, and T1R1/T1R3 can Umami receptor function can be played.The possibility explanation of the function interdependency of T1R1 and T1R3, T1R2 and T1R3 is, with structure phase The GABA of passBSimilar (Jones et al., the Nature 396 of receptor:5316-22(1998)；Kaupmann et al., Nature 396: 683-7(1998)；White et al., Nature 396:679-82(1998)；Kuner et al., Science 283:74-77 (1999)), T1Rs is functioned with heterodimeric compound.However, the interdependency of this function is it is also possible to reflection one Kind necessity but of short duration interaction, the final taste receptors for generating the unrelated monomer or homologous poly of function.
Identification plays sweet taste and the feature of the taste receptors of umami receptor function is meaningful, these receptors will be promoted to know The purposes in the analysis for the compound that sweet taste and delicate flavour are felt (Ke Tiaojie not be enhanced or block).These compounds can be used for improving people With or animal food, beverage, the taste of drug and palatability.It in particular, can be with using the analysis of functional sweet receptor For identifying novel sweetener.
Summary of the invention
The present invention relates to following discovery, i.e. the T1R of various combination can generate response taste stimulus in coexpression Functional taste receptor.The present invention is more particularly directed to following discoveries, that is, co-express T1R2 and T1R3 and generate response sweet taste stimuli Oligomeric taste receptors.The invention further relates to following discoveries, that is, co-express T1R1 and T1R3 and generate response delicate flavour feel thorn Swash the oligomeric taste receptors of object (such as monosodium glutamate).
The invention further relates to the cell lines of coexpression (preferably people) T1R1 and T1R3 or (preferably people) T1R2 and T1R3. In preferred embodiment, these cell line composing types or the increased amount of receptor of inducible expression.These cell lines include instantaneous Or stablize the cell of expression T1R1 and T1R3 or T1R2 and T1R3.
The present invention also provides analysis methods, preferably by the high throughput of T1R2/T1R3 taste receptors or T1R1/T1R3 receptors Screening analysis, is preferably based on the high throughput analysis of cell, and the compound that sweet taste or delicate flavour are felt is adjusted to identify.The present invention also carries For the analysis method including taste test, to confirm that sweet taste is adjusted in these compounds or delicate flavour is felt.
Goal of the invention
For this purpose, it is an object of the present invention to provide the mammalian G Protein coupled receptor families of mediate taste, herein Referred to as T1R.
Another mesh of the present invention is to provide holding for example by sweet taste or umami taste stimuli activation or activity in combination The T1R segment and variant.
Another object of the present invention is to provide the nucleic acid sequences or molecule that encode the T1R, its segment or variant.
Another object of the present invention is to provide expression vectors, wherein including the core for encoding the T1R or its segment or variant Acid sequence, which is operatively connected to less a kind of regulatory sequence, such as positively or negatively gene turns for promoter, enhancer or participation Other sequences of record and/or translation, and/or protein output.
Another object of the present invention is to provide functional expression at least one T1R or its segment or variant, preferably The mankind or nonhuman cells of the combination of T1R or its segment or variant, such as mammal, yeast, worm or insect cell.
Another mesh of the present invention is to provide T1R fused proteins or polypeptide, including at least the piece of at least one T1R Section.
Another object of the present invention is to provide coding T1R polypeptides separation nucleic acid molecules, it includes nucleic acid sequence With the nucleic acid sequence with one of following hT1R nucleic acid sequences and its conservative modification variant at least 50%, preferably 75%, 85%, 90%, 95%, 96%, 97%, 98% or 99% homogeneity.
Another object of the present invention is to provide the nucleic acid molecules of separation, it includes the nucleic acid sequences of coding polypeptide, this is more The amino acid sequence of peptide has extremely with the amino acid sequence selected from one of following T1R amino acid sequences and its conservative modification variant The homogeneity of few 35%-50%, preferably 60%, 75%, 85%, 90%, 95%, 96%, 97%, 98% or 99%, wherein should Fragment length is at least 20, preferably 40,60,80,100,150,200 or 250 amino acid.The optional segment is in combination with anti- The anti-genic fragment of T1R antibody.
Another object of the present invention is to provide the polypeptides of the separation comprising the fragment variant, wherein at most 10, it is excellent It selects and there is variation at 5,4,3,2 or 1 amino acid residues.
Another object of the present invention is to provide T1R1/T1R3 combinations, and wherein T1R1 and/or T1R3 are variant or segment, with And T1R2/T1R3 combinations, wherein T1R2 and/or T1R3 are variant or segment.
It is another object of the present invention to provide the T1R or its segment or the agonists or antagonist of variant.
Another object of the present invention is to provide PDZ structural domains interacting peptide (referred to herein as PDZIP), can promote table Express plasma membrane integral protein matter, particularly GPCR, such as T1R in face.Another object of the present invention is to provide the loads including PDZIP Body, the host cell for expressing the carrier and the method for promoting surface expression using PDZIP.
The present invention's is preferably designed to provide analysis method, particularly high throughput analysis, for identifying the adjustable sense of taste Compound, the compound that especially adjustable sweet taste is felt and delicate flavour is felt.The analysis method preferably by T1R disclosed herein or Its segment or variant or the combination for encoding the T1R or the gene of its segment or variant.The combination most preferably comprises hT1R1/ HT1R3 and hT1R2/hT1R3.
Especially preferred embodiment of present invention be identification be adjusted T1R1/T1R3 or T1R2/T1R3 taste receptors (such as Enhance this receptor response taste stimulus ability) compound.It is such as described below, it has been found that 5'-IMP or 5'-GMP can Enhance the response to Pidolidone delicate flavour (T1R1/T1R3).These, which adjust immunomodulator compounds, can enhance different sweet tastes or delicate flavour feel thorn Swash the activity of object, and provide compound that the specific sweet taste of generation for reducing concentration or delicate flavour feel (can originally be analyzed and identified by using Taste modulator enhances its activity) generate enhancing and/or the identical sense of taste.
Another object of the present invention is to provide the selected analytic method for the sense of taste for evaluating one or more compounds, packets It includes：By one or more compounds and at least one disclosed T1R, its segment or variant, preferably the combination phase of mankind T1R The step of contact.
More specific objective of the present invention is that providing screening has enhancing in mammal, the preferably mankind, simulation, blocks And/or adjust the method that sweet taste feels one or more compounds of ability, it includes by one or more compounds and hT1R2 and Compound the step of being in contact of the combination of hT1R3 or segment comprising hT1R2 and/or hT1R3, chimera or variant.
Another specific purpose of the present invention is that provide screening has enhancing, simulation, resistance in mammal, the preferably mankind It is disconnected and/or adjust the sense of taste, particularly delicate flavour feel ability one or more compounds method, it includes by one or moreization The compound for closing segment of the object with the combination of hT1R1 and hT1R3 or comprising hT1R1 and/or hT1R3, chimera or variant connects Tactile step.
Another specific purpose of the present invention is to generate coexpression hT1R1 and hT1R3 or its segment, variant or chimera Cell, for identifying the compound that can enhance, simulate, block and/or adjust the sense of taste, particularly sweet taste and feel.
Another specific purpose of the present invention is to generate coexpression hT1R1 and hT1R3 or its segment, variant or chimera Cell, for identifying in the analysis that can enhance, simulate, block and/or adjust the compound that the sense of taste, particularly delicate flavour are felt.
Another object of the present invention is to generate that the inhuman dynamic of one or more T1R is expressed or do not expressed through genetic modification Object.
Another object of the present invention is to will utilize T1R, or combinations thereof the compound analyzed and identified be used as food and drink Flavoring ingredients (flavor ingredient) in feed composition.In particular, it is an object of the present invention in food It will be used as sweet taste blocking agent, reinforcing agent, conditioning agent with the compound of hT1R2 and/or hT1R3 interactions in beverage composition for treating dental erosion Or simulant, delicate flavour blocking agent, reinforcing agent, conditioning agent or mould will be used as with the compound of hT1R1 and/or hT1R3 interactions Quasi- agent.
Another object of the present invention is to use T1R, particularly non-human T1R, identifies and animal feed preparation taste is adjusted The compound in road, such as fish aquaculture.
The present invention's is preferably designed to provide the eukaryon, preferably for stablizing coexpression hT1R1/hT1R3 or hT1R2/hT1R3 Mammal or insect cell line, preferably HEK-293 cell lines equally express G-protein such as G α15Albumen or and T1R2/ Other G-proteins of functional taste receptor can be generated when T1R3 or T1R1/T1R3 Combined expressions.
Another be preferably designed to provide of the present invention stablizes expression T1R1/T1R3 or T1R2/T1R3, preferably hT1R1/ The eukaryotic cell lines of hT1R3 or hT1R2/hT1R3, preferably mammal or insect cell.In preferred embodiments, this is thin Born of the same parents include to stablize expression G α15Or it is combined with T1R1/T1R3 or T1R2/T1R3 and can generate functional delicate flavour or sweet taste receptor The HEK-293 cells of other G-proteins.
One object of the present invention also resides in offer under the conditions of composing type or induction type using stable or transient expression The analysis method of the HEK-293 of T1R1/T1R3 or T1R2/T1R3 or other cell lines, preferably high throughput analysis, it is adjustable to identify Save the compound that delicate flavour or sweet taste are felt.
Another object of the present invention is to according to influence Lactitol (lactisole) (a kind of sweet-taste inhibitor) or knot The ability that structure related compound is combined with T1R1/T1R3 (delicate flavour) taste receptors, identification can enhance, simulate, block and/or adjust The compound of T1R1/T1R3 umami receptors.
Brief description
Fig. 1 is people and rat TIR catalogues, and it includes people and rat T1R, people's calcium-sensing receptor and metabolism of rat types (metabotropic) sequence alignment of glutamate receptor.
Fig. 2 includes that RT-PCR expands experimental result, shows and expresses hT1R2 and hT1R3 in taste tissue, is people's tongue epithelium The hT1R2 and hT1R3. expressed in cell can expand cDNA- specific amplifications from the cDNA prepared by the people's profile nipple cut off Product.
Fig. 3 a-3b are the effects that hT1R2/T1R3 plays sweet taste receptor, and it includes the different sweet tastes of various concentration to feel thorn Object is swashed in the stable expression G α for transiently transfecting hT1R2, T1R3 and T1R2/T1R315HEK cells in the functional data that generates (intracellular Ca2+ reaction) (Fig. 3 a)；HT1R2/T1R3 has response (Fig. 3 b) to several sweet taste stimulis；HT1R2/T1R3 exists There are response, endogenous beta 2-adrenergic receptor p-isopropyl kidney in the presence of gurmarin to sucrose in the presence of gurmarin Upper parathyrine has response.Fig. 3 c include the normalized response to different sweeteners.
Fig. 4 indicate T1R2 may control T1R2/T1R3 ligand specificities, it includes transiently transfect hT1R2/hT1R3, RT1R2/rT1R3, hT1R2/rT1R3 and rT1R2/hT1R3's stablizes expression G α15HEK cells for 350mM sucrose, 25mM The intracellular Ca2+ of tryptophan, 15mM radix asparagi sweet extracts (aspartame) and 0.05% monellin (monellin) responds.
Fig. 5 includes based on Fluorescence Plate reactor analytic as a result, will wherein stablize expression G α15HEK cells instantaneously turn HT1R2 and hT1R3 or independent hT1R3 are contaminated, and contact calcium dyestuff Fluo-4 and sweet taste stimuli (12.5mM Cyclohexylamino sulphurs Hydrochlorate (cyclamate)).
Fig. 6 includes standardized dose-response curve, according to itself and various sweet stimulus objects (tryptophan, Cyclohexylamino sulphur Hydrochlorate, sucrose, neotame (neotame), radix asparagi sweet extract (Aspartame), saccharin (saccharin) and dosage Acek) are special Property interaction show hT1R2 and hT1R3 combination play people's sweet receptor function.
Fig. 7 includes the structural information in relation to mGluR1 and T1R1, is shown in and observes crucial ligand in these molecules Wherein in conjunction with residue, the critical ligand combination residue of mGlurR1 is guarded in T1R1.
Fig. 8 a-8c indicate that hT1R1/T1R3 plays delicate flavour (umami taste) function of receptors, it includes functional data, Display transiently transfects stablizing for T1R1/T1R3 and expresses G α15HEK cells in the cell calcium analysis in respond glutamic acid.Fig. 8 a are aobvious Show that intracellular Ca2+ is improved in response to the raising of aminoglutaric acid concentration；Fig. 8 b show intracellular Ca2+ to IMP (2mM), glutamic acid (0.5mM) With the response of 0.2mM IMP；Fig. 8 c show responses of hT1R1/T1R3 in the presence of whether there is or not 0.2mM IMP to glutamic acid.
Fig. 9 a-9b indicate PDZIP promote hT1R2's surface expressions as a result, it is separately included and is marked using Myc- Immuning fluorescent dyeing analysis and the FACS experiment of hT1R2 introduces PDZIP peptides (SEQID No as a result, showing:1) can enhance The immunofluorescence dyeing of expression of the T1R (hT1R2) on plasma membrane, the hT1R2 of wherein A.Myc labels shows that PDZIP is remarkably reinforced The amount of hT1R2's albumen on plasma membrane, the bright identical result of B.FACS analytical datas.Myc marks hT1R2：Green .Myc labels, C. there is the hT1R2 of PDZIP：Black line.
Figure 10 shows that h1TR2/hT1R3 responds different sweet stimulus objects comprising calcium imaging data, and calcium imaging data shows HT1R2/hT1R3 responds many sweet stimulus objects.
Figure 11 utilizes automatic fluorescence imaging, shows the cell line for stablizing expression hT1R1/hT1R3 to umami taste stimuli Response, specially hT1R1+hT1R3, constant IMP (0.5mM).
Figure 12 utilizes automatic fluorescence imaging, shows the cell line for stablizing expression hT1R2/hT1R3 to sweet taste stimuli Response, specially hT1R2+hT1R3 clone B VIPR dose responses.
Figure 13 shows that the cell line of the inducible expression measured using automatic fluorescence imaging hT1R1/T1R3 taste receptors is existed Whether there is or not in the presence of 0.2mM IMP to the dose-response curve of Pidolidone.
The cell line (clone 1-17) of the display inducible expressions of Figure 14 and 15 hT1R1/T1R3 taste receptors is to one group of L- ammonia The response of base acid.Figure 14 detects the different C- amino acid of 10mM in the presence of whether there is or not 1mM IMP.Figure 15 is whether there is or not 0.2mM IMP In the presence of measure active amino acid dose-response.
Figure 16 shows that Lactitol inhibits the receptor active of hT1R2/T1R3 and hT1R1/T1R3 specifically to indicate clarke Inhibit T1R2/T1R3 sweet receptors and T1R1/T1R3 umami receptors and sweet taste and umami taste for alcohol.(left figure) is shown in not In the presence of concentration Lactitol, the HEK-G of T1R1/T1R3 (circle) is transiently transfectedα15Reaction of the cell to 10mM Pidolidones And transiently transfect the HEG-G of T1R2/T1R3 (rectangular)α15Reaction of the cell to 150mM sucrose.(right figure) is shown in 1 and 2mM In the presence of Lactitol, to sweet stimulus object sucrose and D-trp, delicate flavour stimulant Pidolidone (MSG) and Pidolidone+ The increase multiple of the sense of taste detection threshold value of 0.2mMIMP and sodium chloride.Detection threshold is measured according to the method for Schiffman et al. Value.
Detailed description of the invention
The present invention is provided functional by co-expressing combination, preferably T1R1/T1R3 or the T1R2/T1R3 of different T1R Taste receptors, preferably people's taste receptors, and the nucleic acid sequence detached accordingly or its segment, chimera or variant are total to table Functional taste receptor, i.e. sweet taste receptor (T1R2/T1R3) or umami taste receptor (T1R1/T1R3) can be generated after reaching.
Such as document report, the T1R family members of gustatory specificity GPCR are known and by Hoon et al., Cell, 96: 541-551 (1999), WO 00/06592, WO 00/06593 and U.S.Serial No.09/799,629 identifications, it is complete herein Text reference is for reference.
More particularly it relates to co-express different gustatory specificity GPCR.These nucleic acid and its coding Receptor be referred to as " T1R " family member of gustatory specificity GPCR.In the particular embodiment of the present invention, coexpression T1R family members include rT1R1, rT1R2, rT1R3, mT1R1, mT1R2, mT1R3, hT1R1, hT1R2 and hT1R3.It does not wish Prestige is confined to theory, it is believed that these gustatory specificity GPCR is the component of taste transduction pathway, and participate in sweet taste and The sense of taste detection of the taste stimulus of delicate flavour stimulant and/or the other taste modalities of generation.
It proves herein, sweet taste feel and umami taste receptor are exercised in T1R family members and other T1R family members synergy Function.As Examples below is further disclosed in detail, it has proved that the heterologous cells of coexpression hT1R2 and hT1R3 can By sweet taste stimuli in such a way that similar (mirror) people's sweet taste is felt Selective activation.For example, coexpression hT1R2 and hT1R3 HEK-293-G α15Cell-specific responds cyclamate, sucrose, radix asparagi sweet extract and saccharin, the agent of these compounds Amount response is related to body and mind sense of taste detection threshold value.Therefore, the cell of coexpression hT1R2 and hT1R3 can be used for screening, preferred high pass Screening technique is measured, can simulate, adjust, block and/or enhance the compound of sweet tea taste perception with identification.
Equally under the support of embodiment experimental data, it has been shown that the cell of coexpression hT1R1 and hT1R3 can be by glutamic acid (monosodium glutamate) Selective activation in such a way that similar people's delicate flavour is felt with 5'- ribonucleotides.For example, coexpression hT1R1 and The HEK-293-G α of hT1R315Cell-specific responds glutamic acid, is detected to the dose response and the body and mind sense of taste of the umami compound Threshold value is related.In addition, 5'- ribonucleotides such as IMP can enhance the glutamic acid response of T1R1/T1R3 receptors, this is that delicate flavour is felt Synergistic effect feature.Therefore, the cell of coexpression hT1R1 and hT1R3 can be used for screening, preferred high-throughput screening method, with The compound of fresh taste perception can be simulated, adjust, block and/or be enhanced to identification.
In addition, as shown in the experimental data of embodiment, it has been shown that stablize the cell with induction type coexpression T1R1/T1R3 Alternative response umami taste stimuli Pidolidone and L-Aspartic acid, and it is only weaker to other l-amino acids of very high concentration Response, to provide further evidence, it was demonstrated that T1R1/T1R3 receptors can be used for analysis method, to identify adjustable (enhancing Or block) compound of umami taste stimuli.
Equally under the support of the experimental data of embodiment, it has been shown that the cell of coexpression T1R1/T1R3 or T1R2/T1R3 Delicate flavour or sweet stimulus object are responded respectively, and quantitative dose-response mode is further supported to draw a conclusion, i.e. T1R1/ T1R3 and T1R2/T1R3 receptors can be used for identifying receptor stimulating agent and antagonist, such as MSG substituents, delicate flavour blocking agent, novel Artificial and natural sweetener and sweet taste blocking agent.
Equally under the support of the experimental data of embodiment, it has been shown that sweet taste feels that blocking agent Lactitol inhibits T1R2/ T1R3 sweet receptors and T1R1/T1R3 umami receptors.This prompt screening can influence Lactitol combination T1R2/T1R3 or T1R1/ The analysis method of the compound of T1R3, which can be used for identifying, can enhance, simulates, adjusts or block the compound that sweet tea or delicate flavour are felt.Clarke T1R1/T1R3 and T1R2/T1R3 receptors are can inhibit for alcohol, prompt these receptors that may share identical subunit, the subunit and drawing Gram for alcohol and may being combined with other conditioning agents.Therefore, prompt can enhance, simulate, adjust or block certain chemical combination that sweet taste is felt Object may feel there is similar effect to delicate flavour, and vice versa.
Under the further support of embodiment experimental data, using automation fluorescence imaging analysis it has been proved that stablizing altogether The cell line of expression T1R, i.e. T1R1/T1R3 or T1R2/T1R3 effectively respond various sweet tastes and umami taste stimuli, That is degree substantially exceeds the cell of transient transfection.Therefore, these cell lines are particularly suitable for high flux screening point The compound of analysis, blocking adjustable with identification, simulation or enhancing sweet taste or delicate flavour feel.However, the present invention is equally including the use of wink When express a kind of T1R or combinations thereof cell analysis method.
In addition, although it is that the application includes statistics indicate that, certain T1R synergy, especially T1R1/T1R3 and T1R2/ T1R3, and this receptor combination is possibly used for analysis method, preferably high throughput analysis, it is to be understood that the present invention equally wraps Include individually or be used in combination with other oroteins, for example other GPCR the analysis method of T1R1, T1R2 and T1R3.
In this regard, it is predicted that sweet receptor may be only made of T1R2 and/or umami receptor may be only by T1R1 groups At, and T1R3 receptors may have the function of promoting T1R2 or T1R1 surface expressions.
Alternatively, may also sweet receptor and umami receptor may be only made of T1R3, under T1R1 and/or T1R2 controls into The different processing of row.This receptor expression type may look like the RAMP dependences processing of calcitonin associated receptor.
The compound identified using T1R analysis methods can be used for adjusting the taste of food and beverage.It is described in more below Suitable analytical method include such as intact cell analysis and biochemical analysis, including using, different T1R receptors, its is embedding Fit or segment (segment for especially including N-terminal ligand binding domains) combination binds directly analysis.It is detailed below Suitable for the analysis example that the present invention uses, and known to the fields GPCR.
Can be with Design and analysis methods, the mixture and T1R taste receptors or T1R tastes of quantitative different compounds or compound Feel receptor combination or with other heterologous (non-T1R) protein, the knot of the T1R taste receptors of for example other GPCRs Combined expressions The activation of the cell of conjunction or quantitative expression T1R taste receptors.By in heterologous cells, such as HEK-293, CHO and COS cell Middle stabilization or transient expression taste receptors, can accomplish this point.
It is preferable to use express (preferably stablize expression) G-protein (such as G α simultaneously for the analysis method15Or G α16) or it is other mixed The cell of (promiscuous) G-protein of dwelling or G-protein variant or endogenous G proteins.In addition, wherein can also express G β and G γ Albumen.
Using various methods, including uses calcium sensitivity dyestuff, voltage sensitive dyes, cAMP analyses, utilizes fluorescence mark Remember ligand or radioligand for example3H- glutamic acid bind directly analysis or transcription analysis (using suitable report molecule, Such as luciferase or beta-lactamase), the cell or group of expression or the receptor comprising above-mentioned identification or receptor combination can be used It closes object and measures the effect that compound on sweet or delicate flavour are felt.
The analysis method of one or more T1R of the usable present invention includes, such as utilizes point of living cells gene selects Analysis；Utilize the analysis of intact cell or membrane-bound fragment or the T1R protein of purifying；Utilize point of second messenger, such as cAMP and IP3 Analysis；Detection inhibits protein translocation to the analysis of cell surface；It is lost using ligand to be measured detection cell surface receptor expression (interior Change) analysis；Direct ligand binding assays utilize point of In Vitro Translation protein using the competitive binding analysis of inhibitor Analysis detects the analysis of conformation change (such as being proved by protein hydrolysis, fluorescence or NMR) after ligand binding, utilizes expression T1R Or the behavioural analysis of the transgenic nonhuman animal (such as fly, worm or mouse) of T1R combinations, utilize the weight for including T1R genes The analysis of the cell of group virus infection.
Structure-based analysis is similarly positioned within the scope of the invention, wherein measurement T1R or T1R segments (or T1R combinations, Or the combination of T1R and another protein) x-ray crystal structure, and for by molecular modeling techniques predict it is combinable and/or Enhance, simulate, block or adjust the compound of specific T1R receptors or receptor combination.More specifically, the present invention includes measuring The crystal structure and the crystalline substance of T1R1/T1R3 (preferably hT1R1/hT1R3) and/or T1R2/T1R3 (preferably hT1R2/hT1R3) Purposes of the body structure in structure-based design method, the molecule for identifying adjustable T1R receptor actives.
The present invention particularly including using express one or more overall length T1R receptors or segment, preferably T1R1, T1R2 and/or The biochemical analysis that the cell of the N-terminal structural domain of T1R3, such as mammal, yeast, insect or other heterologous cells carry out. Using competitive binding analysis, such as use radioactive glutamic acid or IMP, fluorescence (such as fluorescence polarization, FRET) or GTP γ35S binding analysis can measure effect of the compound in above-mentioned analysis.As described in preferred embodiment, above-mentioned analysis uses Stablize coexpression T1R1/T1R3 or T1R2/T1R3 and suitable G-protein, such as G α15Cell line.Other suitable G-proteins Chimera including the G-protein for being disclosed in U. S. application 09/984,292 and 60/243,770 (being cited in full text herein for reference) And variant.
Furthermore, it is possible to build and express with the change for improving property (such as the surface expression of enhancing or G-protein coupling) Receptor.Can based on cell analysis and biochemical analysis in introduce these T1R variants.
It is envisioned that these discoveries related with people T1R extend to other species, for example, rodent, pig, monkey, dog and Cat, it is also possible to or even nonmammalian is expanded to, such as fish.In this regard, following example 1 identifies several fish T1R Segment.Therefore, present invention can apply to screen the compound for being used in animal feed preparation.
Present invention additionally encompasses different allelic variants for using various T1R and combinations thereof, to which identification can expressed Cause the compound of specific taste perception in the individual of these allelic variants or all individuals.This compound can be used to make food Object is more commonly palatable.
T1R code nucleic acids are specific expressed in gustatory, thus these nucleic acid also provide for identification of taste cell Valuable probe.It is present in lobate, profile and fungiform papilla for example, the probe of T1R peptide and proteins can be used for identifying Gustatory, and the gustatory that is present in geschmackstreifen, oral cavity, GI epithelium and epiglottis.Especially It is the method for detecting T1R, can be used for identifying to sweet taste and/or umami taste stimuli or represent other tastes of other taste modalities Feel the gustatory of stimuli sensitive.For example, according to working it is expected that stable or transient expression T1R2 and/or T1R3 herein Cell can respond sweet taste stimuli.Similarly, it can predict that the cell of expression T1R1 and/or T1R3 can respond delicate flavour and feel thorn Swash object.It, can be from a variety of source separation, genetic engineering, amplification, synthesis, and/or again according to method disclosed in WO 00/035374 The nucleic acid of group expression present invention coding T1R proteins and peptides, which is cited in full text for reference herein.Embodiment carries The inventory of the T1R of the invention that can be expressed is supplied.But it is stressed that the present invention include other specific T1R or segment, variant, Or expression and the purposes of the T1R of the chimera, particularly other species built based on these T1R sequences.
An importance disclosed by the invention is that the conditioning agent for screening these gustatory specificity GPCR is for example living A variety of methods of agent, inhibitor, stimulant, reinforcing agent, agonist and antagonist.These taste transduction conditioning agents are for the sense of taste The adjusting of signal pathway is particularly useful.These screening techniques can be used for the high-affinity agonist of identification of taste cell activity and short of money Anti-agent.These modulating compounds thus can be used for food industry to customize taste, such as adjust the sweet taste and/or delicate flavour of food Feel.
The present invention identifies specificity T 1R and the T1R receptor combination for mediating sweet taste and fresh taste perception, thus corrects for The case where understanding previously is lacked to related sweet taste and delicate flavour feel.Therefore, it is however generally that, this application involves the related T1R of the present inventor Class sense of taste specific G Protein coupled receptor and its relationship in the sense of taste between the discovery of specific function and these discoveries, with It is best understood from the molecular basis of the sense of taste.
Sweet taste is felt and the molecular basis of the taste-of delicate flavour feel-monosodium glutamate is a mystery.Three members are identified recently The sense of taste specific G Protein coupled receptor of type, referred to as T1R.The T1R expression patterns and certification structure of overlapping are relevant GABABReceptor is heterodimer, and T1R is prompted to have the function of heterodimeric taste receptors.In the examples below, of the invention People describes functional coexpression human T1R1, T1R2 and T1R3 in heterologous cells；The cell for co-expressing T1R1 and T1R3 can be by Umami taste stimuli activates；The cell of coexpression T1R2 and T1R3 can be activated by sweet taste stimuli.T1R1/T1R3 and T1R2/ The activity of T1R3 is related to body and mind detection threshold value.It moreover has been found that 5'- ribonucleotides IMP can enhance T1R1/T1R3 receptors to paddy Propylhomoserin responds, this is the synergistic effect feature that delicate flavour is felt.These find the various combination tool of proof, specific T1R, particularly T1R Pleasantly sweet and umami taste receptor function.
It is believed that the hardship of the mankind, sweet tea and delicate flavour feel and mediated by g protein coupled receptor (Lindemann, B., Physiol.Res.76:718-66(1996)).Recently, it measures human genome and discloses T2R classes bitterness receptors (Adler etc. People, Cell 100:613-702(2000)；Chandrasgekar et al., Cell 100:703-11(2000)；Matsunami etc. People, Nature 404:601-604 (2000)), but not yet identify sweet taste and umami receptor.Identify another kind of candidate's recently Taste receptors, T1R.The identification of T1R is the large scale sequencing by the subtractive cDNA library from rat taste tissue first T1R1 is identified, T1R2 (Noon et al., Cell 96 is then identified by the degenerate pcr based on T1R1:541-551 (1999)).The present inventor identifies the 3rd of T1R families in human genome database, may be last recently with other people A member, T1R3 (Kitagawa et al., Biochem Biophys.Res Commun.283 (1):236-42(2001)；Max etc. People, Nat.Genet.28 (1):58-63(2001)；Sainz et al., J.Neurochem.77 (3):896-903(2001)； Montmayeur et al., Nat.Neurosci.4,492-8. (2001)).It most describes the problem, Mouse T1R3, which is positioned at, includes Influence genome interval (Fuller et al., the J.Hered.65 of the locus Sac that mouse sweet taste is felt:33-6(1974)；Li et al. People, Mamm.Genome 12:13-16(2001)).Therefore, prediction T1R3 has the function of sweet taste receptor.Nearest high-resolution Genetic map research strengthens association (Fuller et al., J.Hered.65 (1) between Mouse T1R3 and Sac:33-36 (1974)；Li et al. people, Mammal.Genome 12 (1):13-16(2001)).
What is interesting is, it has been shown that all C family receptors-metabotropic glutamate receptors, the GABA of functional expression so farB Receptor, calcium-sensing receptor (Conigrave, A.D., Quinn, S.J.＆Brown, E.M., Proc Natl Acad Sci U S A 97,4814-9. (2000)), fish olfactory receptor (Speca, D.J. et al., Neuron 23,487-98. (1999))-can be by ammonia Base acid activation.This common trait shows, it is possible to which T1R can recognize that amino acid, and in addition to sweet taste feels amino acid, T1R may be also It is related to the detection of glutamic acid.In addition, it has been suggested that the transcriptional variants of mGluR4 metabotropic glutamate receptors are umami taste receptors, because It is its selective expression in rat taste tissue, and its receptor activation threshold value is similar to the body and mind detection threshold value of glutamic acid (Chaudhari et al., Nat.Neurosci.3:113-119(2000)).The hypothesis and mGIuR4 variants table in taste tissue Up to horizontal extremely low and mGluR4 knock-out mices the glutamic acid sense of taste be not much change be not consistent (Chaudhari and Roper, Ann.N.Y.Acad.Sci.855:398-406(1998)).In addition, the sense of taste variant is insincere in structure, not only lack shape At most of residue of the glutamic acid binding pocket of wild-type receptor, the spherical glutamic acid of N-terminal for also lacking about half combines knot Structure domain (Kunishima et al., Nature407:971-7(2000)).
The comparative analysis of rodent T1R expression patterns proves, T1R2, is also possible to T1R1 and is co-expressed respectively with T1R3 (Hoon et al., Cell 96:541-51(1999)；Kitagawa et al., Biochem Biophy.Res.Commun.283: 236-242(2001)；Max et al., Nat.Genet.28:58-63(2001)；Montmayeur et al., Nat.Neurosci 4: 492-8(2001)；Sainz et al., J.Neurochem 77:896-903(2001)).In addition, Dimerized be revealed as C- families The common trait of receptor：Metabotropic glutamate and calcium-sensing receptor be homodimer (Romomano et al., J.Biol.Chem.271:28612-6(1996)；Okamoto et al., J.Biol.Chem.273:13089-96(1998)；Han Et al., J.Biol.Chem.274:100008-13(1999)；Bai et al., J.Biol.Chem.273:23605-10 (1998)), Relevant GABA in structureBReceptor is heterodimeric (Jones et al., Nature 396:674-9(1998)；Kaupmann etc. People, Nature396:683-687(1998)；White et al., Nature 396:679-682(1998)；Kuner et al., Science 283:74-77(1999)).The present inventor by heterologous cells functional coexpression T1R prove, hT1R2 with HT1R3, which combines, exercises sweet taste receptor function, and hT1R1 combines with hT1R3 exercises umami taste receptor function.
Lack sweet taste with cause to feel analysis method and hamper the exploitation of improved artificial sweetener, discussed herein these It was found that thus especially significant.In fact, 5 kinds of common commercialization artificial sweeteners (activating hT1R2/hT1R3) are all even So find.Similarly, it there is no identification that point for the compound that delicate flavour is felt is adjusted other than this hard method in addition to sensory test Analysis method.Nowadays these difficulties reduce, shown in experimental result as discussed below, identified people sweet taste and delicate flavour by Body, and develop the analysis method of these receptors, especially with stablize expressive function T1R taste receptors, i.e. sweet taste feel or The analysis method of the cell of umami taste receptor.
Based on this, the present invention provides the analysis methods of detection and identification taste modulating compound, wherein T1R family members As in taste bud, sweet taste and delicate flavour as report molecular action in taste modulating compound are felt.Specifically provide mirror Surely the compound that can adjust, simulate, enhance and/or block sweet taste and delicate flavour to feel respectively, and belong within the scope of the invention.Many institutes Known analysis GPCR activity, particularly the method for influencing the active compound activities of GPCR, are suitable for T1R family members of the present invention And its functional combination.Suitable analysis method is specified above.
In particular, the GPCR can be used in analysis, for example, in vivo with in-vitro measurements ligand binding, ion concentration, Film potential, electric current, ionic flux, transcription, receptors ligand interaction, the variation of second messenger's concentration.In another embodiment In, T1R family members can be recombinantly expressed in cell, by measuring Ca++Horizontal and other intracellular couriers such as cAMP, cGMP Or the variation of IP3, analysis pass through adjusting of the GPCR activity to taste transduction.
In certain analyses, the structural domain of T1R polypeptides, such as extracellularly, cross-film or intracellular domain, with heterologous polypeptide Fusion, to form chimeric polyeptides, such as with the active chimeric proteins of GPCR.The present invention particularly including include N-terminal ligand The purposes of T1R1, T1R2 or T1R3 segment of binding structural domain.The protein is particularly useful, such as can be used for identifying T1R receptors Ligand, agonist, antagonist or other conditioning agents analysis in.For example, T1R polypeptides can express in eukaryocyte, with rush It is expressed as Chimerical receptor together into plasma membrane transport or heterologous, chaperone sequence ripe and oriented by secretory pathway.Optionally Heterologous sequence can be PDZ structural domain interacting peptides, such as C-terminal PDZIP segments (SEQ ID NO 1).PDZIP is ER outputs Signal, the present invention is it has been shown that it can promote the surface expression of heterologous protein, T1R receptors for example described herein.Particularly For, in one aspect of the invention, PDZIP can be used for promoting suspicious memebrane protein such as olfactory receptor, T2R taste receptors And the appropriate orientation of T1R taste receptors described herein.
This chimeric T1R receptors can express in any eukaryocyte such as HEK-293 cells.Preferably, these cells Including G-protein, preferably such as G α15Or G α16Or another type of mixed G-protein of dwelling, these protein can by extensive GPCR with Intracellular signaling pathway or signal-proteins such as phospholipase C are associated.Any standard method can be used to detect intracellular this The activation of Chimerical receptor, such as detect the variation of intracellular Ca2+ by detecting intracellular FURA-2 dependences fluorescence.If excellent The host cell of choosing does not express suitable G-protein, the gene of the mixed G-protein of dwelling of coding can be utilized to transfect, such as U. S. application 60/ 243,770, the U.S. Shen that the U. S. application 09/984,297 and on November 21st, 2001 that on October 29th, 2001 submits are submitted Please 09/989, described in 497, be cited in full text herein for reference.
Other methods of analysis taste transduction conditioning agent include external ligand binding experiment, wherein using：T1R polypeptides, its Part is extracellular domain, transmembrane region or combinations thereof or includes the chimeric protein of the one or more structural domains of T1R family members Matter；The egg mother cell or tissue culture cells of expression T1R polypeptides, segment or fused protein；The phosphorylation of T1R family members and Dephosphorylation；In conjunction with the G-protein of GPCR；Ligand binding assays；Voltage, film potential and conductance variation；Ionic flux is analyzed；Cell The variation of interior second messenger such as cGMP, cAMP and InsP3 (IP3)；And the variation of intracellular calcium.
In addition, the present invention provide detection T1R nucleic acid and protein expression method, with carry out taste transduction Regulating study with And the specificity identification of taste receptor cells.T1R family members also provide the nucleic acid probe for parent-offspring and forensic identification.T1R Gene also serves as identification of taste recipient cell, such as lobate, bacterium shape, profile shape, geschmackstreifen and the epiglottis sense of taste by The nucleic acid probe of body cell.T1R receptors can also be used to generate monoclonal and polyclonal antibody for identification of taste recipient cell.
Functionally, T1R polypeptides include the family of a relevant 7 transmembrane G protein coupled receptor, are considered participating in the sense of taste Conduction, may interact with G-protein with mediate taste signal conduction (for example, see, Fong, Cell Signal, 8:217 (1996)；Baldwin,Curr.Opin.Cell Biol.,6:180(1994)).In structure, the nucleotides sequence of T1R family members Related polypeptide of the row coding comprising an extracellular domain, 7 transmembrane domains and a cytoplasmic domains.From other objects In the variant of T1R nucleic acid sequences or its conservative sex modification disclosed in the related T1R family genes and embodiments herein of kind at least about The region of 50 length of nucleotides, optionally 100,200,500 or more length of nucleotides, have at least about 50%, optionally 60%, 70%, 80% or 90% nucleotide sequence homology, or T1R polypeptide sequences disclosed in the polypeptide and Examples below of coding The region of at least about 25 amino acid lengths in the variant of row or its conservative sex modification, optionally 50-100 amino acid length, With at least about 35-50%, optionally 60%, 70%, 80% or 90% amino acid sequence identity.
The characteristic several consensus amino acid sequences of T1R family members or structural domain are identified.For example, T1R families Member generally comprises has at least about 50% with T1R consensus sequences 1 and 2 (are respectively SEQ ID NO2 and 3), optionally 55%, 60%, 65%, 70%, 75%, 80%, 85%, the 90%, sequence of 95-99% or higher homogeneity.These conservative structures Domain thus can be used for identifying by consistency, specific hybridization or amplification or with the specific bond of the antibody generated for structural domain The member of T1R families.T1R consensus sequences include for example following sequence：
T1R families consensus sequence 1：(SEQ ID NO:2)
(TR)C(FL)(RQP)R(RT)(SPV)(VERKT)FL(AE)(WL)(RHG)E
T1R families consensus sequence 2：(SEQ ID NO:3)
(LQ)P(EGT)(NRC)YN(RE)A(RK)(CGF)(VLI)T(FL)(AS)(ML)
The sequence that these consensus sequences are found included in T1R polypeptides described herein, but expectable other organisms T1R family members include and specifically described here consensus sequence have about 75% or higher homogeneity consensus sequence.
The specific regions of T1R nucleotide and amino acid sequence be possibly used for identification T1R family members Polymorphic variant, Inter-species autoploid and allele.The identification can carry out in vitro, such as (for example, making under stringent hybridisation conditions or by PCR With the primer for encoding above-mentioned T1R consensus sequences), or carried out in computer systems using sequence information and other nucleotide sequences Compare.The not iso-allele of T1R genes can also be used for determining whether allelic sequence variation controls kind in single species group Taste perception difference between group's different members.The amplification of classical PCR type and clone technology are very useful for detaching new T1R Place, such as degenerate primer are enough the related gene of detectable substance inter-species.
It, can be in general, by comparing about 25 amino acid or more, the amino acid sequence of such as 50-100 amino acid Carry out the identification of T1R family members Polymorphic variant and allele.About at least about 35-50%, optionally 60%, 70%, 75%, 80%, 85%, 90%, 95-99% or higher amino acid identities generally indicate that protein is T1R family members Polymorphic variant, inter-species autoploid or allele.Following any type sequence comparison algorithms can be used to carry out sequence comparison. Specific binding T1R polypeptides or the antibody of its conserved region can also be used for identification allele, inter-species autoploid and Polymorphic variant.
It can confirm T1R genes by examining the gustatory of the T1R polypeptides estimated or protein specific expressed Polymorphic variant, inter-species autoploid and allele.In general, the T1R polypeptides with amino acid sequence disclosed herein can be used as Positive control, compared with the T1R polypeptides of presumption, to prove the Polymorphic variant of T1R family members or the identity of allele. It is expected that Polymorphic variant, allele and inter-species autoploid remain with 7 transmembrane structures of g protein coupled receptor.In in more detail Hold referring to WO 00/06592, it is disclosed that relevant T1R family members GPCR-B3, content is in a manner of meeting the disclosure It is hereby incorporated by reference.GPCR-B3 receptors are referred to herein as rT1R1 and mT1R1.Turning also now to WO 00/06593, wherein Relevant T1R family members GPCR-B4 is disclosed, content is hereby incorporated by reference in a manner of meeting the disclosure.GPCR- B4 receptors are referred to herein as rT1R2 and mT1R2.As previously mentioned, the present invention is also including the use of T1R or T1R combinations, such as hT1R2/ The structure-based analysis method of the x-ray crystal structure of hT1R3 or hT1R1/hT1R3, the adjustable T1R receptor actives of identification, To adjust the molecule that sweet taste and/or delicate flavour are felt.
The invention likewise provides analysis method, preferably method for high-flux analysis, can be enhanced with identification, be simulated, blocked and/or Adjust the molecule of T1R receptors.In certain analysis methods, by the specific domain of a T1R family member and another T1R families The specific domain of member is used in combination, for example, extracellularly, cross-film or intracellular domain or area.In another embodiment, Extracellular domain, transmembrane region or combinations thereof can in conjunction with solid-phase matrix, be used for for example isolating ligands, agonist, antagonist or In conjunction with T1R polypeptides and/or its active any other molecule can be adjusted.
Various conservative mutations and replacement are all considered within the scope of the invention.For example, utilizing known recombination base Because of technical method, including PCR, gene cloning, cDNA rite-directed mutagenesis, host cell infection and in-vitro transcription, carries out amino acid and replace It changes, within the level in those skilled in the art.So as to screen active variant.
Definition
Unless otherwise specified, following term used herein has its described meaning.
" gustatory " includes neuro-epithelial cell, forms the taste bud of tongue, such as lobate, bacterium shape and profile shape afterwards in groups Cell is (for example, see Roper et al., Ann.Rev.Neurosci.12:329-353(1989)).Gustatory also see jaw and Other tissues, such as oesophagus and stomach.
" T1R " refers to one or more of g protein coupled receptor family member, these receptors gustatory it is for example lobate, Expressed in bacterium shape and profile shape cell and the cell of jaw and oesophagus (for example, see, Hoon et al., Cell, 96:541-551 (1999), it is cited in full text herein for reference).The family member is also referred to as GPCR-B3 and TR1 in WO 00/06592, in WO GPCR-B4 and TR2 is also referred to as in 00/06593.GPCR-B3 is also referred to as rT1R1 herein, and GPCR-B4 is also referred to as rT1R2.The sense of taste by Body cell according to morphology (for example, see, Roper, ibid) or can also pass through the specific expressed protein of gustatory It expresses to identify.T1R family members may have as sweet tea taste transduction receptor or distinguish various other different taste modalities Ability.Examples below identifies representative T1R sequences, including hTlR1, hTlR2 and hTlR3.
" T1R " nucleic acid encode possesses the GPCR families of 7 transmembrane regions, has " g protein coupled receptor activity ", such as it Can be combined with G-protein in response to environmental stimuli object and some enzymes such as phospholipase C and adenyl cyclase (about GPCR tie Under the stimulation description of structure and function, see, for example, Fong, ibid and Baldwin, ibid), promote to generate second messenger such as IP3, cAMP, cGMP and calcium ion.Single gustatory may include many different T1R polypeptides.
Therefore, term " T1R " family refers to Polymorphic variant, allele, mutant and inter-species autoploid,：(1) with The T1R polypeptides that T1R polypeptides, preferred embodiment 1 are identified are in about 25 amino acid, optionally on the window of 50-100 amino acid Compare, have at least about 35-50% amino acid sequence identities, optionally about 60,75,80,85,90,95,96,97,98 or 99% amino acid sequence identity；(2) antibody that specific binding is generated for immunogene, the immunogene include to be preferably selected from reality Apply the amino acid sequence of T1R polypeptide sequences and its conservative modification variant disclosed in example 1；It (3), should by a kind of nucleic acid molecule encoding The sequence for the T1R nucleic acid sequences and its conservative modification variant that molecule can be included under stringent hybridisation conditions and selected from embodiment 1 Row specific hybrid (size at least about 100, optionally at least about 500-1000 nucleotide)；Or (4) include a kind of sequence, it should Sequence and the amino acid sequence for the T1R amino acid sequences identified selected from embodiment 1 have at least about 35-50% consistency.
On topological structure, certain chemoreception GPCR have " N-terminal structural domain ", " extracellular domain ", comprising 7 across " transmembrane domain " of film area and corresponding cytoplasm and extracellular loop；" cytoplasmic domains " and " C-terminal structural domain " (referring to example Such as, Hoon et al., Cell, 96:541-551(1999)；Buck&Axel,Cell,65:175-187(1991)).Use this field Method known to technical staff can identify these structural domains in structure, such as identify hydrophobic and hydrophilic domain sequence point Program is analysed (see, for example, Stryer, Biochemistry, (3rd ed.1988)；See also any sequence based on internet point Program is analysed, such as the program that can be found on dot.imgen.bcm.tmc.edu).These structural domains are fitted into egg for preparing The analyzed in vitro of white matter and the present invention, such as ligand binding assays are particularly useful.
Thus " extracellular domain " refers to the structure that the polypeptides of the T1R on the outside of cell are protruded outward and be exposed to from cell membrane Domain.These structural domains generally comprise " the N-terminal structural domain " being exposed on the outside of the cell of cell, may include optionally being exposed to carefully The extracellular loop part of transmembrane domain on the outside of the cell of born of the same parents, i.e., between transmembrane region 2 and 3, between transmembrane region 4 and 5, transmembrane region Ring between 6 and 7.
" N-terminal structural domain " area starts from N-terminal and extends close to the region that first transmembrane domain starts position. More specifically, in one embodiment of the invention, which starts from N-terminal, and about positioned at 563 ± about 20 Terminate at the conservative glutamic acid of a amino acid position.These extracellular domains are for the external ligand knot in solution and solid phase It is particularly useful to close experiment.In addition, following transmembrane regions and extracellular domain be combined also can binding partner, thus for matching in vitro Body combines experiment also particularly useful.
" transmembrane domain " for including 7 " transmembrane region " refers to the structural domain for the T1R polypeptides being located in plasma membrane, it is also possible to Including corresponding cytoplasm (intracellular) and extracellular loop.In one embodiment, which substantially opens corresponding to T1R family members It starts from the conservative glutaminic acid residue at about 563 ± 20 amino acid positions, terminate at about 812 ± 10 amino acid positions The structural domain of conservative tyrosine residue.Use such as Kyte＆Doolittle, J.Mol.Biol., 157:105-32 (1982), or Standard method described in Stryer (being same as above) can identify 7 transmembrane regions and extracellular and cytoplasm ring.
" cytoplasmic domains " refer to such as " C-terminal structural domain " and cross-film towards the structural domain of the T1R polypeptides of cell interior The intracellular loops of structural domain, such as the intracellular loops between transmembrane region 1 and 2, between transmembrane region 3 and 4 and between transmembrane region 5 and 6. " C-terminal structural domain " refers to the region across the last one transmembrane domain end and c-terminal of protein, is usually located at cytoplasm It is interior.In one embodiment, which starts from the conservative tyrosine residue at about 812 ± 10 amino acid positions, and prolongs Continue to the C-terminal of polypeptide.
Term " ligand binding domain " or " ligand binding domains " refer to from taste receptors, particularly substantially at least Include the sequence of the taste receptors of receptor extracellular domain.In one embodiment, the extracellular structure of ligand binding domain Domain may include N-terminal structural domain, and optionally, a part for transmembrane domain, for example, transmembrane domain extracellular loop. Ligand binding domain may binding partner, especially can enhance, simulate, block and/or adjust the sense of taste, such as sweet taste or delicate flavour feel Compound.
About T1R receptors of the present invention or the term of polypeptide " heteromultimers " or " heterologous polycomplex " refer at least one The function of kind T1R receptors and another receptor, usually another kind T1R receptor polypeptides (or another non-T1R receptor polypeptides) Property joint.Understand for, the application describe T1R functionality interdepend reflect its may with heterodimeric the sense of taste by The function of nanocrystal composition.However, as previously mentioned, functionality, which interdepends, is also possible to reflect indirect interaction.For example, T1R3 May only have the function of promoting T1R1 and T1R2 surface expressions, T1R1 and T1R2 may independently be used as taste receptors.Alternatively, Functional taste receptor may be only made of T1R3, and T1R3 carries out different processing under T1R1 or T1R2 controls, is similar to calcium The RAMP dependences of associated receptor are processed.
It is adjusted in test in the analysis of the compound for the taste transduction that T1R family members mediate, phrase " functionality effect Answer " include it is determining any one directly or indirectly by the parameter of receptor influences, such as functional, physics and chemical effect.It is wrapped Include the ligand binding of internal, external and in vitro (ex vivo), ionic flux, film potential, electric current, transcription, G-protein combination, GPCR Phosphorylation or dephosphorylation, the analysis based on conformation change, signal transduction, receptors ligand interaction, second messenger's concentration are (such as CAMP, cGMP, IP3 or intracellular Ca2+++) variation, further include other physiological effects, such as neurotransmitter or hormone release Increase or decrease.
" determining functionality effect " in analysis refers to the ginseng that analysis can make directly or indirectly to be influenced by T1R family members The compound that number (such as functional, physics and chemical effect) increases or decreases.These functional effects can utilize this field skill Any method known to art personnel measures, such as spectral signature (such as fluorescence, absorption, refractive index), fluid dynamics are (such as Shape), chromatography or dissolution characteristics, Patch-clamp experiments (patch clamping), voltage sensitive dyes, full cell currents, put Injectivity isotope outflow, can induce mark, egg mother cell T1R gene expressions variation；Tissue culture cells T1R expression；T1R bases The transcription activating of cause；Ligand binding assays；Voltage, film potential and conductance variation；Ionic flux analysis, intracellular second messenger are such as The variation of cAMP, cGMP and InsP3 (IP3)；The variation of intracellular calcium；Neurotransmitter regulator, conformational analysis etc..
" inhibitor " of T1R genes or protein, " activator " and " conditioning agent " are used to refer to utilize the internal and external sense of taste Transduction assay identification inhibition, reactivity or modulability molecule, such as ligand, agonist, antagonist and its homologue and Analogies.
Inhibitor refer to for example combine, partially or completely block stimulate, reduce, preventing, postpone activation, inactivate, desensitize or Lower the compound of taste transduction, such as antagonist.Activator refers to for example combinable, and stimulation increases, opens, activation, promoting Into, enhancing activation, enhanced sensitivity or the compound for raising taste transduction, such as agonist.Conditioning agent include for example changeable receptor with The compound of following matter interaction：In combination with extracellular protein (such as the ebnerin and other of activator or inhibitor Hydrophobic carrier family member)；G-protein；Kinases (such as participates in receptor deactivation and the rhodopsin kinase and beta adrenergic of desensitization The analog of energy receptor kinase)；And it can equally make receptor deactivation and the inhibition albumen (arrestin) of desensitization.Conditioning agent includes The T1R family members of genetic modification, for example, activity change, it is naturally occurring and synthesis ligand, antagonist, agonist, Small chemical molecular etc..The analysis of these inhibitor and activator includes, for example, express in cell or cell membrane T1R families at Member, with or without tastant such as sweet tastant, using the adjusting immunomodulator compounds of supposition, then as described above really The fixed functional effect to taste transduction.Including T1R family members sample or analysis by possible activator, inhibitor or After conditioning agent processing compared with without the control sample of activator, inhibitor or conditioning agent, the degree of adjusting is detected.Positive control The opposite T1R activity values of sample (such as being not added with the sweet tastant of conditioning agent) are set to 100%.
The opposite T1R activity values of negative control sample (such as being not added with the buffer solution of taste stimulus) are set to 0%.Work as the positive The mixture of control sample and conditioning agent makes the T1R activity values relative to positive control be about 80%, optionally 50% or 25- When 0%, the inhibition to T1R is just obtained.When relative to positive control sample T1R activity values be 10%, 25%, 50%, 75%, optionally 150%, optionally 150%, optionally 200-500% or 1000-3000% or when higher, just obtain list Only activation of the conditioning agent to T1R.
Terminology used herein " purifying ", " purifying substantially " and " separation " refers to the shape without other different compounds State (these different compounds are usually combined together with the compounds of this invention in its natural state), to by the weight of given sample Gauge, " purifying ", " purifying substantially " and " separation " substance include at least 0.5%, 1%, 5%, the 10% of sample quality Or 20%, most preferably at least 50% or 70%.In a preferred embodiment, these terms refer to comprising (by weight) The compounds of this invention of designated samples at least 95% mass.When being related to nucleic acid or polypeptide, terminology used herein " purifying ", " base This purifying " and " separation " refer to different from a kind of naturally occurring purity or concentration in mammal, particularly human body.It is high In the purity or concentration of naturally occurring any degree in mammal especially human body, including (1) from other dependency structures or In compound purifying or (2) with usually incoherent structure and compound are combined in mammal especially human body, belong to Within " separation " meaning.Nucleic acid or protein or nucleic acid described herein or kinds of protein can be according to those skilled in the art Known a variety of method and process separation, or be connected with the structure of onrelevant under natural conditions and compound.
Term " nucleic acid " or " nucleic acid sequence " refer to either single-stranded or double-stranded form DNA or ribose core Sour oligonucleotides.The term includes nucleic acid, i.e. the oligonucleotides containing analog known to natural nucleotide.The term further includes tool There are the nucleic-acid like structures of synthesis main chain (see, for example, Oligonucleotides and Analogues, a Practical Approach,ed.F.Eckstein,Oxford Univ.Press(1991)；Antisense Strategies,Annals of The N.Y.Academy of Sciences, Vol.600, Eds.Baserga et al. (NYAS 1992)；Milligan, J.Med.Chem.36:1923-1937(1993)；Antisense Research and Applications(1993,CRC Press),WO 97/03211；WO 96/39154；Mata,Toxicol.Appl.Pharmacol.144:189-197(1997)； Strauss-Soukup,Biochemistry36:8692-8698(1997)；Sams tag,Antisense Nucleic Acid Drug Dev,6:153-156(1996))。
Specific nucleic acid sequence is also implied including its conservative modification variant (for example, degenerate codon unless otherwise specified, Replace) and complementary series, and the sequence that clearly indicates.In particular, degenerate codon replacement can be by generating for example wherein The third position of one or more selected codons is mixed the sequence of base and/or deoxyinosine residue replacement to realize (Batzer et al., Nucleic Acid Res., 19:5081(1991)；Ohtsuka et al., J.Biol.Chem., 260: 2605-2608(1985)；Rossolini et al., Mol.Cell.Probes, 8:91-98(1994)).Term nucleic acid can be with base Cause, cDNA, mRNA, oligonucleotides and polynucleotides are used alternatingly.
Term " polypeptide ", " peptide " and " protein " is used interchangeably herein, refers to amino acid residue polymer.The term is suitable Be for one or more of which amino acid residue the artificial chemical analogue of corresponding naturally occurring amino acid amino acid it is more Polymers and naturally occurring amino acid polymer and non-naturally occurring amino acid polymer.
Term " domain plasma membrane translocation structure (plasma membrane translocation domain) " or referred to as " indexing Structural domain " refer to when it mixes polypeptid coding sequence, can be effectively heterozygosis (" fusion ") compared with structural domain Albumen " with (chaperone) " or " indexing " to cytoplasma membrane polypeptide domain.For example, " transposition structural domain " can derive from The amino terminal of ox rhodopsin receptor polypeptides (7 transmembrane receptors).It is however possible to use the rhodopsin of any mammal Matter, and other indexings promote sequence.Therefore, transposition structural domain is especially effective in indexable 7 transmembrane fusion proteins to plasma membrane, And including the protein (such as taste receptor polypeptide) of amino terminal transposition structural domain is more than the protein without the structural domain Easily and efficiently it is transported on plasma membrane.But if polypeptide N-terminal structural domain is active in combination, such as the present invention T1R receptors, then preferably using other transposition structural domains.For example, PDZ structural domains described herein can be used to interact Peptide.
" transposition structural domain " described herein, " ligand binding domains " and chimeric receptors compositions also include having and example sequence Arrange almost the same structure and active " analog " or " examples of conservative variations " and " analogies " (" peptide mimics " (peptidomimetic)).Therefore, term " examples of conservative variations " or " analog " or " analogies " refer to the amino acid with modification The polypeptide of sequence, it is this kind of to change (examples of conservative variations) structure and/or activity for not substantially changing defined herein polypeptide.This Including amino acid sequence conservative sex modification variation, i.e., for the amino acid substitution of the inessential residue of protein active, add Add or lacks or the amino acid substitution of kin residue (such as acid, alkalinity, positive electricity or negative electricity, polarity or nonpolarity Deng) so that or even replace crucial amino acid and also not substantially change structure and/or activity.
More specifically, " guard the variant of sex modification " it is suitable for amino acid and nucleic acid sequence.For specific nucleic acid sequence Row, conservative modification variant refer to encoding the nucleic acid of identical or essentially identical amino acid sequence, or if the nucleic acid is not compiled Code amino acid sequence, then refer to essentially identical sequence.Due to the degeneracy of genetic codon, a large amount of functional identical nucleic acid can Encode any specific protein.
For example, codon GCA, GCC, GCG and GCU all encoding alanines.Therefore, every in the alanine determined by codon The codon can be changed into any corresponding codon by one position, the polypeptide without changing coding.
The variance is " silent variant ", is a kind of variation of conservative sex modification.The core of each coding polypeptide herein Acid sequence also illustrates each possible silent variant of the nucleic acid.It should be recognized by those skilled in the art that each of nucleic acid is close Numeral is (in addition to the unique codon AUG of methionine under normal conditions, and the unique codon of tryptophan under normal conditions TGG the identical molecule of function) can be generated through modification.Therefore, each silent variant for encoding the nucleic acid of polypeptide is implicit In the sequence of each description.
The conservative substitution table for being capable of providing intimate amino acid is generally well-known in the art.For example, a selection The exemplary guideline that conservative is replaced includes (Original Residue is followed by example replacement)：Ala/gly or ser；arg/lys；asn/ Gln or his；asp/glu；cys/ser；gln/asn；gly/asp；Gly/ala or pro；His/asn or gln；Ile/leu or val；Leu/ile or val；Lys/arg or gln or glu；Met/leu or tyr or ile；Phe/met or leu or tyr；ser/ thr；thr/ser；trp/tyr；Tyr/trp or phe；Val/ile or leu.Another exemplary guideline uses following six groups, every group of packet The amino acid replaced containing mutual conservative：1) alanine (A), serine (S), threonine (T)；2) aspartic acid (D), glutamic acid (E)；3) asparagine (N), glutamine (Q)；4) arginine (R), lysine (I)；5) isoleucine (I), leucine (L), Methionine (M), valine (V)；With 6) phenylalanine (F), tyrosine (Y), tryptophan (W)；(see, for example, Creighton,Proteins,W.H.Freeman and Company(1984)；Schultz and Schimer, Principles of Protein Structure,Springer-Vrlag(1979)).It should be understood by those skilled in the art that above-mentioned replacement is not Only possible conservative is replaced.For example, for certain purposes, people may conservative be replaced each other by all Charged acids Body, but regardless of being positive electricity or negative electricity.In addition, changing, adding or lacking single amino acids or fraction ammonia in coded sequence Each replacement, missing or the addition of base acid are also considered as " conservative sex modification variation ".
Term " analogies " and " peptide mimics " refer to the chemical compound of synthesis, it has and the basic phase of polypeptide of the present invention Same structure and/or functional characteristic, such as transposition structural domain, ligand binding domains or Chimerical receptor.Analogies can be complete Be made of entirely the non-natural amino acid analogs synthesized or part natural peptide amino acids and some non-natural amino acids seemingly The chimeric molecule of object.Analogies can also have any number of natural amino acid conservative to replace, as long as the replacement is also basic On do not change the structure and/or activity of analogies.
For the polypeptide of examples of conservative variations of the present invention, routine experiment will determine whether analogies belong to the scope of the present invention, That is its structure and/or activity does not change substantially.Peptidomimetics component may include the combination of any non-natural structural component, It is usually from three building stones：A) residue linkage groups in addition to natural amide bond (" peptide bond ") connects；B) non-natural Residue substitutes naturally occurring amino acid residue；Or c) can induce the residue of Sketch of secondary structure object, that is, induce or stablize two level Residue of the structure such as β-bend, γ corners, β-pleated sheet piece and alpha helical conformation.When whole or certain residues of polypeptide are to pass through When chemical means rather than native peptides are keyed, which can be accredited as analogies.Each peptide simulation object residue can lead to Cross peptide bond, other chemical bonds or coupling means connection, such as glutaraldehyde, N-hydroxy-succinamide ester, difunctional maleimide Amine, N, N'- dicyclohexylcarbodiimides (DCC) or N, N'- diisopropylcarbodiimide (DIC).Traditional amide bond can be used as The linking group of the substitute of (" peptide bond ") includes, such as ketone methylene (such as-C (=O)-CH2- replacements-C (=O)-NH-), Ammonia methylene (CH2-NH), ethylene, alkene (CH=CH), ether (CH2-O), thioether (CH2-S), tetrazolium, thiazole, Retroamide, thioamides or ester are (see, for example, Spatola, Chemistry and Biochemistry of Amino Acids,Peptides and Proteins,Vol.7,pp267-357,“Peptide Backbone Modifications,” Marcell Dekker,NY(1983)).Including whole or certain non-natural residues substitute the more of naturally occurring amino acid residue Peptide can also be accredited as analogies；There is detailed description to non-natural residues in science and patent document.
" label " or " detectable part " be can be by spectrum, photochemistry, biochemistry, immunochemistry or chemical hand The component that section detects.For example, useful label includes32P, fluorescent dye, electron-dense reagents, enzyme (such as are commonly used for ELISA's), biotin, digoxin or haptens and (such as by mixing radioactive label in peptide) can be detected or can Protein for detecting the antibody that specific can be reacted with peptide.
" nucleic acid probe or oligonucleotides of label " refers to or by the covalent bond of connector or chemical bond, or by from Son, Van der Waals force, electrostatic or hydrogen bond Non-covalent binding and the nucleic acid or oligonucleotides that are combined with marker, pass through inspection in this way Combined on probing needle label can detection probe presence.
" nucleic acid probe or oligonucleotides " used herein is defined as can be by the chemical bond of one or more types, generally It is the complementary base pairing formed by hydrogen bond, and the nucleic acid combined with the target nucleic acid of complementary series.Probe used herein can wrap Include natural (i.e. A, G, C or T) or modified base (7- denitrogenations guanylic acid, inosine etc.).In addition, the base in probe can be by phosphoric acid Connection except diester linkage is connected, as long as not influencing hybridization.Thus, for example probe can be its form base by peptide bond and The connected peptide nucleic acid of non-phosphodiester bond.It will be understood by those of skill in the art that according to the preciseness of hybridization conditions, probe can To combine the target sequence not fully complementary with probe sequence.The optionally direct label isotope of probe, chromophore, illuminophore, chromogen, Or indirect labelling such as then can be in conjunction with the biotin of Streptavidin compound.By the presence or absence of analysis probe, people can examine It surveys with the presence or absence of selection sequence or subsequence.
When using term " heterologous " for nucleic acid moiety, indicate that the nucleic acid is included under native state without same mutual Two or more subsequences of relationship.For example, the nucleic acid generally recombinates generation, two or more irrelevant gene orders are organized New functional nucleic acid is formed, for example, the code area in the promoter in source and another source.Similarly, different Source protein matter indicate the protein be included in native state under do not have same correlation two or more subsequences (for example, Fused protein).
" promoter " is defined as instructing the nucleic acid sequence of transcribed nucleic acid.Promoter used herein is included in transcription initiation site Neighbouring necessary nucleic acid sequence, such as the TATA elements in polymerase Il type promoter.Promoter also optionally includes remote End enhancer checks subcomponent, can be located at from the up to thousands of base-pairs of transcription initiation site." composing type " promoter be All active promoter under most of environment and developmental condition.
" induction type " promoter is promoter active under environment or growth adjustment.Term " effectively connection " refers to Function of the expression of nucleic acid control sequence (such as promoter or Binding site for transcription factor array) between second nucleic acid sequence Property connection, wherein expression control sequence instruct second sequence corresponding to nucleic acid transcription.
" recombination " used herein refers to the polynucleotides (for example, " recombination of polynucleotide ") of synthesis or other manipulation in vitro, And the method for using recombination of polynucleotide to generate gene outcome in cell or other biosystems, or refer to by recombination multinuclear glycosides The polypeptide (" recombinant protein ") of acid encoding." recombination method " also includes by the various code areas with separate sources or structural domain Or the nucleic acid of promoter sequence is connected in expression cassette or carrier and expresses, such as induction type or constitutive expression include that the present invention is easy The fusion protein of the nucleic acid sequence of nuclear localization sequence and use primer amplification of the present invention.
It is used herein " stable cell lines " refer to stablize, i.e. long-term expression heterologous nucleic acid sequence, i.e. T1R or G-protein is thin Born of the same parents system.In preferred embodiments, the linearized vector for including T1R expression constructs, i.e. T1R1, T1R2 and/or T1R3 is utilized Suitable cell, typically mammalian cell, such as HEK-293 cells are transfected, this stable cell lines are generated.Most preferably Ground, two kinds of linearization plasmids of hT1R1 and hT1R3 or hT1R2 and hTlR3 are expressed by cotransfection, and pass through the properly side of screening Method generates the cell line of wherein these genes of stable integration, to generate this stable cell lines.Most preferably, the cell line is also Stablize expression G-protein, such as G α15。
Phrase " selectivity (or specificity) hybridization " refers under stringency hybridisation conditions, a kind of molecule only be present in complexity The specific nucleotide sequence of (for example, total cell or library DNA or RNA) combines, forms double-strand or hybridization in mixture.
Phrase " stringency hybridisation conditions " refer in such a situa-tion, probe only be typically found in nucleic acid COMPLEX MIXED Target sequence hybridization in object, without hybridizing with other sequences.Stringent conditions are sequence dependents, and in varied situations It will be different.Long sequence specific hybrid at relatively high temperatures.The detailed guide of related nucleic acid hybridization referring to Tijssen, Techniques in Biochemistry and Molecular Biology-Hybridization with Nucleic Probes,“Overview of Principles of Hybridization and the Strategy of Nucleic Acid Assays”(1993).In general, the stringent conditions bit sequencing of selection is listed in the heat under specified ionic strength pH Melting temperature (Tm) is about 5-10 DEG C low.Tm refer to (under specified ionic strength, pH and nucleic acid concentration) balance after with target sequence There is 50% temperature when hybridizing with target sequence (when target sequence excess, at Tm, 50% to be utilized after balance in complementary probe Probe).Stringent conditions are that salinity is dense less than about 1.0M sodium ions, typically about 0.01-1.0M sodium ions (or other salt) At least about 30 DEG C of temperature, long probe (are greater than 50 cores when degree, pH 7.0-8.3, short probe (such as 10-50 nucleotide) Thuja acid) when at least about 60 DEG C of temperature.Addition goes stable reagent such as formamide that can also form stringent conditions.For selectivity Or specific hybrid, positive signal are at least twice of background, optional 10 times of hybrid contexts.Typical stringency hybridisation conditions are such as Under：50% formamide, 5xSSC and 1%SDS, 42 DEG C incubate or 5xSSC, 1%SDS, 65 DEG C of incubations, in 0.2xSSC and 0.1%SDS, it washs at 65 DEG C.When the hybridization and washing step can carry out such as 1,2,5,10,15,30,60 minute or longer Between.
If the polypeptide of nucleic acid encode is substantially related, the nucleic acid not hybridized mutually under stringent conditions is still substantially It is related.For example, maximum Codon degeneracy under being allowed using genetic code will this thing happens when generating nucleic acid copy. In these cases, nucleic acid usually hybridizes under intermediate stringency hybridization conditions.Typically " intermediate stringency hybridization conditions " are wrapped Include 40% formamide, 1M NaCl, 1%SDS buffer solution in, in 37 DEG C hybridization, and in 1xSSC, 45 DEG C washing.The hybridization Can be carried out such as 1,2,5,10,15,30,60 minute with washing step or longer time.Positive hybridization is at least background value Twice.Those of ordinary skill is it is readily appreciated that other items for hybridizing preciseness similar with wash conditions offer can be utilized Part.
" antibody " refers to the polypeptide of the ramework region comprising immunoglobulin gene or its segment, be may specifically bind And identify antigen.Generally acknowledged immunoglobulin gene includes κ, λ, α, γ, δ, ε and μ constant region gene and many immune globulins White variable region gene.Light chain can be classified as κ or λ.Heavy chain can be classified as γ, μ, α, δ or ε, they determine immune respectively successively Globulin type IgG, IgM, IgA, IgD and IgE.
Typical immunoglobulin (antibody) structural units includes the tetramer.Each tetramer is by two pairs of identical polypeptide chains Composition, each pair of polypeptide chain have " light " chain (about 25kDa) and " weight " chain (about 50-70kDa).Every chain N-terminal is true The variable region for having determined about 100-110 or more amino acid, is mainly responsible for antigen recognizing.Term " variable light " (VL) and " Variable heavy chain " (VH) respectively refer to these light chains and heavy chain.
" chimeric antibody " refers to a kind of antibody molecule, wherein (a) constant region or part thereof is changed, replaces or exchanges, with It is connected as the constant region that antigen binding site (variable region) is different from classification, effector function and/or species or is changed, or With can assign chimeric antibody with the entirely different molecule (such as enzyme, toxin, hormone, growth factor, drug etc.) of new characteristic It is connected；Or (b) variable region or part thereof is changed, replaced or is exchanged into the variable of antigentic specificity that is different or changing Area.
" anti-T1R " antibody is the antibody or antibody for the polypeptide that can specifically bind T1R genes, cDNA or its subsequence coding Segment.
Term " immunoassay " is the analysis using antibody specificity combination antigen.Immunoassay is characterized in that using spy The specific binding characteristics of antibody are determined to detach, orient and/or quantify antigen.
When being related to protein or peptide, phrase and antibody " specificity (or selectivity) combines " or " specificity (or selection Property) carry out immune response " refer to that can determine to combine existing for protein in the group of heterologous protein and other biological products Reaction.Therefore, under the conditions of specified immunoassay, the combination of specified antibody and specific protein is at least the two of background value Times, and obviously do not combined with other oroteins present in sample substantially.Under the conditions of these with the specific binding of antibody The specificity for specific protein may be needed to carry out the antibody of selection.For example, may be selected for particular species such as rat, small The polyclonal antibody that mouse or people T1R family members generate, with obtain those only with T1R polypeptides or its immunogen portion rather than its Specificity occurs for its protein (except T1R polypeptide orthologous objects (ortholog) or Polymorphic variant and allele) The polyclonal antibody of immune response.This selection course can pass through removal and other species T1R molecules or other T1R molecules The antibody of cross reaction is completed.It can also select the antibody of only identification T1R GPCR family members rather than other family GPCR.
The antibody that panimmunity analytical form can be used for selecting that specific immune response occurs with specific protein.Example Such as, generally use solid phase ELISA immunoassays selection with protein occur specific immune response antibody (for example, see, Harlow＆Lane, Antibodies, A Laboratory Manual, (1988), wherein related can be used for determining that specificity is exempted from The description of epidemic disease reactive immunoassay formats and condition).Typical specificity or selective reaction are at least background signal or make an uproar Twice of sound, more preferably greater than 10-100 times of background.
Phrase " selective binding " refer to nucleic acid with another nucleic acid as described above the ability of " selective cross " or antibody with The ability of protein " selectivity (specificity) combines " as described above.
Term " expression vector " refers to that be intended to any cell include that protokaryon, yeast, fungi, plant, insect or lactation are dynamic In object cell, any recombinant expression system of the nucleic acid sequence of in vivo or in vitro, composing type or the inducible expression present invention.The art Language includes linear or circular expression systems.The term includes the expression system for keeping episome or being integrated into host cell gene group System.Expression system can have the of self-replication capacity, or not have, i.e., only drive transient expression in the cell.The term includes weight Group expression " box ", wherein including only the minimum element needed for recombinant nucleic acid transcription.
" host cell " refers to including expression vector and supporting the cell of expression vector duplication or expression.Host cell can Be prokaryotic cell such as Escherichia coli or eukaryocyte such as yeast, insect, amphibian, worm or mammalian cell such as CHO, Hela and HEK-293 etc., such as the cell of culture, explant (explants) and internal cell.
The separation and expression of T1R polypeptides
The separation and expression of T1R of the present invention or its segment or variant can be completed as described below.It can be expanded using PCR primer The nucleic acid for encoding taste receptor ligand combined area, optionally establishes the library of these nucleic acid.Can use each expression vector or Expression vector library is infected or transfection host cell, is used for these nucleic acid of functional expression or library.These genes and carrier can It prepares and expresses in vitro or in vivo.Technical staff should be understood that (such as to be opened by adjusting gene and nucleic acid in carrier of the present invention Mover, enhancer etc.) expression or activity, can be changed and control the desired phenotypes of expression of nucleic acid.It can use described For increasing or decreasing expression or active any known method.It can implement in conjunction with any of method in this field or scheme The present invention, these methods or scheme have detailed description in science and patent document.
The nucleic acid sequence of the present invention and other nucleic acid molecules for carrying out the present invention, no matter RNA, cDNA, genome DNA, carrier, virus or its heterozygote, can be from a variety of source separation, genetic engineering, amplification, and/or recombinant expression.It can make Include such as bacterium, yeast, insect or botanical system in addition to mammalian cell with any recombinant expression system.
In addition, these nucleic acid molecules, such as Carruthers are synthesized in vitro using following well known chemical synthesising technologies, Cold Spring Harbor Symp.Quant.Biol.47:411-418(1982)；Adams,Am.Chem.Soc.105:661 (1983)；Belousov,Nucleic Acids Res.25:3440-3444(1997)；Frenkel,Free Radic.Biol.Med.19:373-380(1995)；Blomers,Biochemistry33:7886-7896(1994)； Narang,Meth.Enzymol.68:90(1979)；Brown,Meth.Enzymol.68:109(1979)；Beaucage, Tetra.Lett.22:1859(1981)；Described in United States Patent (USP) 4,458,066.Then or by synthesizing complementary strand and appropriate Under the conditions of anneal together, or complementary strand is added by using suitable primer sequence and archaeal dna polymerase, double-stranded DNA can be obtained Segment.
Nucleic acid manipulation technology, such as generate series jump, subclone, label probe, sequencing, hybridization etc., in science and specially There is detailed description in sharp document.See, for example, Sambrook, ed., Molecular Cloning:a Laboratory Manual(2nd ed),Vols.1-3,Cold Spring Harbor laboratory(1989)；Current Protocols in Molecular Biology,Ausubel,ed.John Wiley&Sons,Inc.,New York(1997)； Laboratory Techniques in Biochemistry and Molecular Biology:Hybridization with Nucleic Acid Probes,Part I,Theory and Nucleic Acid Preparation,Tijssen, ed.Elsevier,N.Y.(1993)。
Nucleic acid, carrier, capsid (capsids), polypeptide etc. can be by the way that well known to a person skilled in the art numerous general sides Any one in method is analyzed and is quantified.Including, such as analytical biochemistry method such as NMR, spectrophotometry, radiation be aobvious Shadow, electrophoresis, Capillary Electrophoresis, high performance liquid chromatography (HPLC), thin-layer chromatography (TLC) and super diffusion chromatography etc., various immunologys Method such as fluid or the reaction of gel precipitation element, Immune proliferation, immunoelectrophoresis, radiommunoassay (RIA), Enzyme-linked Immunosorbent Assay point Analyse (ELISA), immunofluorescence analysis, Southern analyses, Northern analyses, point engram analysis, gel electrophoresis (such as SDS- PAGE), RT-PCR, quantitative PCR, other nucleic acid or target substance or signal amplification method, radioactive label, scinticounting and parent And chromatography.
Oligonucleolide primers can be used for the nucleic acid fragment of amplification coding taste receptor ligand combined area.Nucleic acid described herein Clone or quantitative measurment can also be carried out using amplification technique.Amplification method is also known in the art, including such as polymerase chain Reaction, PCR (PCR Protocols, a Guide to Methods and Applications, ed.Innis.Academic Press, N.Y. (1990) and PCR Strategies, ed.Innis, Academic Press, Inc., N.Y. (1995), connect Meet enzyme chain reaction (Ligase chain reaction, LCR, for example, see Wu, Genomics 4:560(1989)； Landegren,Science 241:1077,(1988)；Barringer,Gene 89:117(1990))；Transcription amplification (such as Referring to Kwoh, Proc.Natl.Acad.Sci.USA 86:1173(1989))；It is automatic maintain sequence replicating (for example, see, Guatelli,Proc.Natl.Acad.Sci.USA 87:1874(1990))；Q- β duplications enzymatic amplification (for example, see, Smith, J.Clin.Microbiol.35:1477-1491(1997))；Automatic Q- β replicase Amplification Analysis (for example, see, Burg, Mol.Cell.Probes 10:257-271 (1996)) and other RNA polymerases mediate technology (such as NASBA, Cangene,Mississauga,Ontario)；Referring further to Berger, Methods Enzymol.152:307-316(1987)； Sambrook；Ausubel；United States Patent (USP) 4,683,195 and 4,683,202；Sooknanan,Biotechnology13:563- 564(1995).Primer can be designed to retain the original series of " donor " 7 membrane receptor.It is replaced alternatively, primer can encode conservative (such as hydrophobic residue changes hydrophobic residue into, referring to above-mentioned) or functionally advantageous replacement are changed (for example, plasma membrane is not interfered to insert Enter, cause Isopeptidase cleavage, cause receptor it is improper fold etc.) amino acid residue.After amplification, if it is desired, using conventional Nucleic acid is cloned into any carrier by molecular biology method one by one or in the form of library according to methods known in the art；Amplification The body outer clone method of nucleic acid is as described in United States Patent (USP) 5,426,039.
Primer pair can be designed to the ligand binding domain of selective amplification T1R family members.For different ligand or rush Taste agent, these regions may be varied from.Thus, it may be possible to a kind of minimum calmodulin binding domain CaM of tastant, for second of rush taste Agent may then be limited to very much.Accordingly, it is possible to expand the different size of ligand binding domain for including different extracellular structure domain structures.
Design degenerate primer pair example be it is well known in the art that.For example, COnsensus-DEgenerate Hybrid Oligonucleotide Primer (CODEHOP) strategic computer program can be from http://blocks.fhcrc.org/ Codehop.html is obtained, and is directly connected to from Blockmaker Multiple Sequence Alignments address, by a set of relevant protein sequence Taste receptor ligand combined area as is known starts, and carries out hybrid primer prediction (for example, see Rose, Nucleic Acids Res.26:1628-1635(1998)；Singh,Biotechniques 24:318-319(1998)).
The means of synthetic oligonucleotide primers pair are known in the art." natural " base-pair or synthesis base can be used It is right.For example, being capable of providing operation primer sequence using artificial nucleobases, generating more complicated amplified production mixture Universal method.Multiple families of artificial nucleobases can be rotated by interior keys by take a variety of hydrogen bond directions, to carry For the method for degeneracy molecular recognition.The single locus of these analogs incorporation PCR primer can generate the complicated library of amplified production. For example, see Hoops, Nucleic Acids Res.25:4866-4871(1997).It can also be imitated using nonpolar molecule The shape of natural DNA bases.The non-hydrogen key-shaped shape analogies of adenine can efficiently and selectively be directed to the non-pole of thymidine The analogies of property shape are replicated (for example, see Morales, Nat.Struct.Biol.5:950-954(1998)).Example Such as, two degeneracy bases can be pyrimidine bases 6H, 8H-3,4- dihydroxy-pyrimidine simultaneously [4,5-c] [1,2] piperazine -7- ketone or purine Base N6- methoxyl groups -2,6- diaminopurine (for example, see, Hill, Proc.Natl.Acad.Sci, USA 95:4258- 4263(1998)).Exemplary degenerate primers of the present invention are mixed with nucleobase (nucleobase) analog 5'- dimethoxy triphen first Base-N- benzoyl -2'- deoxycytidines, 3'- [(2- cyanoethyls)-(N, N- diisopropyl)]-phosphoramidite (see above sequence Term " P " in row).This pyrimidine analogue and purine, including A and G residues form hydrogen bond.
Essentially identical Polymorphic variant, allele and inter-species autoploid, can make with taste receptors disclosed herein It is detached with above-mentioned nucleic acid probe.Alternatively, same to expressing by using the antiserum or antibody purification prepared for T1R polypeptides Source body carries out immunology detection (its same identification and selective binding T1R autoploids), and expression library clone T1R can be used more Peptide and its Polymorphic variant, allele and inter-species autoploid.
Degenerate primer can be used to amplification (such as PCR) suitable nucleic acid sequence, obtain the ligand knot of coding taste receptors Close the nucleic acid in area.The nucleic acid being amplified can come from the genomic DNA of any cell or tissue or be expressed from taste receptors The mRNA or cDNA of cell.
In one embodiment, the hybrid protein of the nucleic acid comprising the coding T1R merged with transit sequences can be built Matter coded sequence.It is also provided that include translocation motif and the rush of chemosensory receptor, particularly the other families of taste receptors The heterozygosis T1Rs of taste agent binding structural domain.These nucleic acid sequences can effectively connect transcription or translation control element, such as transcribe With translation initiation sequence, promoter and enhancer, transcription and translation terminator, polyadenylation sequence and DNA is transcribed into Other sequences useful RNA.In recombinant expression cassettes, carrier and the composition of transgenic animals, it can be referred to using promoter fragment Purpose nucleic acid is led to express in all purposes cell or tissue.
In another embodiment, fused protein may include C-terminal or N-terminal transit sequences.In addition, fusion protein Matter can include other element, such as protein detection, purifying or other application.Promote the structural domain of detection and purifying Including, for example, metal chelating peptide such as polyhistidine sequence section, histidine-tryptophan modules or it is other can be in the metal of immobilization On the structural domain that is purified；Maltose-binding protein；The albumin A knot that can be purified on the immunoglobulin of immobilization Structure domain；Or the structural domain (Immunex Corp, Seattle WA) used in FLAGS extensions/affinity purification system.
Between transposition structural domain (being expressed for effective plasma membrane) and the rest part of new translation polypeptide, including can be broken Attachment sequence, such as Xa factor is (for example, see Ottavi, Biochimie 80:289-293 (1998)), hay bacillus Protease identifies motif (for example, see Polyak, Protein Eng.10:615-619 (1997)), enterokinase (Invitrogen, San Diego, CA) etc., it is advantageously possible for promote purifying.For example, a construct may include one and 6 The connected polypeptide encoding nucleic acid sequence of a histidine residues, be followed by thioredoxin, enterokinase cleavage site (for example, see, Williams,Biochemistry 34:1787-1797 (1995))) and a C-terminal transposition structural domain.The histidine is residual Base can promote to detect and purify, and enterokinase cleavage site provides the hand that target protein is purified from remaining fusion protein Section.The technology of carrier and fused protein application in relation to encoding fusion protein matter has detailed in science and patent document Description, for example, see Kroll, DNA Cell.Biol.12:441-53(1993).
Include expression vector (the either single expression vector or expression vector of ligand binding domains coded sequence Library) it can be introduced into genome or cytoplasm or the nucleus of cell, and expressed using various routine techniques, this is in science And have detailed description in patent document.For example, see Roberts, Nature 328:731(1987)；Berger is same as above； Schneider,Protein Expr.Purif.6435:10(1995)；Sambrook；Tijssen；Ausubel.Biological reagent The information for closing known biological method also is provided with the product information of experimental facilities producer.Carrier can be from natural origin point From, obtain from the suchlike source in the libraries ATCC or GenBank or prepared by synthesis or recombination method.
Nucleic acid can use stablize in the cell or the expression cassette of transient expression (such as episome expression system), carrier or Virus is expressed.Selection marker can be introduced into expression cassette and carrier, to assign transformed cells and sequence may be selected Phenotype.For example, selection marker can be encoded for episome Maintenance and Replication, so that host genome need not be integrated into In.For example, mark may encode antibiotic resistance (such as chloramphenicol, kanamycins, G418, bleomycin, hygromycin) or remove Careless agent resistance (such as chlorosulfuron or Basta), with selection converted target DNA sequence cell (for example, see, Blondelet-Rouault,Gene 190:315-317(1997)；Aubrecht,J.Pharmacol.Exp.Ther.281: 992-997(1997)).Selected marker gene due to assigning neomycin or this kind of substrate resistance of hygromycin is only used for tissue training It supports, chemo-resistance gene can also be used as the selective key of in vitro and in vivo.
Chimeric nucleic acid sequence codified is located at the T1R ligand binding domains inside any 7 transmembrane polypeptide.Due to 7 cross-films Receptor polypeptides have similar primary sequence and two level and tertiary structure, and structure can be easily identified by sequence analysis Domain (such as extracellular domain, TM structural domains, cytoplasmic domains etc.).For example, homology modeling, Fourier analysis and spiral week Phase detects 7 structural domains that can identify and characterize 7 transmembrane receptor sequences.Fast Fourier transform (FFT) algorithm can be used for commenting Valence characterization is analyzed the dominant periods of sequence hydrophobicity and changeability overview.Cycle detection enhances and α spiralization cycles index can be with For example, by Donnelly, Protein Sci.2:The method of 55-70 (1993) is completed.It is ability that other comparisons and mould, which build algorithm, Well known to domain, for example, see Peitsch, Receptors Channels 4:161-164(1996)；kyte&Doolittle, J.Md.Bio.,157:105-132(1982)；Cronet,Protein Eng.6:59-64(1993).
The invention also includes DNA and protein with designated nucleotide and amino acid sequence, and include DNA fragmentation, Especially such as 40,60,80,100,150,200 or 250, or more nucleotide segment, and such as 10,20,30,50, , or more 70,100 or 150 the protein fragments of amino acid.Optionally, nucleic acid fragment codified can with for T1R families at The antigenic polypeptide that the antibody that member generates is combined.In addition, the present invention protein fragments be optionally can with for T1R family The anti-genic fragment that the antibody that family member generates is combined.
The invention also includes chimeric protein, it includes at least one T1R polypeptides described herein at least 10,20,30, , or more 50,70,100 or 150 amino acid, with all or part for representing another GPCR, preferably 7 cross-film superfamily members Other amino acid sequence be mutually coupled.These chimeric sons can thus locate receptor and prepared by another GPCR, or two kinds of combination Or a variety of T1R receptors and prepare.In one embodiment, a part for the chimeric son is corresponding or from the present invention The extracellular domain of T1R polypeptides.In another embodiment, a part for the chimeric son is corresponding or from described herein The extracellular domain of T1R polypeptides and one or more transmembrane domains, rest part come from another GPCR.Chimerical receptor It is well known in the art, it technology of preparing and introduces the selection of g protein coupled receptor structural domain therein or segment and defines Also well known in the art.Therefore, the knowledge of those skilled in the art's this respect is easily used for preparing this Chimerical receptor.Make Can provide a kind of such as taste selectivity characteristic of specifically disclosed receptor herein with this Chimerical receptor, and with another by The signal transduction feature of body is mutually coupled, such as the well known receptor used in prior art analysis system.
As described above, the analog of this chimera, natural T1R receptors or natural T1R receptor combinations or joint can be tied Close the molecule or activated by it for usually influencing that sweet taste is felt or delicate flavour is felt.The T1R receptors or receptor combination of functional chimeric are these The molecule of sample can combine when combining individually or with other T1R or other GPCR (its own may be exactly chimera) expression To taste stimulus, particularly sweet tea (T1R2/3) or fresh (T1R1/3) taste stimulus, or by its activation.Sweet taste is caused to be felt Molecule includes natural and artificial sweetener, such as sucrose, radix asparagi sweet extract (aspartame), xylitol, cyclamate Deng, cause delicate flavour feel molecule include glutamic acid and the like and in combination with natural T1R1 and/or T1R3 other chemical combination Object, such as 5'- nucleotide.
For example, such as ligand binding domains, extracellular domain, transmembrane domain, transmembrane domain, cytoplasmic structure Domain, N-terminal structural domain, C-terminal structural domain or its structural domain arbitrarily combined, can be covalently attached to heterologous protein.For example, T1R extracellular domains can be connected to heterologous GPCR transmembrane structural domain or heterologous GPCR extracellular domains can be connected to T1R across Spanning domain.Other optional heterologous proteins, such as egfp can be used.
The host cell for expressing T1R of the invention, segment, chimera or variant is also within the scope of the invention.In order to Obtain clone gene or nucleic acid (such as encode the present invention T1R, segment or variant cDNA) high level expression, technology people Purpose nucleic acid sequence is usually subcloned to the expression vector of the strong promoter comprising guidance transcription, transcription/translation termination by member In, for the nucleic acid of coding protein, wherein also including the ribosome bind site for translation initiation.Suitable promoters It is well known in the art, such as described in Sambrook et al..However bacterium or eukaryotic expression system can be used.
Any well known method that exogenous nucleotide sequence is introduced into host cell can be used.Including using phosphoric acid Calcium transfection, polybrene, protoplast fusion, electroporation, liposome, microinjection, cytoplasm carrier (plasma vector), disease Poisonous carrier and cloned genomic dna, cDNA, synthetic DNA or other exogenous genetic materials are introduced other of host cell What known method (for example, see Sambrook et al.).Only require that specific gene engineering method used can successfully will at least One nucleic acid molecules is introduced into the host cell that can express purpose T1R, segment or variant.
After expression vector is introduced cell, transfectional cell is under conditions of suitable purpose receptor, segment or variant are expressed Culture, is then recycled it using standard technique from culture.The example of this technology is known in the art.For example, see WO 00/06593, it is quoted in the way of meeting present disclosure, it is for reference.
The detection of T1R polypeptides
Other than using nucleic acid hybridization technique detection T1R genes and gene expression, it can also be detected using immunoassay T1R, such as identification of taste recipient cell and the variant of T1R family members.Immunoassay qualitative or quantitative analysis can be used T1R.The summary of applicable technology is referring to Harlow＆Lane, Antibodies:A Laboratory Manual(1988).
The antibody of 1.T1R family members
It is art technology to prepare with the monoclonal antibody of T1R family member's specific reactions and the method for polyclonal antibody (for example, see Coligan, Current Protocols in Immunology (1991) known to personnel；Harlow&Lane, supra；Goding,Monoclonal Antibodies:Principles and Practice(2d ed.1986)；And Kohler&Milstein,Nature,256:495-497(1975)).Such technology includes by from bacteriophage or similar substrates Recombinant antibodies library in selection antibody prepare monoclonal antibody and Duo Ke to prepare antibody, and by immune rabbit or mouse Grand antibody is (for example, see Huse et al., Science, 246:1275-1281(1989)；Ward et al., Nature, 341:544- 546(1989))。
Immunogene of many comprising T1R can be used for preparing the antibody with T1R family member's specific reactions.For example, can be such as Separation recombination T1R polypeptides described herein or its anti-genic fragment.Suitable antigenicity area includes, such as identifying T1R families The consensus sequence of member.Recombinant protein can express in eukaryon or prokaryotic cell as described above, and as described in generally above It is purified.Recombinant protein is the preferred immunogene for preparing monoclonal or polyclonal antibody.In addition, deriving from sequence disclosed herein It arranges and the synthetic peptide of conjugate vectors protein may be used as immunogene.The naturally occurring of pure or impure form can also be used Protein.It then will be in injections to the animal that can generate antibody.Monoclonal or polyclonal antibody can be generated, is subsequently used for Immunoassay is to measure protein.
The method for preparing polyclonal antibody is known to those skilled in the art.For example, being helped using standard adjuvant such as Freund Agent and standard immunization protocol, with Western Immuno inbred mouse (such as BALB/C mice) or rabbit.By take a blood sample and measure with The reaction titre of T1R monitors immune response of the animal to immunogen preparation.When acquisition is suitably directed to the high titre of immunogene After antibody, collects animal blood and prepare antiserum.If desired, separation antiserum can be further classified to be enriched with and albumen The antibody (referring to Harlow＆Lane, ibid) of qualitative response.
Monoclonal antibody is obtained using multiple technologies familiar to the person skilled in the art.In brief, generally go through with Myeloma cell is merged, can make purpose antigen be immunized animal immortalizing spleen cells (referring to kohler＆Milstein, Eur.J.Immunol.,6:511-519(1976)).The other method immortalized is including the use of epstein-barr virus (Epstein Barr Virus), oncogene or other methods Retrovirus transformed or well known in the art.Screening is by single immortalized cells shape At clone, with generate for antigen have it is required specificity and compatibility antibody, can improve these using multiple technologies The yield for the monoclonal antibody that cell generates, including it is injected into the intraperitoneal of vertebrate host.Alternatively, according to Huse et al., Science,246:The conventional method that 1275-1281 (1989) is summarized can detach coding by screening human B cell DNA library The DNA sequence dna of monoclonal antibody or its binding fragment.
It collects monoclonal antibody and polyclonal serum and is titrated using immunogen protein in immunoassay, such as using It is fixed on the solid-phase immunoassay that the immunogene of solid support carries out.Being typically chosen has 104Or more high titre is polyclonal Antiserum, and competitive binding immunoassay is used, it is tested to non-T1R polypeptides or even other T1R family members or other The cross reaction of other related proteins of organism.It is extremely that specific polyclonal antiserum and monoclonal antibody, which usually combine Kd, Few about 0.1mM, more generally at least about 1pM, optionally at least about 0.1pM or more preferable, and optionally 0.01pM or more preferable.
Once obtaining T1R family member's specific antibodies, then panimmunity analysis method can be utilized to detect each T1R eggs White matter and protein fragments.Summary in relation to immunology and immunoassay method, referring to Basic and Clinical Immunology(Stites&Terr eds.,7th ed.1991).In addition, the immunoassay of the present invention can be by several forms It completes, is specified in Enzyme Immuncassay (Maggio, ed., 1980)；And Harlow＆Lane, ibid.
2. immunology binding analysis
(such as join using any well known immunology binding analysis detection and/or quantitative T1R albumen, segment and variant See United States Patent (USP) 4,366,241；4,376,110；4,517,288；With 4,837,168).The summary of general immunoassay referring further to Methods in Cell Biology Autibodies in Cell Biology, volume 37 (Asai, ed.1993)； Basic and Clinical Immunology(Stiters&Terr,eds.,7th ed.1991).Immunology binding analysis (or immunoassay) (is herein T1R family members or its antigen usually using that can specifically bind selected protein or antigen Property subsequence) antibody.Can use well known to a person skilled in the art and a variety of methods as described above in any system Standby antibody (such as anti-T1R).
Marker is also commonly used in immunoassay, specifically binds the compound that simultaneously labelled antibody and antigen are formed.Label Object itself may be a part comprising antibody/antigen compound.Therefore, marker can be the T1R polypeptides or mark of label The anti-T1R antibody of note.Alternatively, marker can be third part, such as specifically bind the second of antibody/T1R compounds Antibody (secondary antibody is usually special to the antibody of the species in first antibody institute source).It being capable of specific bond immunoglobulin The other oroteins of constant region, as albumin A or Protein G also are used as marker.The immune ball of these protein and many species Albumen constant region show strong non-immunogenic reactivity (for example, see, Kronval et al., J.Immunol., 111: 1401-1406(1973)；Akerstrom et al., J.Immunol., 135:2589-2542(1985)).Using detectable portion Divide modification marker, such as biotin, can be combined with another molecular specificity, such as Streptavidin.A variety of detectable parts It is well known to those skilled in the art.
Through entire analysis, each binding reagents are required for incubation and/or rinse step.Incubation step from about 5 seconds to A few houres etc., optionally -24 hours about 5 minutes.But incubative time depends on analytical form, antigen, liquor capacity, dense Degree etc..Analysis usually carries out at room temperature, although can be carried out within the temperature range of such as 10 DEG C -40 DEG C.
A. noncompetitive analytical form
The immunoassay of T1R polypeptides in sample is detected either competitive or noncompetitive.Noncompetitive immune point Analysis is the direct analysis for measuring amount of antigen.Such as in one preferred " sandwich " (sandwich) analysis, anti-T1R antibody can be straight It connects and is incorporated on its fixed solid-phase matrix of institute.Then the T1R polypeptides in these immobilized antibodies capture sample.T1R polypeptides It is thus immobilized, then in conjunction with marker, such as with markd 2nd T1R antibody.Alternatively, secondary antibody may not marked Note, but the third antibody that it can be marked with combines, which is directed to the species in secondary antibody institute source Antibody.It, can be with another molecule such as chain usually using detectable part modification secondary antibody or third antibody, such as biotin Mould Avidin specific binding, to provide detectable part.
B. competitive analysis form
In competitive analysis, replace from anti-T1R antibody that (competition is gone by T1R polypeptides unknown in sample by measuring Except) known to additional (external source) T1R polypeptides amount, to indirectly measure sample in T1R polypeptides amount.In a competitive analysis In, the T1R polypeptides of known quantity are added in sample, are then contacted sample with the antibody that can specifically bind T1R.With antibody knot T1R peptide concentrations are inversely proportional in the amount and sample of the external source T1R polypeptides of conjunction.In an especially preferred embodiment, antibody It is immobilized on solid-phase matrix.By the amount of T1R polypeptides in measurement T1R/ antibody complexes, or measures and do not form compound The amount of remaining protein, it may be determined that the amount of the T1R polypeptides combined with antibody.By providing the T1R molecules of label, can detect The amount of T1R polypeptides.
Another preferred competitive analysis is haptens inhibition analysis.In the analysis, it is known that T1R polypeptides be immobilized On solid-phase matrix.The anti-T1R antibody of known quantity is added in sample, is then contacted sample with the T1R of immobilization.With it is known The amount of anti-T1R antibody that combines of immobilization T1R polypeptides and the amount of T1R polypeptides in sample be inversely proportional.Equally, by detecting antibody Immobilization part or solution in remaining antibody moiety, the amount of immobilized antibody can be detected.Antibody can be straight when labeled Detection is connect, or the above-mentioned mark part for specifically binding antibody is then added and carries out indirect detection.
C. cross reactivity measures
The immunoassay of competitive binding form can also be used to measure cross reactivity.For example, can be by one kind at least partly Thus locate the protein immobilization of disclosed nucleic acid sequence encoding on solid-phase matrix.By protein (such as T1R polypeptides and homologous Object) it is added in analysis system, with immobilized antigen competitive binding antiserum.The protein of addition is competed with immobilized protein Property combine sero-fast ability, and thus locate the T1R polypeptides of disclosed nucleic acid sequence encoding compared with the ability that itself is competed. The cross reactivity percentage of above-mentioned protein is calculated using canonical algorithm.It selects and merges each above-mentioned protein with addition Cross reactivity be less than 10% antiserum.By the way that specific protein, for example remote source (distantly related) is added together Source object carries out immunosorbent, and the antibody of cross reaction is removed optionally from combined antiserum.In addition, for identifying T1R families The peptide for including the amino acid sequence for representing Conserved motifs of member, can be used for measuring cross reactivity.
Through immunosorbent and combined antiserum then can be used for above-mentioned competitive binding immunoassay, will be deemed likely to be Thus the allele of T1R family members or the second protein of Polymorphic variant (locate disclosed core with immunogen protein The T1R polypeptides of sequences code) it compares.To be compared, in a wide concentration range to two kinds of protein respectively into Row analysis, determines the amount for inhibiting antiserum to be combined each required protein with immobilized protein 50%.If inhibited 50% combines the amount of required second protein to inhibit needed for 50% combination than thus locating the protein of disclosed nucleic acid sequence encoding Amount it is 10 times low, it is judged that second protein can specifically bind the polyclonal antibody generated for T1R immunogenes.
For T1R Conserved motifs generate antibody can also be used for prepare only with T1R families GPCR specific binding, without The antibody combined with the GPCR of other families.
Cut down removal cross reacting antibody by using other T1R family members, it is specific that specific binding can be prepared The polyclonal antibody of T1R family members.Species specificity polyclonal antibody can be prepared in a similar manner.For example, being gone by abatement Except the antibody with orthologous object sequence such as rat T1R1 or mouse T1R1 cross reactions, it is special hT1R1 can be prepared Property antibody.
D. other analytical forms
Westem traces (immunoblotting) analysis can be used for detecting and quantify T1R polypeptides present in sample.The technology one As comprising being supported to suitable solid phase according to molecular weight separating sample proteins, by the Protein transfer of separation using gel electrophoresis Object (such as nitrocellulose filter, nylon membrane or derivative nylon membrane), by sample and the antibody temperature that T1R polypeptides can be specifically bound It educates.Anti- T1R polypeptide antibodies can specifically bind the T1R polypeptides on solid support.These antibody may be marked directly, or with It is detected afterwards using the labelled antibody (such as sheep anti-mouse antibodies of label) that can specifically bind anti-T1R antibody.
Other analytical forms include liposome immunization analysis (LIA), (such as anti-in combination with specific molecular using being designed to Body) and discharge coated reagent or mark liposome.Then according to standard technique detection release reagent (referring to Monroe et al., Amer.Clin.Prod.Rev., 5:34-41(1986)).
E. the reduction of non-specific binding
It will be appreciated by those skilled in the art that it is frequently desirable to non-specific binding is minimized in immunoassay.Especially It is when the analysis is related to being fixed on antigen or antibody on solid-phase matrix, it is desirable to drop the amount in non-specific binding to matrix It is extremely minimum.The method for reducing the non-specific binding is known to those skilled in the art.In general, the technology is related to using egg The component of white matter property is coated with matrix.In particular, it is widely used protein component, such as bovine serum albumin(BSA) (BSA), de- Fat milk powder, gelatin, most preferably milk powder.
F. marker
The particular marker or detectable group used in analysis is not the key factor of the present invention, as long as its not notable shadow Ring the specific binding of antibody in analysis.Detectable group can have detectable physically or chemically any Substance.This kind of detectable label is perfect in the development of immunoassay field, under normal circumstances, for most of marks in these methods Remember that object can be applied to the present invention.Therefore, marker is available spectrum, photochemistry, biochemistry, immunochemistry, electricity, light Any component of or chemical method detection.The useful marker of the present invention includes magnetic bead (such as DYNABEADSTM), fluorescent dye (such as fluorescein isothiocynate, texas Red (Texas red), rhodamine etc.), radioactively labelled substance are (such as3H、125I、14C 、35S), enzyme (such as horseradish peroxidase, alkaline phosphatase and other enzymes commonly used in ELISA) and chromogenic label, Such as colloidal gold or stained glass or plastic bead (such as polystyrene, polystyrene, latex).
According to methods known in the art, marker can be directly or indirectly coupled with the purpose component of analysis.As described above, It is provided according to required sensitivity, the difficulty being conjugated with compound, stability requirement, existing instrument and disposition, can select to make With various markers.
Usually non-radioactive marker is connected using indirect method.In general, ligand molecular (such as biotin) is covalently tied It closes on molecule.Then another molecule of the ligand binding (such as Streptavidin), the molecule itself can be detected or be covalently attached To signal system, such as detectable enzyme, fluorescent chemicals or chemiluminescence compound.Aglucon and its target substance can be with knowledges The antibody of other T1R polypeptides or the secondary antibody of the anti-T1R of identification are applied in combination with any suitable.
The compound for generating signal can also be directly conjugated in molecule, such as conjugated with enzyme or fluorogen.As marker Purpose enzyme is mainly hydrolase, especially phosphatase, lipase and glycosidase or oxidizing ferment, especially peroxidase.It is Fluoresceinated It includes fluorescein and its derivative, rhodamine and its derivative, red sulfonyl compound, umbelliferone etc. to close object.Chemiluminescence chemical combination Object includes luciferin (luciferin) and 2,3- dihydrophenazines diones (2,3-dihydrophthalazinediones), example Such as luminol (luminol).The various labels or signal generation system that may be used are referring to the comprehensive of United States Patent (USP) 4,391,904 It states.
The method of detection marker is well known to those skilled in the art.Thus, for example when marker is radio-labeled object When, detection method includes scintillation counter or light-sensitive surface autoradiograph.When marker is fluorescent marker, conjunction may be passed through It fits the light excitation fluorchrome of wavelength and the fluorescence for detecting generation is detected.Fluorescence can be examined by light-sensitive surface, using electronics It surveys device such as charge coupled device (CCD) or photoelectric multiplier etc. and carries out visual detection.Similarly, enzyme marker can pass through offer The suitable substrates of enzyme simultaneously detect reaction product to be detected.Finally, simple chromogenic label can pass through observation and label The relevant color of object carries out easy detection.Therefore, in various test strips (dipstick) analysis, conjugated gold is usually aobvious pink Color, and various conjugated globules show the color of the globule.
Certain analytical forms need not use labeling component.For example, agglutination test can be used for detecting the presence of target antibody. In this case, antigen coat particle can be aggregated by the sample comprising target antibody.In the form, each component is not necessarily to label, leads to The presence of target antibody can be detected by crossing the simple observation of naked eyes.
The detection of conditioning agent
Determine whether untested compound can specifically bind composition and the side of the T1R receptors of the present invention in vitro and in vivo Method is as described below.Many aspects that stechiology can be monitored, to evaluate the effect of ligand binding T1R polypeptides of the present invention.It can With to the intact cell for expressing chemosensory receptor, permeabilized cell or the film component for utilizing standard method to generate or in vitro The protein of de novo formation carries out these analyses.
In vivo, taste receptors combination tastant and cause chemical irritant transduction be electric signal.Activation or the G eggs inhibited Bai Yici changes the property of target enzyme, channel or other effect protein matter.Some examples have, such as are turned by vision system It leads plain (transducin) and activates cGMP phosphodiesterases, by stimulating physical property G-protein activation adenyl cyclase, passing through Gq Different channels is adjusted with other similar G-protein activation phospholipase Cs and by Gi and other G-proteins to adjust.Also under can detect Trip is then caused the mobilization of calcium as a result, for example by the diacylglycerol and IP3 of phospholipase C generation by IP3.
T1R protein or polypeptide in the analysis preferably have the T1R polypeptide sequences disclosed in the embodiment 1 polypeptide, Or its segment or conservative modification variant.Optionally, the segment and variant can be the antigenic pieces that can combine anti-T1R antibody Section and variant.Optionally, the segment and variant can be in conjunction with sweetener or delicate flavour tastant or by its activation.
Alternatively, T1R protein or polypeptide in the analysis can derive from eukaryotic host cell, may include and embodiment T1R polypeptides or its segment or conservative modification variant disclosed in 1 have the amino acid sub-sequences of amino acid sequence identity.One As for, the amino acid sequence identity be at least 35-50% or optional 75%, 85%, 90%, 95%, 96%, 97%, 98% or 99%.Optionally, the T1R albumen in the analysis or polypeptide can include the structural domain of T1R albumen, such as extracellular knot Structure domain, transmembrane region, transmembrane domain, cytoplasmic domains, ligand binding domains etc..In addition as described above, T1R protein or its Structural domain can be covalently attached heterologous protein, to generate the chimeric protein that can be used for analysis described herein.
It is whether recombinating or naturally-produced using above-mentioned T1R protein or polypeptide, to test T1R receptor actives Conditioning agent.It can detach, coexpression, coexpression, the total table in tissue or animal on the film from cell in cell Up to recombination or naturally-produced T1R albumen or polypeptide.For example, tongue slice, the cell, transformed cells or the film that are detached from tongue all may be used To use.Adjustment effect can be tested using one of analysis in vitro or in vivo described herein.
For example, disclosed in the experiment of following article embodiment, it has been found that certain 5 ' nucleotide, such as 5 ' IMP or 5 ' GMP, it can Enhance the activity of Pidolidone activation umami taste receptor or blocks umami taste stimuli, such as Pidolidone and L-Aspartic acid Activation to umami taste receptor.
1. external binding analysis
It can be with vitro detection taste transduction using soluble or solid phase reaction using the T1R polypeptides of the present invention.Specific In embodiment, T1R ligand binding domains can be used, ligand binding is analyzed with vitro in soluble or solid phase reaction.
For example, the N-terminal structural domain of prediction T1R participates in ligand binding.Specifically, T1R belongs to GPCR subfamilies, it is special Sign is with big, about 600 amino acid extracellular N-terminal segment.These N-terminal segments are it is believed that form ligand binding Structural domain, thus can be used for identifying the biochemical analysis of T1R agonists and antagonist.Ligand binding domain may be by extracellularly tying The other part in structure domain is formed, for example, transmembrane domain extracellular loop.
It is used for utilizing the external binding analysis with the relevant other GPCR of T1R, such as metabotropic glutamate receptor (for example, see, Han and Hampson, J.Biol.Chem.274:10008-10013(1999)).These analyses may relate to replace The ligand for changing radioactivity or fluorescent marker measures the variation of intrinsic fluorescence or the variation of proteolytic susceptibility etc..
It can be detected in solution, duplicature (optional to be attached to solid phase), lipid monolayer or vesica and T1R polypeptides of the present invention The ligand that heterologous polycomplex combines.Such as spectral signature variation (such as fluorescence, absorption, refractive index), fluid can be used The combination of mechanics (such as shape), chromatography or dissolubility test conditioning agent.
In another embodiment of the present invention, GTP γ can be used35S is analyzed.As described above, the case where GPCR is activated Under, stimulate the G α subunits of G-protein compound, combining GDP to be exchanged for GTP.In biochemical analysis, what measurement was added Radioactive label GTP γ35S it is assumed that ligand in the presence of and G-protein combination, can measure it is ligand-mediated to G-protein hand over Change active stimulation.In general, the film comprising purpose chemosensory receptor is mixed with G-protein compound.Add in analysis Enter potential inhibitor and/or activator and GTP γ35S measures the GTP γ combined with G-protein35S.It is counted by liquid scintillation Or other methods known in the art including scintillation proximity assay (SPA), combination can be measured.In other analysis shapes In formula, the GTP γ S of fluorescent marker can be used.
2. fluorescence polarization assay
In another embodiment, the analysis based on fluorescence polarization (" FP ") can be used to detect and monitor ligand binding. Fluorescence polarization is the versatile laboratory technique for measuring balance combination, nucleic acid hybridization and enzymatic activity.Fluorescence polarization assay is homogeneity , because it does not need separating step, such as centrifugation, filtering, chromatography, precipitation or electrophoresis.These analyses are directly real in the solution Shi Jinhang does not need immobilization phase.Polarization value is repeatable to be measured, and measurement polarization is very quick after reagent is added, so will not Destroy sample.It is arrived down to picomole (picomolar) in general, this technology can be used for measuring fluorogen (fluorophore) The polarization value of micromole (micromolar) level.This section describes how it is simple quantitatively use fluorescence polarization measurement ligand with The combination of T1R polypeptides of the present invention.
When fluorescent tag molecule is excited by linearly polarized light, send out the light with certain polarization degree, polarization degree with Molecule rotation is inversely proportional.Big fluorescent tag molecule keeps opposing stationary in excitation state (being 4 nanoseconds for fluorescein), The polarization of light is kept relative constant between excitation and transmitting.Small fluorescent tag molecule is quickly rotated in excitation state, excitation and The polarization significant changes of light between transmitting.Therefore, small molecule polarization value is low, and macromolecular polarization value is high.For example, single-stranded fluorescein mark The polarization value of the oligonucleotides of note is relatively low, but when it is with complementary strand thereof, just has higher polarization value.When using FP When may activate to detect and monitor or the tastant of chemosensory receptor of the present invention is inhibited to combine, fluorescent marker can be used Tastant or autofluorescence tastant.
Fluorescence polarization (P) is defined as：
Wherein ∏ be with the parallel emitted luminescence intensity of excitation optical plane, Int ⊥ are the transmitting light vertical with excitation optical plane Intensity.P is intensity ratio, is a nondimensional number.For example, Beacon and Beacon 2000TMSystem can be with these Analysis joint uses.This kind of system generally indicates polarization value (1 Polarization Unit=1000mP units) with milli Polarization Unit.
Perrin equations describe the relationship between molecule rotation and size, and reader can refer to Jolley, M.E. (1991) in Journal of Analytical Toxicology, 236-240 pages, there is shown the detailed solutions of party's formula It releases.Summarily, Perrin equations identification polarization is revolved with spin relaxation (rotational relaxation) time, i.e. molecule It is directly proportional the time required to turning about 68.5 ° of angles.Rotational relaxation time by following equation and viscosity (η), absolute temperature (T), Molecular volume (V) is related to gas constant (R)：
Rotational relaxation time is for small molecule (such as fluorescein) very little (1 nanoseconds of ≈), for macromolecular (such as immune globulin Very big (100 nanoseconds of ≈) in vain).If viscosity and temperature are kept constant, Rotational relaxation time, namely polarization, straight with molecular size Connect correlation.The variation of molecular volume may be due to fluorescent tag molecule and the interaction of other molecules, dissociation, polymerize, drop Caused by solution, hybridization or conformation change.For example, fluorescence polarization have been used to measure protease, DNA enzymatic and RNA enzyme enzymatic lysis it is big Fluorescein-labeled polymer.It is additionally operable to measure protein/protein interaction, antibody/antigen combination and DNA/ eggs The combination balance that white matter combines.
A. solid phase and soluble high throughput assays
In another embodiment, the present invention provides use oligomeric T1R polypeptide complexes or coexpression T1R more The soluble analysis that the cell or tissue of peptide carries out.Preferably, which includes to stablize coexpression functionality T1R1/T1R3 (fresh) The cell line of taste receptors or T1R2/T1R3 (sweet tea) taste receptors.In another embodiment, the present invention provides high-throughput shapes The analyzed in vitro based on solid phase of formula, wherein by T1R polypeptides or express T1R polypeptides cell or tissue be attached to solid-phase matrix, Or taste stimulating compound, and be in contact with T1R receptors, the antibody generated using suitable label or for the T1R receptors is examined Survey combination.
It, may screening up to thousands of kinds different conditioning agents or ligand within one day in the high throughput analysis of the present invention. In particular, the independent analysis for selected potential conditioning agent can be carried out with each hole of microwell plate, or if wanted Concentration or incubative time effect are observed, a kind of conditioning agent can be tested per 5-10 hole.Therefore, one piece of standard microplate can divide Analyse about 100 kinds of (such as 96 kinds) conditioning agents.If using 1536 orifice plates, one block of plate can easily analyze about 1000-1500 The different compound of kind.Multiple compounds may also be analyzed in each plate well.The different tablet of several piece may be analyzed daily； It may the Analysis and Screening up to about different compound of 6,000-20,000 kinds using the integrated system of the present invention.Latest developments examination The microfluidics process (microfluidic approaches) of agent operation.
Molecules of interest can directly or indirectly be combined with solids fraction by covalently or non-covalently key (such as passing through label). Label can be various ingredients any one.In general, by solid phase is fixed in combination with the molecule (label combination) of label On support, by the interaction of label and label combination, by the molecules of interest of marking (such as taste transduction purpose point Son) it is attached to solid support.
According to the known interaction of molecules that document is described in detail, a variety of labels and label combination can be used.For example, such as Fruit label has natural binder, such as biotin, albumin A or Protein G, it can be (affine with suitable label combination Element, Streptavidin, neutral Avidin, immunoglobulin fc region etc.) it is used in combination.Also broadly available for natural knot The antibody of the molecule (such as biotin) of object is closed, this is also suitable label combination (referring to SIGMA Immunochemicals 1998Catalogue Sigma,St.Louis MO)。
Similarly, any haptens or antigenic compound all can with antibody combination appropriate, to form label/label knot Close object pair.It can be commercialized and obtain thousands of kinds of antibody, document describes many other antibody.For example, in a kind of common shape In formula, label is first antibody, and it is the secondary antibody for identifying first antibody to mark and sign conjugate.In addition to antibody-antigene phase interaction With outer, receptor-ligand interaction also is suitable as label and label combination pair.For example, the agonist of cell-membrane receptor and Antagonist (such as cell receptor-ligand interaction, as transferrins, c-kit, viral receptor ligands, cytokine receptor, Chemokine receptors, interleukin-2-receptor, immunoglobulin receptor and antibody, cadherin family, integrins group, selection Albumen (selectin) family etc., for example, see Pogott＆Power, The Adhesion Molecule Facts Book I (1993)).Similarly, toxin and venom (venoms), virus epitopes, hormone (such as opiate, steroids etc.), intracellular Receptor (for example, it mediates the effect of various small ligands, including steroids, thyroid hormone, biostearin (retinoids) and Vitamin D；Peptide), drug, agglutinin, sugar, nucleic acid (linear and cyclic polymer configurations), oligosaccharides, protein, phosphatide and antibody, It can be acted on various cell receptors.
Synthetic polymer, such as polyurethanes, polyester, makrolon, polyureas, polyamide, polyethyleneimine, poly- virtue Sulfide, polysiloxanes, polyimides, poly- acetic acid esters (polyacetates) are supportted, suitable label or label can also be formed Conjugate.Those skilled in the art when browsing present disclosure it is clear that many other label/label combinations to It can be used for analysis system described herein.
Common connector, such as peptide, polyethers etc., are also used as label, including polypeptide sequence, for example, about 5-200 amino The polyglycine sequences of acid.This kind of flexible joint is known to those skilled in the art.For example, poly(ethylene glycol) connector can derive from Shearwater Polymers,Inc.Huntsville,Alabama.These connectors optionally have amido bond, mercapto key or different Source function key.
Label combination can be fixed on solid-phase matrix using existing various methods.The usual whole of solid-phase matrix or portion Divide and be exposed to chemical reagent, the chemical group that can be reacted with a part for label combination is fixed on surface, thus into Row derivatization or functionalization.For example, the group being suited for attachment on long-chain moiety includes amine, hydroxyl, mercaptan and carboxylic group.Ammonia Base alkyl silane (aminoalkylsilanes) and hydroxyalkylsilanes (hydroxyalkylsilanes) can be used for a variety of surfaces Functionalization, such as glass surface.Document details the method for building such solid phase biopolymer arrays.For example, see Merrifield,J.Am.Chem.Soc.,85:2149-2154 (1963) (synthesis in solid state for describing such as peptide)；Geysen etc. People, J.Immun.Meth., 102:259-274 (1987) (describes the solid phase components synthesis on needle)；Frank&Doring, Tetrahedron,44:6031-6040 (1988) (is described and is synthesized various peptide sequences on cellulose disk)；Fodor et al., Science,251:767-777(1991)；Sheldon et al., Clinical Chemistry, 39 (4):718-719(1993)； And Kozal et al., Nature Medicine, 2 (7):753-759 (1996) (describes the life being fixed on solid-phase matrix Object polymer array).Include other common methods by the non-chemical method that label combination is fixed in matrix, such as heating, UV crosslinkings with radiation etc..
3. the analysis based on cell
It is in preferably processing embodiment, T1R protein or polypeptide group name is instantaneous or stable total in eukaryocyte It is expressed as unmodified form or chimera, variant or truncated receptor, is carried or preferably without it being promoted ripe and fixed To the heterologous chaperone sequence by secretory pathway.This kind of T1R polypeptides can express in any eukaryocyte, such as HEK-293 thin Born of the same parents.Preferably, which includes functional G Protein such as G α15Or aforementioned chimeric G protein or Chimerical receptor can be coupled to cell The other protein of interior signal pathway or signal protein such as phospholipase C.Further preferably generate can stablize coexpression T1R1/T1R3 or The cell of T1R2/T1R3, it has been found that the response to taste stimulus of (as shown in EXPERIMENTAL EXAMPLE) cell display enhancing (cell of T1R combinations identical compared to transient expression).Any standard method can be used to detect the work of T1R receptors in these cells Change, such as the variation of intracellular Ca2+ is detected by Fluo-4 dependences fluorescence in detection cell.This alanysis is the application institute Show the basis that experiment is found.
The GPCR receptors of activation are typically the bottom for the kinases for making receptor C terminal tail portion (may also other sites) phosphorylation Object.Therefore, activator can promote32P is transferred to receptor from radiolabeled ATP, using scintillation counter analysis.C-terminal The phosphorylation of tail portion can promote to inhibit the combination of albumen (arrestin) sample protein, and interfere the combination of G-protein.GPCR signals The summary of conduction and signal transduction analysis method see, e.g. Methods in Enzymology, 237 and 238 (1994) of volume with And 96 (1983) of volume；Bourne et al., Nature, 10:349:117-27(1991)；Bourne et al., Nature, 348:125- 32(1990)；Pitcher et al., Annu.Rev.Biochem., 67:653-92(1998).
By the response for the T1R polypeptides that will be handled through the T1R conditioning agents of presumption with untreated control sample or comprising Know " positive " response of control sample makes comparisons, to analyze T1R adjustment effects.The T1R conditioning agents of this kind of presumption may include Inhibit or activate the molecule of T1R polypeptide actives.In one embodiment, by control sample (at activator or inhibitor Reason) opposite T1R activity values be set to 100.If the T1R activity values relative to control are about 90%, optionally 50%, optionally 25-0% then obtains the inhibition of T1R polypeptides.If relative to control T1R activity values be 110%, optionally 150%, 200- 500% or 1000-2000% then obtains the activation of T1R polypeptides.
Ionic polarization (i.e. potential) variation that the cell or film of T1R polypeptides are expressed by measurement, can evaluate ionic flux Variation.A method for measuring polarization variation is to utilize voltage-pincers (voltage-clamp) and patch-pincers (patch- Clamp) technology measures curent change (to detect polarized variation) (for example, see " cell attachment " pattern, " interior-outer " mould Formula and " full cell " pattern, such as Ackerman et al., New Engl.J Med., 336:1575-1595(1997)).Utilize mark Quasi- method can easily measure full cell currents.Other known analysis includes：Radiolabeled ion fluxes analysis with And use the fluorescence analysis of voltage sensitive dyes (for example, see Vestergarrd-Bogind et al., J.Membrane Biol.,88:67-75(1988)；Gonzales&Tsien,Chem.Biol.,4:269-277(1997)；Daniel et al., J.Pharmacol.Meth.,25:185-193(1991)；Holevinsky et al., J.Membrane Biol., 137:59-70 (1994))。
By detecting any of the above parameters, effect of the untested compound to polypeptide function can be measured.It can use The active any physiology variations appropriate of GPCR are influenced to evaluate influence of the untested compound to polypeptide of the present invention.When having used When whole cell or animal determine function result, various effects can also be measured, such as the release of tramsmitter release, hormone, known or not Identify transcription variation (such as Northern traces), the cell metabolism variation (such as cell growth or pH variation) of genetic marker with And intracellular second messenger such as Ca2+, IP3, cGMP or cAMP variation.
Preferred GPCR analyses include with ion or voltage sensitive dyes with the cell of report receptor activity.Measure this Receptoroid is active to analyze the known agonist that other g protein coupled receptors can also be used and antagonist as a contrast, to evaluate The activity of untested compound.In the analysis of identification modulating compound (such as agonist, antagonist), use ion quick respectively Cytoplasm intermediate ion is horizontal or the variation of film potential to monitor for perception or film potential fluorescence indicator.The ion-sensitive that may be used Indicator and voltage probe, which disclose, sees Molecular Probes 1997Catalog.It is mixed to dwell (promiscuous) G-protein such as Gα15With G α16It can be used for selection analysis g protein coupled receptor (Wilkie et al., Proc.Natl.Acad.Sci., 88:10049- 10053(1991))。
Receptor activation starts subsequent intracellular events, such as second messenger increases.The activation of certain g protein coupled receptors By phospholipase C mediate phosphatidylinositols hydrolysis stimulate to be formed InsP3 (IP3) (Berridge＆Irvine, Nature,312:315-21(1984)).IP3 then stimulates the release of intracellular calcium inventory.Therefore, cytoplasmic calcium ionized water The variation of flat variation or second messenger such as IP3 levels can be used for evaluating the function of g protein coupled receptor.Due to intracellular library It deposits the release of calcium and effect that extracellular Ca2＋ is entered by plasma membrane ion channels, expresses the cell of such g protein coupled receptor It may show that calcium ion level improves in cytoplasm.
In preferred embodiments, by stablize in heterologous cells or it is instantaneous, preferably stablize coexpression T1R genes and Receptor can be connected to the mixed G-protein of dwelling of phospholipase C signal pathway, can measure T1R polypeptides activity (referring to Offermanns&Simon,J.Biol.Chem.,270:15175-15180(1995)).In preferred embodiments, the cell System is HEK-293 (it does not express T1R genes usually), which is G α15(Offermanns＆Simon, ibid).Cell Interior Ca2+Level can respond by giving the T1R signal transduction paths adjusted in combination with the molecule of T1R polypeptides and change, and pass through survey Measure intracellular Ca2+Horizontal variation can analyze the adjusting of taste transduction.Optionally use fluorescence Ca2+Indicator dye and fluorescence Imaging measures Ca2+It is horizontal.
In another embodiment, phosphatidylinositols (PI) hydrolysis can be analyzed according to United States Patent (USP) 5,436,128, It is hereby incorporated by reference.In brief, which includes using3H- inositols mark cell 48 hours or the longer time.Label Cell is handled 1 hour using untested compound.It is extracted by the cell cracking of processing and with chloroform-methanol-water, thereafter by ion Displacement chromatography detaches phosphoinositide, quantitative by scinticounting.It calculates there are cpm when agonist and there are when buffer control Cpm ratio, determine stimulation multiple.Equally, it calculates there are cpm when antagonist and there are buffer controls (may contain Or do not contain agonist) when cpm ratio, determine inhibit multiple.
Other receptor assays may include the level for measuring Levels of Intracellular Cyclic Nucleotides, such as cAMP or cGMP.In receptor activation In the case of causing levels of cyclic nucleotides to reduce, it may be preferred to, will before cell is added in receptor-activating compound in analysis Cell is exposed in the reagent that can increase Levels of Intracellular Cyclic Nucleotides level such as Forskolin (Forskolin).In an embodiment party In case, the variation of immune analysis determination intracellular cAMP or cGMP can be used.Offermanns&Simon, J.Biol.Chem.,270:15175-15180 (1995) the method can be used for measuring cAMP horizontal.Equally, Felley- Bosco et al., Am.J.Resp.Cell and Mol.Biol., 11:159-164 (1994) the method can be used for measuring CGMP is horizontal.In addition, United States Patent (USP) 4,115,538 describe measure cAMP and/or cGMP assay kit, be hereby incorporated with For reference.
In another embodiment, transcriptional level can be measured, to evaluate effect of the untested compound to signal transduction.It will Including the host cell of purpose T1R polypeptides contacts the sufficiently long time with untested compound, to realize any interaction, then Measure gene expression dose.The time quantum for realizing these interactions can be empirically determined, such as execute a time course And measure function of the transcriptional level as the time.Proper method well known by persons skilled in the art can be used to measure transcription amount. It is, for example, possible to use the mRNA expression of Northern trace testing goal albumen, or identify its polypeptide product using immunoassay. Alternatively, the analysis based on transcription using reporter gene can be used, such as United States Patent (USP) 5, described in 436,128, be hereby incorporated with For reference.Reporter gene can be, for example, chloramphenicol acetyltransferase, luciferase, beta galactosidase, beta-lactamase and Alkaline phosphatase.In addition, by being attached to the second report molecule such as green fluorescent protein, it is informed of a case between destination protein being used as Accuse molecule (for example, see, Mistili＆Spector, Nature Biotechnology, 15:961-964(1997)).
Then transcription amount is compared with transcription amount of the same cell when lacking untested compound, or with it is essentially identical Transcription amount of the cell when lacking purpose T1R polypeptides compares.Essentially identical cell can derive from Prepare restructuring cell Same cell, but be not introduced into allogeneic dna sequence DNA and modified.Any difference of transcription amount means untested compound in some sense Change the activity of purpose T1R polypeptides.
4. expressing the non-human transgenic animal of chemosensory receptor
The non-human animal of expression T1R taste receptor sequences combination of the present invention can also be used for receptor assay.By that will stablize Or the non-human animal of transient transfection encoding chemosensory receptors or its ligand binding domain nucleic acid contacts with untested compound, and really Whether the fixed animal is reacted by specifically binding receptor polypeptide complex with untested compound, so as to use this kind of table Whether up to determining untested compound mammalian taste transmembrane receptor complex is specifically bound in vivo,
It can be used for identifying using carrier of the present invention transfected or infected animal and characterize in combination with specific or receptor in groups In the analysis of taste stimulus.This kind of animal for being infected through carrier, can expressing mankind's taste receptor sequences, can be used for screening in vivo Taste stimulus and its to such as stechiology (such as to sense of taste neuron), CNS or the effect of behavior.In addition, expression T1R Or combinations thereof stable cell lines can be used as nucleic acid donor, stablize the transgene clone for expressing specific T1R or combinations thereof to generate Animal.The method that the cloned animal of expression purpose exogenous DNA is generated using nucleic acid transfer technique, is to authorize University of Massachusetts (licensing to Advanced Cell Technology, Inc.) and Roslin Institute (are licensed to Geron Corp.) several mandates United States Patent (USP) theme.
The method of infection/express nucleic acid and carrier is well known in the art in the form of independent or library.It can utilize a variety of Method measures the parameter of various individual cells, organ or whole animal.By using infectious agent, such as adenovirus expression carrier It transports, the T1R sequences of the present invention can be co-expressed in such as animal taste tissues.
Endogenous gustatory receptor genes can keep functional, and still (natural) activity of performance wild type.In other situations Under, if it is desired to all taste receptor activities are generated by introducing external source hybrid receptors, it is preferable to use knocking out system.It builds non- Non-human transgenic animal, particularly transgenic mice, and select and prepare generate transformed cells recombinant precursor method for It is well known in the art.
Structure " knockout " cell and animal according to premise be, by that will be used to interfere gene part DNA sequences to be suppressed The new DNA sequence dna of row is introduced into genome, can reduce or completely eliminate the expression of specific gene in mammalian cell. Equally, " gene trap insertion " (gene trap insertion) can be used for destroying host gene, mouse embryonic stem (ES) cell It can be used for generating the transgenic animals knocked out (for example, see Holzschu, Transgenic Res.6:97-106(1997)). The insertion of exogenous array is usually carried out using the homologous recombination between complementary nucleic acid sequences.Exogenous array is certain of target gene to be finished A part, such as exon, introne or transcriptional regulatory sequences, or any genome sequence of expression of target gene level can be influenced Row；Or combinations thereof.Pluripotent embryonic stem cells carry out gene targeting by homologous recombination, can accurately modify target gene group sequence Row.Any technology can be used in generating, screening, breed knock-out animal, for example, see Bijvoet, Hum.Mol.Genet.7:53- 62(1998)；Moreadith,J.Mol.Med.75:208-216(1997)；Tojo,Cytotechnology 19:161-165 (1995)；Mudgett,Methods Mol.Biol.48:167-184(1995)；Longo,Transgenic Res.6:321- 328(1997)；United States Patent (USP) 5,616,491；5,464,764；5,631,153；5,487,992；5,627,059；5,272, 071；WO 91/09955；WO 93/09222；WO 96/29411；WO 95/31560；WO 91/12650.
The nucleic acid of the present invention also is used as generating " knockout " people's cell and its reagent of offspring.Similarly, core of the invention Acid also is used as generating the reagent of mouse " knocking in (knock-ins) ".People or rat T1R gene orders can replace murine genes Orthologous T1R in group.The mouse of expression people or rat T1R can be generated by means of which.The mouse then can Function for analyzing people or rat T1R, and identify the ligand of this kind of T1R.
A. regulator
The compound tested as T1R family member's conditioning agents can be any small chemical compound or biology Learn entity, such as protein, nucleic acid or lipid.The example includes 5 ' IMP and 5 ' GMP.Substantially any chemical compound all may be used Potential conditioning agent or ligand in being analyzed for use as the present invention, although most often test dissolves in the compound in aqueous solution.This A little analyses can be designed to through automated analysis step and provide from any compound for facilitating source, carry out big change Learn the screening in library；These analysis usually it is parallel carry out (for example, robot analysis in, on microtiter plate with micro drop Determine mode to carry out).It should be appreciated that one of a variety of chemical reactions (such as Senomyx proprietary can be utilized Chemistries chemistry library) is synthesized.In addition also have many chemical compound suppliers, including Sigma (St.Louis, MO), Aldrich(St.Louis,MO)、Sigma-Aldrich(St.Louis,MO)、Fluka Chemika-Biochemica Analytika (Buchs, Switzerland) etc..
In a preferred embodiment, high-throughput screening method includes providing comprising potentially large number of organoleptic effect chemical combination The combination chemistries or peptide library of object (potential conditioning agent or ligand compound).Then it is screened in above-mentioned one or more analyses This kind of " combination chemistries library " or " ligand library " show that the library constructs of required characteristic activities (especially change with identification Learn type or subclass).The compound identified in this way can be used as conventional " lead compound (lead compounds) " or its Body can be used as possible or actual taste modulator.
Preferred pin to stablize expression T1R or T1R combinations, i.e. T1R1/T1R3 or T1R2/T1R3 and preferably suitably G-protein, Such as G α15Cell or cell line, screen this kind of library.Shown in following article embodiment, the cell line of this kind of stabilization pierces the sense of taste Swash object, such as delicate flavour is felt or sweet taste stimuli shows the response of highly significant.However, the one or more T1R of transient expression Cell and cell line can also be used in this alanysis.
Combination chemistries library is the set of the different chemical compounds generated by chemical synthesis or biosynthesis, through a large amount of Chemical " structural member (building blocks) " such as reagent is composed.For example, linear combinatorial chemical library, such as Polypeptide libraries are by various possible modes by a series of chemical building blocks (amino acid) with appointed compound length (i.e. polypeptide Close the number of the amino acid in object) it is composed.By the combined hybrid of this chemical building blocks, can synthesize on thousands of Million kinds of chemical compounds.
The preparation and screening in combination chemistries library are known to those skilled in the art.This kind of combination chemistries library packet Include but be not limited to peptide library (for example, see, United States Patent (USP) 5,010,175, Furka, Int.J.Pept.Prot.Res., 37:487-493 (1991) and Houghton et al., Nature, 354:84-88(1991)).Generation chemistry can also be used more Other chemical substances in sample library.These chemical substances include but are not limited to：Class peptide (peptoid) (such as PCT is public Open WO 91/19735), the peptide (such as PCT Publication WO 93/20242) of coding, random biological oligomer (PCT Publication WO 92/ 00091), benzodiazepine (such as United States Patent (USP) 5,288,514), heteropolymer (diversomers) such as hydantoins and benzene And diaza and dipeptides (Hubbs et al., Proc.Nat.Acad.Sci., 90:6906-6913 (1993)), bivinyl (vinylogous) polypeptide (Hagihara et al., J.Amer.Chem.Soc., 114:6568 (1992)), have glucose holder Non-peptide property peptide mimics (nonpeptidal petidomimetics) (Hirschmann et al., J.Amer.Chem.Soc., 114:9217-9218 (1992)), similar organic synthesis small molecule compound libraries (Chen et al., J.Amer.Chem.Soc.,116:2661 (1994)), few carbamate (oligocarbamates) (Cho et al., Science,261:1303 (1993)), peptidyl phosphonate ester (Campbell et al., J.Org.Chem., 59:658 (1994)), core Sour library (Ausubel, Berger and Sambrook are same as above), peptide nucleic acid library (United States Patent (USP) 5,539,083), antibody text Library (Vaughn et al., Nature Biotechnology, 14 (3):309-314 (1996) and PCT/US96/10287), carbohydrate Library (Liang et al., Science, 274:1520-1522 (1996) and United States Patent (USP) 5,593,853), organic molecule library (benzodiazepine, Baum, C＆EN, Jan page 18,33 (1993)；Thiazolidone (thiazolidinones) and thiazine alkanone (metathiazanones), United States Patent (USP) 5,549,974；Pynrolidines, United States Patent (USP) 5,525,735 and 5,519, 134；Morpholino compounds, United States Patent (USP) 5,506,337；Benzodiazepine, 5,288,514 etc.).
The device for preparing combinatorial libraries is commercially available (for example, see 357MPS, 390MPS (Advanced Chem Tech,Louisville KY),Symphony(Rainin,Woburn,MA),433A(Applied Biosystems,Foster City,CA),9050Plus(Millipore,Bedford,MA)).In addition, numerous combinatorial libraries itself are also commercially available (for example, see ComGenex, Princeton, NJ；Tripos,Inc.,St.Louis,MO；3D Pharmaceuticals, Exton,PA；Martek Biosciences；Columbia, MD etc.).
In one aspect of the invention, T1R conditioning agents can be used for any food, candy, pharmaceutical composition or its ingredient, from And the taste of product, composition or ingredient is adjusted in desired manner.For example, the T1R tune that can enhance sweet tea taste perception can be added Agent is saved, product or composition is made to sweeten, and the T1R conditioning agents that can enhance fresh taste perception are added in food, can be increased delicious Taste.Further, it is possible to use T1R Antagonist blocks sweet taste and/or delicate flavour are felt.
B. kit
T1R genes and its autoploid are identification chemosensory receptor cell, medical jurisprudence and paternity test and the inspection sense of taste The useful tool of conduction.The T1R family member's specific reagents that can hybridize with T1R nucleic acid specificities, such as T1R probes and draw Object, and the T1R specific reagents of T1R polypeptides, such as T1R antibody can be specifically bound, it can be used for examining gustatory table It is adjusted up to taste transduction.
Determine in sample there are the foranalysis of nucleic acids of T1R family members DNA and RNA to include crowd well known by persons skilled in the art More technologies, for example, Southern analysis, Northern analysis, point trace, RNA enzyme protection, S1 analyses, amplification technique such as PCR, And in situ hybridization.Such as in hybridizing in situ, target nucleic acid is released from its intracellular environment, so as in cell Inside hybridized, while keeping cellular morphology, for subsequent explanation and analysis.Following article provides the general of hybridization in situ technique Condition：Singer et al., Biotechniques, 4:230-250(1986)；Haase et al., Methods in Virology, volume VII189-226 pages (1984)；And Nucleic Acid Hybridization:A Practical Approach(Names Et al., eds.1987).Furthermore it is possible to detect T1R polypeptides using above-mentioned panimmunity analytical technology.Test sample is usually simultaneously It makes comparisons with positive control (for example, sample of expression recombination T1R polypeptides) and negative control.
The present invention also provides the kits for screening T1R family member's conditioning agents.The kit can be from being easy to obtain It is prepared in the material and reagent that obtain.For example, the kit can include any one or more of llowing group of materials：T1R nucleic acid Or protein, reaction tube and the test active specifications of T1R.Optionally, the kit include biological activity T1R by Body can be stablized or the cell line of the taste receptors of transient expression T1R containing biological activity.It can be prepared according to the present invention more Kind kit and component, this depends on the specific user of the kit and the specific needs of user.
Embodiment
While the present invention as detailed above, following embodiment is provided and illustrates its preferred embodiment.These embodiments are anticipated In the range for illustrating, being not intended to limit the present invention.
Here in shown protein sequence, one-letter code X or Xaa refer to appointing in 20 kinds of common amino acid residues What is a kind of.Here in shown DNA sequence dna, one-letter code N or n refer to appointing in four kinds of common nucleotide bases A, T, C or G What is a kind of.
Embodiment 1
Prepare the hT1R expression constructs of intronless
The method based on cDNA and genomic DNA of combination, clones the hT1R expression constructs of intronless.It is complete in order to generate Long hT1R1 expression constructs encode two 5' that (accession number #AL159177) is identified in the intervals hT1R1 of clone outer Aobvious son is combined by PCR- overlappings, is then attached in the truncated TESTIS cDNA clones of 5'-.From the hT1R2 of part sequencing HT1R2 expression constructs are generated in genome interval.It is cloned using the probe screening for corresponding to coded sequence from rat The shotgun libraries (shotgun libraries) at genome interval identify the two hT1R2 5' exons lacked.Then pass through PCR- overlapping combinations encoded exons, generate the expression construct of overall length.It is overlapped between the hT1R3 genomes of sequencing by PCR- (accession number #AL139287) generates hT1R3 expression constructs in.Use what is generated by the degenerate pcr based on hT1R3 RT1R3 exon segments, rT1R3 is detached from the cDNA library in rat taste tissue source.From Dr.Charles Zuker (University of California, San Diego) obtains part hT1R1cDNA, rT1R2cDNA and part hT1R2 Sequence.
The following institute of nucleic acid and amino acid sequence of the T1R cloned sequences of above-mentioned identification and other overall lengths and fractional t1 R sequences Show：
SEQ ID NO：4
Amino acid sequence rT1R3
MPGLAILGLSLAAFLELGMGSSLCLSQQFKAQGDYILGGLFPLGTTEEATLNQRTQPNGILCTRFSPLG LFLAMAMKMAVEEINNGSALLPGLRLGYDLFDTCSEPVVTMKPSLMFMAKVGSQSIAAYCNYTQYQPRVLAVIGPHS SELALITGKFFSFFLMPQVSYSASMDRLSDRETFPSFFRTVPSDRVQLQAVVTLLQNFSWNWVAALGSDDDYGREGL SIFSGLANSRGICIAHEGLVPQHDTSGQQLGKVVDVLRQVNQSKVQVVVLFASARAVYSLFSYSILHDLSPKVWVAS ESWLTSDLVMTLPNIARVGTVLGFLQRGALLPEFSHYVETRLALAADPTFCASLKAELDLEERVMGPRCSQCDYIML QNLSSGLMQNLSAGQLHHQIFATYAAVYSVAQALHNTLQCNVSHCHTSEPVQPWQLLENMYNMSFRARDLTLQFDAK GSVDMEYDLKMWVWQSPTPVLHTVGTFNGTLQLQHSKMYWPGNQVPVSQCSRQCKDGQVRRVKGFHSCCYDCVDCKA GSYRKHPDDFTCTPCGKDQWSPEKSTTCLPRRPKFLAWGEPAVLSLLLLLCLVLGLTLAALGLFVHYWDSPLVQASG GSLFCFGLICLGLFCLSVLLFPGRPRSASCLAQQPMAHLPLTGCLSTLFLQAAEIFVESELPLSWANWLCSYLRGPW AWLVVLLATLVEAALCAWYLMAFPPEVVTDWQVLPTEVLEHCRMRSWVSLGLVHITNAVLAFLCFLGTFLVQSQPGR YNRARGLTFAMLAYFIIWVSFVPLLANVQVAYQPAVQMGAILFCALGILATFHLPKCYVLLWLPELNTQEFFLGRSP KEASDGNSGSSEATRGHSE
SEQ ID NO：5
Amino acid sequence hT1R1
MLLCTARLVGLQLLISCCWAFACHSTESSPDFTLPGDYLLAGLFPLHSGCLQVRHRPEVTLCDRSCSFN EHGYHLFQAMRLGVEEINNSTALLPNITLGYQLYDVCSDSANVYATLRVLSLPGQHHIELQGDLLHYSPTVLAVIGP DSTNRAATTAALLSPFLVPMISYAASSETLSVKRQYPSFLRTIPNDKYQVETMVLLLQKFGWTWISLVGSSDDYGQL GVQALENQATGQGICIAFKDIMPFSAQVGDERMQCLMRHLAQAGATVVVVFSSRQLARVFFESVVLTNLTGKVWVAS EAWALSRHITGVPGIQRIGMVLGVAIQKRAVPGLKAFEEAYARADKKAPRPCHKGSWCSSNQLCRECQAFMAHTMPK LKAFSMSSAYNAYRAVYAVAHGLHQLLGCASGACSRGRVYPWQLLEQIHKVHFLLHKDTVAFNDNRDPLSSYNIIAW DWNGPKWTFTVLGSSTWSPVQLNINETKIQWHGKDNQVPKSVCSSDCLEGHQRVVTGFHHCCFECVPCGAGTFLNKS DLYRCQPCGKEEWAPEGSQTCFPRTVVFLALREHTSWVLLAANTLLLLLLLGTAGLFAWHLDTPVVRSAGGRLCFLM LGSLAAGSGSLYGFFGEPTRPACLLRQALFALGFTIFLSCLTVRSFQLIIIFKFSTKVPTFYHAWVQNHGAGLFVMI SSAAQLLICLTWLVVWTPLPAREYQRFPHLVMLECTETNSLGFILAFLYNGLLSISAFACSYLGKDLPENYNEAKCV TFSLLFNFVSWIAFFTTASVYDGKYLPAANMMAGLSSLSSGFGGYFLPKCYVILCRPDLNSTEHPQASIQDYTRRCG ST
SEQ ID NO：6
Amino acid sequence hT1R2
MGPRAKTICSLFFLLWVLAEPAENSDFYLPGDYLLGGLFSLHANMKGIVHLNFLQVPMCKEYEVKVIGY NLMQAMRFAVEEINNDSSLLPGVLLGYEIVDVCYISNNVQPVLYFLAHEDNLLPIQEDYSNYISRVVAVIGPDNSES VMTVANFLSLFLLPQITYSAISDELRDKVRFPALLRTTPSADHHVEAMVQLMLHFRWNWIIVLVSSDTYGRDNGQLL GERVARRDICIAFQETLPTLQPNQNMTSEERQRLVTIVDKLQQSTARVVVVFSPDLTLYHFFNEVLRQNFTGAVWIA SESWAIDPVLHNLTELGHLGTFLGITIQSVPIPGFSEFREWGPQAGPPPLSRTSQSYTCNQECDNCLNATLSFNTIL RLSGERVVYSVYSAVYAVAHALHSLLGCDKSTCTKRVVYPWQLLEEIWKVNFTLLDHQIFFDPQGDVALHLEIVQWQ WDRSQNPFQSVASYYPLQRQLKNIQDISWHTVNNTIPMSMCSKRCQSGQKKKPVGIHVCCFECIDCLPGTFLNHTED EYECQACPNNEWSYQSETSCFKRQLVFLEWHEAPTIAVALLAALGFLSTLAILVIFWRHFQTPIVRSAGGPMCFLML TLLLVAYMVVPVYVGPPKVSTCLCRQALFPLCFTICISCIAVRSFQIVCAFKMASRFPRAYSYWVRYQGPYVSMAFI TVLKMVIVVIGMLATGLSPTTRTDPDDPKITIVSCNPNYRNSLLFNTSLDLLLSVVGFSFAYMGKELPTNYNEAKFI TLSMTFYFTSSVSLCTFMSAYSGVLVTIVDLLVTVLNLLAISLGYFGPKCYMILFYPERNTPAYFNSMIQGYTMRRD
SEQ ID NO：7
Amino acid sequence hT1R3
MLGPAVLGLSLWALLHPGTGAPLCLSQQLRMKGDYVLGGLFPLGEAEEAGLRSRTRPSSPVCTRFSSNG LLWALAMKMAVEEINNKSDLLPGLRLGYDLFDTCSEPVVAMKPSLMFLAKAGSRDIAAYCNYTQYQPRVLAVIGPHS SELAMVTGKFFSFFLMPQVSYGASMELLSARETFPSFFRTVPSDRVQLTAAAELLQEFGWNWVAALGSDDEYGRQGL SIFSALAAARGICIAHEGLVPLPRADDSRLGKVQDVLHQVNQSSVQVVLLFASVHAAHALFNYSISSRLSPKVWVAS EAWLTSDLVMGLPGMAQMGTVLGFLQRGAQLHEFPQYVKTHLALATDPAFCSALGEREQGLEEDVVGQRCPQCDCIT LQNVSAGLNHHQTFSVYAAVYSVAQALHNTLQCNASGCPAQDPVKPWQLLENMYNLTFHVGGLPLRFDSSGNVDMEY DLKLWVWQGSVPRLHDVGRFNGSLRTERLKIRWHTSDNQKPVSRCSRQCQEGQVRRVKGFHSCCYDCVDCEAGSYRQ NPDDIACTFCGQDEWSPERSTRCFRRRSRFLAWGEPAVLLLLLLLSIALGLVLAALGLFVHHRDSPLVQASGGPLAC FGLVCLGLVCLSVLLFPGQPSPARCLAQQPLSHLPLTGCLSTLFLQAAEIFVESELPLSWADRLSGCLRGPWAWLVV LLAMLVEVALCTWYLVAFPPEVVTDWHMLPTEALVHCRTRSWVSFGLAHATNATLAFLCFLGTFLVRSQPGRYNRAR GLTFAMLAYFITWVSFVPLLANVQVVLRPAVQMGALLLCVLGILAAFHLPRCYLLMRQPGLNTPEFFLGGGPGDAQG QNDGNTGNQGKHE
SEQ ID NO：8
Nucleic acid sequence hT1R1
ATGCTGCTCTGCACGGCTCGCCTGGTCGGCCTGCAGCTTCTCATTTCCTGCTGCTGGGCCTTTGCCTGC CATAGCACGGAGTCTTCTCCTGACTTCACCCTCCCCGGAGATTACCTCCTGGCAGGCCTGTTCCCTCTCCATTCTGG CTGTCTGCAGGTGAGGCACAGACCCGAGGTGACCCTGTGTGACAGGTCTTGTAGCTTCAATGAGCATGGCTACCACC TCTTCCAGGCTATGCGGCTTGGGGTTGAGGAGATAAACAACTCCACGGCCCTGCTGCCCAACATCACCCTGGGGTAC CAGCTGTATGATGTGTGTTCTGACTCTGCCAATGTGTATGCCACGCTGAGAGTGCTCTCCCTGCCAGGGCAACACCA CATAGAGCTCCAAGGAGACCTTCTCCACTATTCCCCTACGGTGCTGGCAGTGATTGGGCCTGACAGCACCAACCGTG CTGCCACCACAGCCGCCCTGCTGAGCCCTTTCCTGGTGCCCATGATTAGCTATGCGGCCAGCAGCGAGACGCTCAGC GTGAAGCGGCAGTATCCCTCTTTCCTGCGCACCATCCCCAATGACAAGTACCAGGTGGAGACCATGGTGCTGCTGCT GCAGAAGTTCGGGTGGACCTGGATCTCTCTGGTTGGCAGCAGTGACGACTATGGGCAGCTAGGGGTGCAGGCACTGG AGAACCAGGCCACTGGTCAGGGGATCTGCATTGCTTTCAAGGACATCATGCCCTTCTCTGCCCAGGTGGGCGATGAG AGGATGCAGTGCCTCATGCGCCACCTGGCCCAGGCCGGGGCCACCGTCGTGGTTGTTTTTTCCAGCCGGCAGTTGGC CAGGGTGTTTTTCGAGTCCGTGGTGCTGACCAACCTGACTGGCAAGGTGTGGGTCGCCTCAGAAGCCTGGGCCCTCT CCAGGCACATCACTGGGGTGCCCGGGATCCAGCGCATTGGGATGGTGCTGGGCGTGGCCATCCAGAAGAGGGCTGTC CCTGGCCTGAAGGCGTTTGAAGAAGCCTATGCCCGGGCAGACAAGAAGGCCCCTAGGCCTTGCCACAAGGGCTCCTG GTGCAGCAGCAATCAGCTCTGCAGAGAATGCCAAGCTTTCATGGCACACACGATGCCCAAGCTCAAAGCCTTCTCCA TGAGTTCTGCCTACAACGCATACCGGGCTGTGTATGCGGTGGCCCATGGCCTCCACCAGCTCCTGGGCTGTGCCTCT GGAGCTTGTTCCAGGGGCCGAGTCTACCCCTGGCAGCTTTTGGAGCAGATCCACAAGGTGCATTTCCTTCTACACAA GGACACTGTGGCGTTTAATGACAACAGAGATCCCCTCAGTAGCTATAACATAATTGCCTGGGACTGGAATGGACCCA AGTGGACCTTCACGGTCCTCGGTTCCTCCACATGGTCTCCAGTTCAGCTAAACATAAATGAGACCAAAATCCAGTGG CACGGAAAGGACAACCAGGTGCCTAAGTCTGTGTGTTCCAGCGACTGTCTTGAAGGGCACCAGCGAGTGGTTACGGG TTTCCATCACTGCTGCTTTGAGTGTGTGCCCTGTGGGGCTGGGACCTTCCTCAACAAGAGTGACCTCTACAGATGCC AGCCTTGTGGGAAAGAAGAGTGGGCACCTGAGGGAAGCCAGACCTGCTTCCCGCGCACTGTGGTGTTTTTGGCTTTG CGTGAGCACACCTCTTGGGTGCTGCTGGCAGCTAACACGCTGCTGCTGCTGCTGCTGCTTGGGACTGCTGGCCTGTT TGCCTGGCACCTAGACACCCCTGTGGTGAGGTCAGCAGGGGGCCGCCTGTGCTTTCTTATGCTGGGCTCCCTGGCAG CAGGTAGTGGCAGCCTCTATGGCTTCTTTGGGGAACCCACAAGGCCTGCGTGCTTGCTACGCCAGGCCCTCTTTGCC CTTGGTTTCACCATCTTCCTGTCCTGCCTGACAGTTCGCTCATTCCAACTAATCATCATCTTCAAGTTTTCCACCAA GGTACCTACATTCTACCACGCCTGGGTCCAAAACCACGGTGCTGGCCTGTTTGTGATGATCAGCTCAGCGGCCCAGC TGCTTATCTGTCTAACTTGGCTGGTGGTGTGGACCCCACTGCCTGCTAGGGAATACCAGCGCTTCCCCCATCTGGTG ATGCTTGAGTGCACAGAGACCAACTCCCTGGGCTTCATACTGGCCTTCCTCTACAATGGCCTCCTCTCCATCAGTGC CTTTGCCTGCAGCTACCTGGGTAAGGACTTGCCAGAGAACTACAACGAGGCCAAATGTGTCACCTTCAGCCTGCTCT TCAACTTCGTGTCCTGGATCGCCTTCTTCACCACGGCCAGCGTCTACGACGGCAAGTACCTGCCTGCGGCCAACATG ATGGCTGGGCTGAGCAGCCTGAGCAGCGGCTTCGGTGGGTATTTTCTGCCTAAGTGCTACGTGATCCTCTGCCGCCC AGACCTCAACAGCACAGAGCACTTCCAGGCCTCCATTCAGGACTACACGAGGCGCTGCGGCTCCACCTGA
SEQ ID NO 9
Nucleic acid sequence hT1R3
ATGCTGGGCCCTGCTGTCCTGGGCCTCAGCCTCTGGGCTCTCCTGCACCCTGGGACGGGGGCCCCATTGTGCCTGTC ACAGCAACTTAGGATGAAGGGGGACTACGTGCTGGGGGGGCTGTTCCCCCTGGGCGAGGCCGAGGAGGCTGGCCTCC GCAGCCGGACACGGCCCAGCAGCCCTGTGTGCACCAGGTTCTCCTCAAACGGCCTGCTCTGGGCACTGGCCATGAAA ATGGCCGTGGAGGAGATCAACAACAAGTCGGATCTGCTGCCCGGGCTGCGCCTGGGCTACGACCTCTTTGATACGTG CTCGGAGCCTGTGGTGGCCATGAAGCCCAGCCTCATGTTCCTGGCCAAGGCAGGCAGCCGCGACATCGCCGCCTACT GCAACTACACGCAGTACCAGCCCCGTGTGCTGGCTGTCATCGGGCCCCACTCGTCAGAGCTCGCCATGGTCACCGGC AAGTTCTTCAGCTTCTTCCTCATGCCCCAggtcagCTACGGTGCTAGCATGGAGCTGCTGAGCGCCCGGGAGACCTT CCCCTCCTTCTTCCGCACCGTGCCCAGCGACCGTGTGCAGCTGACGGCCGCCGCGGAGCTGCTGCAGGAGTTCGGCT GGAACTGGGTGGCCGCCCTGGGCAGCGACGACGAGTACGGCCGGCAGGGCCTGAGCATCTTCTCGGCCCTGGCCGCG GCACGCGGCATCTGCATCGCGCACGAGGGCCTGGTGCCGCTGCCCCGTGCCGATGACTCGCGGCTGGGGAAGGTGCA GGACGTCCTGCACCAGGTGAACCAGAGCAGCGTGCAGGTGGTGCTGCTGTTCGCCTCCGTGCACGCCGCCCACGCCC TCTTCAACTACAGCATCAGCAGCAGGCTCTCGCCCAAGGTGTGGGTGGCCAGCGAGGCCTGGCTGACCTCTGACCTG GTCATGGGGCTGCCCGGCATGGCCCAGATGGGCACGGTGCTTGGCTTCCTCCAGAGGGGTGCCCAGCTGCACGAGTT CCCCCAGTACGTGAAGACGCACCTGGCCCTGGCCACCGACCCGGCCTTCTGCTCTGCCCTGGGCGAGAGGGAGCAGG GTCTGGAGGAGGACGTGGTGGGCCAGCGCTGCCCGCAGTGTGACTGCATCACGCTGCAGAACGTGAGCGCAGGGCTA AATCACCACCAGACGTTCTCTGTCTACGCAGCTGTGTATAGCGTGGCCCAGGCCCTGCACAACACTCTTCAGTGCAA CGCCTCAGGCTGCCCCGCGCAGGACCCCGTGAAGCCCTGGCAGCTCCTGGAGAACATGTACAACCTGACCTTCCACG TGGGCGGGCTGCCGCTGCGGTTCGACAGCAGCGGAAACGTGGACATGGAGTACGACCTGAAGCTGTGGGTGTGGCAG GGCTCAGTGCCCAGGCTCCACGACGTGGGCAGGTTCAACGGCAGCCTCAGGACAGAGCGCCTGAAGATCCGCTGGCA CACGTCTGACAACCAGAAGCCCGTGTCCCGGTGCTCGCGGCAGTGCCAGGAGGGCCAGGTGCGCCGGGTCAAGGGGT TCCACTCCTGCTGCTACGACTGTGTGGACTGCGAGGCGGGCAGCTACCGGCAAAACCCAGACGACATCGCCTGCACC TTTTGTGGCCAGGATGAGTGGTCCCCGGAGCGAAGCACACGCTGCTTCCGCCGCAGGTCTCGGTTCCTGGCATGGGG CGAGCCGGCTGTGCTGCTGCTGCTCCTGCTGCTGAGCCTGGCGCTGGGCCTTGTGCTGGCTGCTTTGGGGCTGTTCG TTCACCATCGGGACAGCCCACTGGTTCAGGCCTCGGGGGGGCCCCTGGCCTGCTTTGGCCTGGTGTGCCTGGGCCTG GTCTGCCTCAGCGTCCTCCTGTTCCCTGGCCAGCCCAGCCCTGCCCGATGCCTGGCCCAGCAGCCCTTGTCCCACCT CCCGCTCACGGGCTGCCTGAGCACACTCTTCCTGCAGGCGGCCGAGATCTTCGTGGAGTCAGAACTGCCTCTGAGCT GGGCAGACCGGCTGAGTGGCTGCCTGCGGGGGCCCTGGGCCTGGCTGGTGGTGCTGCTGGCCATGCTGGTGGAGGTC GCACTGTGCACCTGGTACCTGGTGGCCTTCCCGCCGGAGGTGGTGACGGACTGGCACATGCTGCCCACGGAGGCGCT GGTGCACTGCCGCACACGCTCCTGGGTCAGCTTCGGCCTAGCGCACGCCACCAATGCCACGCTGGCCTTTCTCTGCT TCCTGGGCACTTTCCTGGTGCGGAGCCAGCCGGGCTGCTACAACCGTGCCCGTGGCCTCACCTTTGCCATGCTGGCC TACTTCATCACCTGGGTCTCCTTTGTGCCCCTCCTGGCCAATGTGCAGGTGGTCCTCAGGCCCGCCGTGCAGATGGG CGCCCTCCTGCTCTGTGTCCTGGGCATCCTGGCTGCCTTCCACCTGCCCAGGTGTTACCTGCTCATGCGGCAGCCAG GGCTCAACACCCCCGAGTTCTTCCTGGGAGGGGGCCCTGGGGATGCCCAAGGCCAGAATGACGGGAACACAGGAAAT CAGGGGAAACATGAGTGA
SEQ ID NO：10
Nucleic acid sequence hT1R2
ATGGGGCCCAGGGCAAAGACCATCTGCTCCCTGTTCTTCCTCCTATGGGTCCTGGCTGAGCCGGCTGAG AACTCGGACTTCTACCTGCCTGGGGATTACCTCCTGGGTGGCCTCTTCTCCCTCCATGCCAACATGAAGGGCATTGT TCACCTTAACTTCCTGCAGGTGCCCATGTGCAAGGAGTATGAAGTGAAGGTGATAGGCTACAACCTCATGCAGGCCA TGCGCTTCGCGGTGGAGGAGATCAACAATGACAGCAGCCTGCTGCCTGGTGTGCTGCTGGGCTATGAGATCGTGGAT GTGTGCTACATCTCCAACAATGTCCAGCCGGTGCTCTACTTCCTGGCACACGAGGACAACCTCCTTCCCATCCAAGA GGACTACAGTAACTACATTTCCCGTGTGGTGGCTGTCATTGGCCCTGACAACTCCGAGTCTGTCATGACTGTGGCCA ACTTCCTCTCCCTATTTCTCCTTCCACAGATCACCTACAGCGCCATCAGCGATGAGCTGCGAGACAAGGTGCGCTTC CCGGCTTTGCTGCGTACCACACCCAGCGCCGACCACCACGTCGAGGCCATGGTGCAGCTGATGCTGCACTTCCGCTG GAACTGGATCATTGTGCTGGTGAGCAGCGACACCTATGGCCGCGACAATGGCAGCTGCTTGGCGAGCGCGTGGCCCG GCGCGACATCTGCATCGCCTTCCAGGAGACGCTGCCCACACTGCAGCCCAACCAGAACATGACGTCAGAGGAGCGCC AGCGCCTGGTGACCATTGTGGACAAGCTGCAGCAGAGCACAGCGCGCGTCGTGGTCGTGTTCTCGCCCGACCTGACC CTGTACCACTTCTTCAATGAGGTGCTGCGCCAGAACTTCACGGGCGCCGTGTGGATCGCCTCCGAGTCCTGGGCCAT CGACCCGGTCCTGCACAACCTCACGGAGCTGGGCCACTTGGGCACCTTCCTGGGCATCACCATCCAGAGCGTGCCCA TCCCGGGCTTCAGTGAGTTCCGCGAGTGGGGCCCACAGGCTGGGCCGCCACCCCTCAGCAGGACCAGCCAGAGCTAT ACCTGCAACCAGGAGTGCGACAACTGCCTGAACGCCACCTTGTCCTTCAACACCATTCTCAGGCTCTCTGGGGAGCG TGTCGTCTACAGCGTGTACTCTGCGGTCTATGCTGTGGCCCATGCCCTGCACAGCCTCCTCGGCTGTGACAAAAGCA CCTGCACCAAGAGGGTGGTCTACCCCTGGCAGCTGCTTGAGGAGATCTGGAAGGTCAACTTCACTCTCCTGGACCAC CAAATCTTCTTCGACCCGCAAGGGGACGTGGCTCTGCACTTGGAGATTGTCCAGTGGCAATGGGACCGGAGCCAGAA TCCCTTCCAGAGCGTCGCCTCCTACTACCCCCTGCAGCGACAGCTGAAGAACATCCAAGACATCTCCTGGCACACCG TCAACAACACGATCCCTATGTCCATGTGTTCCAAGAGGTGCCAGTCAGGGCAAAAGAAGAAGCCTGTGGGCATCCAC GTCTGCTGCTTCGAGTGCATCGACTGCCTTCCCGGCACCTTCCTCAACCACACTGAAGATGAATATGAATGCCAGGC CTGCCCGAATAACGAGTGGTCCTACCAGAGTGAGACCTCCTGCTTCAAGCGGCAGCTGGTCTTCCTGGAATGGCATG AGGCACCCACCATCGCTGTGGCCCTGCTGGCCGCCCTGGGCTTCCTCAGCACCCTGGCCATCCTGGTGATATTCTGG AGGCACTTCCAGACACCCATAGTTCGCTCGGCTGGGGGCCCCATGTGCTTCCTGATGCTGACACTGCTGCTGGTGGC ATACATGGTGGTCCCGGTGTACGTGGGGCCGCCCAAGGTCTCCACCTGCCTCTGCCGCCAGGCCCTCTTTCCCCTCT GCTTCACAATTTGCATCTCCTGTATCGCCGTGCGTTCTTTCCAGATCGTCTGCGCCTTCAAGATGGCCAGCCGCTTC CCACGCGCCTACAGCTACTGGGTCCGCTACCAGGGGCCCTACGTCTCTATGGCATTTATCACGGTACTCAAAATGGT CATTGTGGTAATTGGCATGCTGGCCACGGGCCTCAGTCCCACCACCCGTACTGACCCCGATGACCCCAAGATCACAA TTGTCTCCTGTAACCCCAACTACCGCAACAGCCTGCTGTTCAACACCAGCCTGGACCTGCTGCTCTCAGTGGTGGGT TTCAGCTTCGCCTACATGGGCAAAGAGCTGCCCACCAACTACAACGAGGCCAAGTTCATCACCCTCAGCATGACCTT CTATTTCACCTCATCCGTCTCCCTCTGCACCTTCATGTCTGCCTACAGCGGGGTGCTGGTCACCATCGTGGACCTCT TGGTCACTGTGCTCAACCTCCTGGCCATCAGCCTGGGCTACTTCGGCCCCAAGTGCTACATGATCCTCTTCTACCCG GAGCGCAACACGCCCGCCTACTTCAACAGCATGATCCAGGGCTACACCATGAGGAGGGACTAG
SEQ ID NO 11
Nucleic acid sequence rT1R3
ATGCCGGGTTTGGCTATCTTGGGCCTCAGTCTGGCTGCTTTCCTGGAGCTTGGGATGGGGTCCTCTTTG TGTCTGTCACAGCAATTCAAGGCACAAGGGGACTATATATTGGGTGGACTATTTCCCCTGGGCACAACTGAGGAGGC CACTCTCAACCAGAGAACACAGCCCAACGGCATCCTATGTACCAGGTTCTCGCCCCTTGGTTTGTTCCTGGCCATGG CTATGAAGATGGCTGTAGAGGAGATCAACAATGGATCTGCCTTGCTCCCTGGGCTGCGACTGGGCTATGACCTGTTT GACACATGCTCAGAGCCAGTGGTCACCATGAAGCCCAGCCTCATGTTCATGGCCAAGGTGGGAAGTCAAAGCATTGC TGCCTACTGCAACTACACACAGTACCAACCCCGTGTGCTGGCTGTCATTGGTCCCCACTCATCAGAGCTTGCCCTCA TTACAGGCAAGTTCTTCAGCTTCTTCCTCATGCCACAGGTCAGCTATAGTGCCAGCATGGATCGGCTAAGTGACCGG GAAACATTTCCATCCTTCTTCCGCACAGTGCCCAGTGACCGGGTGCAGCTGCAGGCCGTTGTGACACTGTTGCAGAA TTTCAGCTGGAACTGGGTGGCTGCCTTAGGTAGTGATGATGACTATGGCCGGGAAGGTCTGAGCATCTTTTCTGGTC TGGCCAACTCACGAGGTATCTGCATTGCACACGAGGGCCTGGTGCCACAACATGACACTAGTGGCCAACAATTGGGC AAGGTGGTGGATGTGCTACGCCAAGTGAACCAAAGCAAAGTACAGGTGGTGGTGCTGTTTGCATCTGCCCGTGCTGT CTACTCCCTTTTTAGCTACAGCATCCTTCATGACCTCTCACCCAAGGTATGGGTGGCCAGTGAGTCCTGGCTGACCT CTGACCTGGTCATGACACTTCCCAATATTGCCCGTGTGGGCACTGTTCTTGGGTTTCTGCAGCGCGGTGCCCTACTG CCTGAATTTTCCCATTATGTGGAGACTCGCCTTGCCCTAGCTGCTGACCCAACATTCTGTGCCTCCCTGAAAGCTGA GTTGGATCTGGAGGAGCGCGTGATGGGGCCACGCTGTTCACAATGTGACTACATCATGCTACAGAACCTGTCATCTG GGCTGATGCAGAACCTATCAGCTGGGCAGTTGCACCACCAAATATTTGCAACCTATGCAGCTGTGTACAGTGTGGCT CAGGCCCTTCACAACACCCTGCAGTGCAATGTCTCACATTGCCACACATCAGAGCCTGTTCAACCCTGGCAGCTCCT GGAGAACATGTACAATATGAGTTTCCGTGCTCGAGACTTGACACTGCAGTTTGATGCCAAAGGGAGTGTAGACATGG AATATGACCTGAAGATGTGGGTGTGGCAGAGCCCTACACCTGTACTACATACTGTAGGCACCTTCAACGGCACCCTT CAGCTGCAGCACTCGAAAATGTATTGGCCAGGCAACCAGGTGCCAGTCTCCCAGTGCTCCCGGCAGTGCAAAGATGG CCAGGTGCGCAGAGTAAAGGGCTTTCATTCCTGCTGCTATGACTGTGTGGACTGCAAGGCAGGGAGCTACCGGAAGC ATCCAGATGACTTCACCTGTACTCCATGTGGCAAGGATCAGTGGTCCCCAGAAAAAAGCACAACCTGCTTACCTCGC AGGCCCAAGTTTCTGGCTTGGGGGGAGCCAGCTGTGCTGTCACTTCTCCTGCTGCTTTGCCTGGTGCTGGGCCTGAC ACTGGCTGCCCTGGGGCTCTTTGTCCACTACTGGGACAGCCCTCTTGTTCAGGCCTCAGGTGGGTCACTGTTCTGCT TTGGCCTGATCTGCCTAGGCCTCTTCTGCCTCAGTGTCCTTCTGTTCCCAGGACGACCACGCTCTGCCAGCTGCCTT GCCCAACAACCAATGGCTCACCTCCCTCTCACAGGCTGCCTGAGCACACTCTTCCTGCAAGCAGCCGAGATCTTTGT GGAGTCTGAGCTGCCACTGAGTTGGGCAAACTGGCTCTGCAGCTACCTTCGGGGCCCCTGGGCTTGGCTGGTGGTAC TGCTGGCCACTCTTGTGGAGGCTGCACTATGTGCCTGGTACTTGATGGCTTTCCCTCCAGAGGTGGTGACAGATTGG CAGGTGCTGCCCACGGAGGTACTGGAACACTGCCGCATGCGTTCCTGGGTCAGCCTGGGCTTGGTGCACATCACCAA TGCAGTGTTAGCTTTCCTCTGCTTTCTGGGCACTTTCCTGGTACAGAGCCAGCCTGGTCGCTATAACCGTGCCCGTG GCCTCACCTTCGCCATGCTAGCTTATTTCATCATCTGGGTCTCTTTTGTGCCCCTCCTGGCTAATGTGCAGGTGGCC TACCAGCCAGCTGTGCAGATGGGTGCTATCTTATTCTGTGCCCTGGGCATCCTGGCCACCTTCCACCTGCCCAATGC TATGTACTTCTGTGGCTGCCAGAGCTCAACACCCAGGAGTTCTTCCTGGGAAGGAGCCCCAAGGAAGCATCAGATGG GAATAGTGGTAGTAGTGAGGCAACTCGGGGACACAGTGAATGA
Equally, following conceptual translations are corresponding to the C-terminal of two orthologous gene pairs of fish T1R, source In the genome sequence that do not deliver, it is provided in this.Fugu T1RA derive from accession number ' scaffold164 '；Fugu T1RB come Derived from accession number LPC61711；Tetradon T1RA derive from accession number AL226735；Tetradon T1RB are from login Number AL222381.Ambiguity (ambiguity) (' X ') in conceptual translations comes from the ambiguity of database sequence.
SEQ ID NO 12
T1RA Fugu
PSPFRDIVSYPDKIILGCFMNLKTSSVSFVLLLLLCLLCFIFSYMGKDLPKNYNEAKAITFCLLLLILT WIIFTTASLLYQGKYIHSLNALAVLSSIYSFLLWYFLPKCYIIIFQPQKNTQKYFQGLIQDYTKTISQ
SEQ ID NO 13
T1RA Tetradon
FAVNYNTPVVRSAGGPMCFLILGCLSLCSISVFFYFERPTEAFCILRFMPFLLFYAVCLACFAVRSFQI VIIFKIAAKFPRVHSWWMKYHGQWLVISMTFVLQAVVIVIGFSSNPPLPYXXFVSYPDKIILGCDVNLNMASTSFFL LLLLCILCFTFSYMGKDLPKNYNEAKAITFCLLLLILTWIIFATAFMLYHGKYIHTLNALAVLSSAYCFLLWYFLPK CYIIIFQPHKNTQKYFQLS
SEQ ID NO 14
T1RB Fugu
KKQGPEVDIFIVSVTILCISVLGVAVGPPEPSQDLDFYMDSIVLECSNTLSPGSFIELCYVCVLSVLCF FFSYMGKDLPANYNEAKCVTFSLMVYMISWISFFTVYLISRGPFTVAAYVCATLVSVLAFFGGYFLPKIYIIVLKPQ MNTTAHFQNCIQMYTMSKQ
SEQ ID NO 15
T1RB Tetradon
APKSSQRXLRRTRLXLEWDHPMSVALLFFLVCCLLMTSSSAVILLLNINTPVAKSAGGXTCXLKLAALT AAAMSSXCHFGQPSPLASKLKQPQFTFSFTVCLACNRCALATGHLHFXIRVALPPAYNXWAKNHGPXATIFIASAAI LCVLCLRVAVGPPQPSQBLBFXTNSIXLXXSNTLSPGSFVELCNVSLLSAVCFVFSXMGKBLPANYNEAKCVTFSLM VNXISWISFFTVY
In addition, mouse and rat T1R and its relevant accession number of public structural domain allelic variant and reference are such as Shown in lower：
RT1R1 (accession number AAD18069) (Hoon et al., Cell 96 (4)：541-51(1999))；RT1R2 (accession number AAD18070) (Hoon et al., Cell 96 (4)：541-59(1999))；MT1R1 (accession number AAK39437)；MT1R2 (is logged in Number AAK 39438)；MT1R3 (accession number AAK 55537) (Max et al., Nat.Genet.28 (I)：58-63(2001))； RT1R1 (accession number AAK7092) (Li et al. people, Mamm.Genome (12 (I)：13-16(2001))；MT1R1 (accession number NP 114073)；MT1R1 (accession number AAK07091) (Li et al. people, Mamm.Genome (121):13-16(2001))；RT1R2 (is logged in Number AAD18070) (Hoon et al., Cell 9664):541-551(1999))；MT1R2 (accession number NP114079)；MT1R3 (is stepped on Record AAK39436)；MT1R3 (accession number BAB47181)；(Kitagawa et al., Biochem.Biophys.Res.Comm.283(1):236-42(2001))；MT1R3 (accession number NP114078)；MT1R3 (is stepped on Record AAK55536) (Max et al., Nat.Genet.28 (1):58-63(2001))；And mT1R3 (accession number AAK01937).
Embodiment 2
The sequence alignment of people and rat T1R
It is compared with the corresponding T1R of rat selected from above-mentioned identified T1R cloned sequences.As shown in Figure 1, hT1R1, people (rT1R1 and accession number that accession number is AAD18069 are AAD18070 with aforementioned T1R for T1R2 and hT1R3 and rT1R3 RT1R2), rat mGluR1 metabotropic glutamate receptors (accession number P23385)；And people's calcium sensitivity receptor (accession number P41180 it) is compared.Compare for clarity, mGluR1 and calcium sensitivity receptor C terminal are truncated.Blue box can for 7 The transmembrane segment of energy.Red boxes are the residue that glutamate side chain carbutylate is contacted in mGluR1 crystal structures, green Box is the residue for contacting glutamic acid a-amino acid part.Purple circle is the mGluR1 for participating in being formed based on intersubunit disulfide bonds With calcium sensitivity receptor cysteine residues.These cysteines are not guarded in T1R1 and T1R2, but under comparing Area is dropped, wherein comprising potential similar T1R3 cysteine residues, is also drawn in circle.
Embodiment 3
RT-PCR proves that hT1R2 and hT1R3 is expressed in taste tissue
As shown in Fig. 2, hT1R2 and hT1R3 are expressed in taste tissue：It is detected in people's profile needle biopsy using RT-PCR The expression of two genes.
Embodiment 4
The method of heterogenous expression T1R in heterologous cells
Stablize expression G α15HEK-293 derived cells (Chandrashekar et al., Cell100 (6):703-11 (2000)), in addition 10%FBS, MEM nonessential amino acid (Gibco BRL) and 3 μ g/ml blasticidins (blasticidin) Dulbecco's Modified Eagle Medium (DMEM, Gibco BRL) in grow and maintain.In calcium imaging experiment In, first by cell inoculation in 24 hole tissue culturing plates (about 0.1x106/ hole), pass through liposome transfection Mirus Transit-293(PanVera).To minimize the desensitized effect of glutamate induction and glucose induction, about 24 after transfection Hour replaces the DMEM being added using low glucose DMEM/GlutaMAX (Gibco BRL).After 24 hours, cell is added In Dulbecco's PBS buffer solution (DPBS, GibcoBRL) containing 3 μM of calcium dyestuff Fluo-4 (Molecular Probes), room Temperature 1.5 hours.After replacing 250 μ l DPBS, the 200 μ l DPBS containing taste stimulus are added, are stimulated at room temperature.It utilizes AxiovertS100TV microscopes (Zeiss) monitor the mobilization of calcium by 4.0 softwares of Imaging Workbench (Axon). The response of T1R1/T1R3 and T1R2/T1R3 is very of short duration-and calcium increases seldom can keep being more than 15 seconds-and be asynchronous.Response The quantity of cell is thus relative constant at any time, therefore, by set time point, generally 30 seconds after stimulant is added, hand Work counting response cell number, can quantify cellular response.
Embodiment 5
HT1R2/T1R3 has the function of sweet taste receptor
It is transiently transfected using hT1R2, T1R3 and T1R2/T1R3 and stablizes expression G α15HEK cells, analyze intracellular Ca2+ It is improved in response to the raising of sucrose concentration (Fig. 3 (a)).Equally, the T1R2/T1R3 dose responses of several sweet taste stimulis are measured (Fig. 3 (b)).The response cell largest percentage of different sweeteners is different, is 10-30%.For clarity, dose response is pressed Response cell largest percentage is standardized.Fig. 3 numerical value represents the average value ± s.e. of 4 separate responses.X- axis circle marks The body and mind detection threshold value determined by taste test is shown.Gurmarin (gymnema sylvestre (the Gymnema of 10g/l filterings Sylvestre) 50 times of dilutions of water extract) responses of the T1R2/T1R3 to 250mM sucrose is can inhibit, but do not inhibit endogenous The response (Fig. 3 (b)) of property 20 μM of isoproterenols of beta 2-adrenergic receptor pair.Fig. 3 (c) is total to table comprising T1R2/T1R3 Normalized response up to cell line to different sweeteners (sucrose, radix asparagi sweet extract, D-trp and saccharin).
Embodiment 6
Rat T1R2/T1R3 also has the function of sweet taste receptor
It is transiently transfected using hT1R2/hT1R3, rT1R2/rT1R3, hT1R2/rT1R3 and rT1R2/hT1R3 and stablizes table Up to G α15HEK cells.Then analyze these transfectional cells response 350mM sucrose, 25mM tryptophans, 15mM radix asparagi sweet extracts and 0.05% monellin (monellin) and improve intracellular Ca2+.Fig. 4 includes sucrose and radix asparagi sweet extract as a result, showing RT1R2/rT1R3 also has the function of sweetener receptor.It is special that these results also prompt T1R2 that may control T1R2/T1R3 ligands Property.
Embodiment 7
T1R2/T1R3 is measured using fluorescence-based automated analysis to respond
It is transiently transfected using hT1R2 and hT1R3 and stablizes expression G α15HEK cells.Calcium dyestuff Fluo- is added in these cells 4, measure its response to sweetener using fluorescence plate reader.Cells of the Fig. 5 comprising expression hT1R2/hT1R3 and only table Up to response of the cell (J19-22) to cyclamate (cyclamate, 12.5mM) of hT1R3.Gained fluorescence results show The cell of only expression hT1R2/hT1R3 responds these taste stimulus.Fig. 6 includes that standardized dosage-response is bent Line, the results showed that, it interacts according to the dosage specificity of a variety of taste stimulus, hT1R2 and hT1R3 play tasty jointly Feel the function of receptor.The special agent comprising to sucrose, tryptophan and other a variety of sweeteners that acquisition can be commercialized of Fig. 6 Amount-response.These results indicate that since gained sorts in analysis and threshold value reflects the numerical value of people's sweet taste feel, T1R2/ closely T1R3 is people's sweetener receptor.
Embodiment 8
The ligand-binding residues of mGluR1 are guarded in T1R1
As shown in fig. 6, the critical ligand combination residue of mGluR1 is guarded in T1R1.According to color side identical with Fig. 1 Several Key residues that case highlights show the interaction of glutamic acid and mGluR1.
Embodiment 9
HT1R1/T1R3 has the function of umami taste receptor
It is transiently transfected using hT1R1, T1R3 and T1R1/T1R3 and stablizes expression G α15HEK cells, analyze intracellular Ca2+ Improved in response to the raising of aminoglutaric acid concentration (Fig. 8 (a)), 0.5mM glutamic acid), 0.2mM IMP, and 0.5mM glutamic acid add 0.2mM IMP (Fig. 8 (b)).Dose response (the Fig. 8 of hT1R1/T1R3 to glutamic acid is measured in the presence of whether there is or not 0.2mM IMP (c)).Cell largest percentage to glutamic acid response is about 5%, and glutamic acid is about 10% plus IMP.For clarity, Dose response is standardized by response cell largest percentage.Numerical value represents the average value ± s.e. of 4 separate responses.X- Axis circle denotes the sense of taste detection threshold value determined by taste test.
Embodiment 10
PDZIP is as output sequence
By 6 residue PDZIP sequences (SVSTW (SEQ ID NO:1) it) is merged with the C-terminal of hT1R2, the Chimerical receptor is (i.e. HT1R2-PDZIP HEK-293 host cells) are transfected.Then the surface of immunofluorescence and FACS scan datas monitoring hT1R2 is used Expression.As illustrated in figures 9a and 9b, including PDZIP sequences can be with respect to the surface expression for improving hT1R2-PDZIP for hT1R2. More specifically, Fig. 9 A show the immunofluorescence dyeing of the hT1R2 of myc- labels, show that PDZIP is remarkably improved hT1R2 albumen Quantity of the matter on plasma membrane.Fig. 9 B show facs analysis data, show identical result-dotted line and indicate expression myc- labels The cell of hT1R2, solid line indicate the cell of the hT1R2-PDZIP of expression myc- labels.In particular, Figure 10 A, which are shown, is in HBS The G α of untransfected in buffer solution15Stable host cell, Figure 10 B, which are shown, is in sweet agent mixture no.5 (saccharin, hexamethylene ammonia Base sodium sulfonate, acesulfame K (Acesulfam K) and each 20mM of radix asparagi sweet extract-, in the HBS buffer solutions) in transfection The G α of hT1R2-PDZIP15Stable host cell, Figure 10 C show the transfection T1R3- in sweet agent mixture no.5 The G α of PDZIP15Stable host cell, Figure 10 D show the cotransfection hT1R2-PDZIP/ in sweet agent mixture no.5 The G α of hT1R3-PDZIP15Stable host cell.In addition, Figure 10 E-10H show the G α of hT1R2/hT1R3 cotransfections15Stablize Host cell to the dose dependent response-E of sucrose:0mM, in HBS buffer solutions；F:30mM；G:60mM；H:250mM.Figure 10I-10L shows the G α of hT1R2/hT1R3 cotransfections15Response-I of the stable host cell to various sweeteners:Radix asparagi sweet extract (1.5mM)；J:Acesulfame K (1mM)；K:Neotame (Neotame, 20mM)；L:Sodium Cyclamate (20mM).The calcium of Figure 10 Imaging show hT1R2 and hT1R3 be sweet stimulus object cause activity it is required.
Embodiment 11
Generate the cell line for stablizing coexpression T1R1/T1R3 or T1R2/T1R3
To separately include hT1R1 or hT1R2 expression constructs (T1R1 be plasmid SAV2485, T1R2 SAV2486) and The PEAK10 of the linearisation of hT1R3 (T1R3 is plasmid SXV550) derives (Edge Biosystems) carrier and pCDNA 3.1/ZEO derives (Invitrogen) carrier and is transfected into expression G α15Cell line in, obtain stablizing coexpression human T1R2/T1R3 Or the human cell line of hT1R1/T1R3.Specifically, by by SAV2486 the and SXV550 cotransfections of linearisation to stablizing table Up to G α15Aurora Bioscience's HEK-293 cell lines in, generate T1R2/T1R3 stable cell lines.By will be linear SAV2485 the and SXV550 cotransfections of change express G α to stable15Identical HEK-293 cell lines in, generate T1R1/T1R3 stablize Cell line.After transfecting S AV2485/SXV550 and SAV2486/SXV550, selection, amplification puromycin (puromycin) are anti- Property and zeocin resistance clones, and pass through its response to sweet taste or umami taste stimuli of calcium image checking.It is adding GlutaMAX, 10% dialysis FBS and 0.003mg/ml blasticidins (blasticidin) low glucose DMEM in, utilize 0.0005mg/ml puromycins (CALBIOCHEM) and 0.1mg/ml zeocin (Invitrogen) select cell in 37 DEG C.Expand Increase resistance clone, its response to sweet taste stimuli is evaluated by fluorescence microscopy.In order to utilize VIPR-II instruments (Aurora Biosciences) carries out automation fluorescence imaging analysis, will stablize the cell inoculation of expression T1R2/T1R3 first In 96 orifice plates (about 100,000 cells/well).After 24 hours, the dyestuff of calcium containing 0.005mM Fluo-3-AM is added in cell In the PBS buffer solution of (Molecular Probes), room temperature 1 hour.After replacing 70 μ l PBS, addition contains taste stimulus 70 μ l PBS, are stimulated at room temperature.It is measured before compound is added, correcting background fluorescence, to be carried to 0.001mM calcium ions The response of body ionomycin (ionomycin, CALBIOCHEM) is standardized, be added compound after fluorescence (480nm excite, 535nm emits) average response 20-30 seconds.
These cell lines contact sweet taste or delicate flavour stimulant, then observe that the cell of active clone general 80% can respond Taste stimulation.Surprisingly, the response intensity of each cell is significantly higher than the cell of transient transfection.
On the basis of observing herein, the present inventor utilizes Aurora Bioscience's VIPR instruments to pass through certainly as described above Dynamicization fluorescence imaging tests the T1R activity of stable cell lines.Two kinds of T1R1/T1R3 and a kind of T1R2/T1R3 cell lines Response is as shown in FIG. 11 and 12 respectively.
It is worth noting that, joint, which improves response cell number and response intensity, leads to the cell relative to transient transfection, Activity improves 10 times.(ionomycin for the cell that by comparing, with optimal conditions, T1R2/T1R3 is transiently transfected responds percentage Than being about 5%.) in addition, stablize the dose response that expression hT1R2/T1R3 and T1R1/T1R3 is obtained feels detection threshold with tasty Value is related.The powerful T1R activity of these stable cell lines prompts, and is highly suitable for high flux screening chemistry library, with identification Sweet taste or umami taste receptor is adjusted, to adjust, enhance, block or simulate the compound that sweet taste or delicate flavour are felt, such as small point Son.
Embodiment 12
Generate the cell line of the induction type coexpression T1R1/T1R3 of selective response umami taste stimuli
293 cell lines of T1R1/T1R3HEK of stable expression umami taste receptor show stronger than the cell of transient transfection Activity.But the disadvantage is that, its rapid loss activity in proliferation process.
These discoveries also support the present inventor's it is assumed that i.e. (i) T1R1/T1R3 is umami taste receptor, and (ii) is high to express The cell line of T1R1/T1R3, preferably stable and/or inducible expression T1R1/T1R3 cell line can be used in analysis, preferably use In high flux screening chemistry library, to identify novel fresh taste modulator.The conditioning agent that enhancing delicate flavour may be used to feel.
To overcome the unstability for the cell line for stablizing expression T1R1/T1R3, GeneSwitch systems have been used (Invitrogen) by HEK-G α15Cell transform inducible expression T1R1/T1R3 as.HT1R1 and T1R3 derived from pGene Zeocin resistance expression vectors (T1R1 is plasmid SXV603, and T1R3 is plasmid SXV611) and carrying GeneSwitch protein Puromycin-resistant pSwitch derivative vectors (plasmid SXV628) it is linearized, cotransfection to HEK-G α15Cell line. The clone of Zeocin resistances and puromycin-resistant is chosen, expands, and utilizes the mifepristone of different number (mifepristone) it induces, passes through its response to umami taste stimuli of calcium imaging test.
Inducible expression T1R1/T1R3 generates high activity.For example, about 80% inducing cell can respond Pidolidone, and It transiently transfects cell and there was only about 10%；More specifically, Zeocin resistance tables derived from the pGene of hT1R1 and hT1R3 are expressed Linearized, the cotransfection to G up to carrier and the puromycin-resistant pSwitch derivative vectors for carrying GeneSwitch protein α15In cell.In the Dulbecco's Modified of addition GlutaMAX, 10% dialysis FBS and 3 μ g/ml blasticidins In Eagle Medium, 0.5 μ g/ml puromycins (CAL BIOCHEM) and 100 μ g/ml Zeocin (Invitrogen) are utilized Cell is selected in 37 DEG C.Resistance clone is through amplification and with 10-10After the induction of M mifepristones, according to Li et al. people, PNAS 99 (7): The method of 4692-4696 (2002), its response to umami taste stimuli is measured using fluorescence microscopy.
In order to carry out automation fluorescence imaging using FLIPR instruments (Molecular Device), a clone will be come from The cell of (referred to as cloning 1-17) is 10-10It is inoculated in 96 orifice plates (about 80,000 cells/well), incubates in the presence of M mifepristones 48 hours.Then cell is added in the PBS buffer solution containing 3 μM of calcium dyestuff Fluo-4-AM (Molecular Probes), room temperature 1.5 hour.
After replacing 50 μ l PBS, the 50 μ l PBS containing different stimulated object are added, are stimulated at room temperature.It is needed with previous The instantaneous T1R1/T1R3 umami receptors expression system (Li of T1R1/T1R3 receptor actives is quantified by counting response cell one by one Et al., PNAS 99 (7):4692-4696 (2002)) it is compared (since its receptor active is relatively low), this inducible expression system obtains The activity of clone 1-17 greatly improve so that can (480nm swashs by measuring the total maximum fluorescence enhancing in the imaging cells visual field Hair, 535nm transmittings) quantify receptor active.It is measured before compound is added, correcting background fluorescence, with to 0.002mM ions The response of mycin (CALBIOCHEM) is standardized, and the maximum fluorescence that 4 times independently measure is averaged.
Figure 13 contains these results.Figure 13 is specifically included in that whether there is or not measured in the presence of 0.2mM IMP to Pidolidone Dose response curve.Each numerical value represents 4 average total maximum fluorescences (correcting background fluorescence) independently measured in the figure. These dose-response curves correspond to the surveyed curve of cell that T1R1/T1R3 is transiently transfected.
It is also screened using different l-amino acids, to evaluate the selectivity that delicate flavour feels T1R1/T1R3 receptors.Acquired results Show that T1R1/T1R3 can be by l-amino acid (Pidolidone and L-Aspartic acid) Selective activation of delicate flavour.
The experimental result of the obtained responses of test 1-17 clones in the presence of different l-amino acids of Figure 14 and 15.Figure 14 Show the experimental result for the different l-amino acids that 1-17 cell lines exposure concentration in the presence of whether there is or not 1mM IMP is 10mM.
Figure 15 is included in the dose-response curve of the active amino acid measured in the presence of 0.2mM IMP.Each numerical value represents 4 average values independently measured.
These experiment eligible results confirm that the umami taste receptor has specificity and selectivity to umami taste stimuli.It is fresh Taste stimulus Pidolidone and L-Aspartic acid significantly activated under various concentration T1R1/T1R3 receptors (referring to Figure 14 and 15), can activate hT1R1/T1R3 receptors other l-amino acids only under very high concentration faint activation this receptor.
Therefore, these results confirm that T1R1/T1R3 receptors stablize the selectivity of umami taste stimuli and the induction type Expression system is suitable for analyzing using the high flux screening of automation phosphorimager, and the change of umami taste receptor can be activated with identification Object, such as Pidolidone or L-Aspartic acid are closed, or the active compound of Pidolidone activation umami taste receptor, example can be enhanced Such as 5'-IMP or 5'-GMP, or the umami taste receptor by umami taste stimuli such as Pidolidone and L-Aspartic acid can be blocked The compound of activation.
The compound identified with these analysis methods possibly serves for the fragrance of food and beverage composition, to simulate or block Umami taste stimuli.
Embodiment 13
The receptor active and sweet taste and delicate flavour of Lactitol inhibition hT1R2/T1R3 and T1R1/T1R3 is felt
A kind of aralkyl carboxylic acid, Lactitol, it is considered to be selective sweet taste agent inhibitor is (referring to Lindley (1986) United States Patent (USP) 4,567,053；And Schiffman et al. Chem Senses 24:439-447(1999)).It determines Transiently transfect the HEK-G α of T1R2/T1R315Response of cell in the presence of various concentration Lactitols to 150mM sucrose.Clarke Inhibit hT1R2/T1R3 activity, IC for alcohol50It is 24 μM.
T1R1/T1R3 delicate flavours are felt and T1R2/T1R3 sweet taste receptors may have common subunit.Therefore inference, can press down The Lactitol of T1R2/T1R3 sweetener receptors processed may have similar effect to T1R1/T1R3 umami taste receptors.The present invention People tests the effect that Lactitol responds hT1R1/T1R3 10mM Pidolidones.It is similar with T1R2/T1R3 sweet receptors, Lactitol inhibits the IC of T1R1/T1R350It is 165 μM.Lactitol inhibits to may reflect the antagonism to T1R receptors, Rather than for example non-specific inhibition G α15The signal of mediation occurs, because mAChR is not pressed down by Lactitol System.
Then the present inventor has rated the effect that Lactitol feels people's delicate flavour.According to Schiffman et al. (Chem.Senses 24:439-447 (1989)) method, measure in the presence of 1 and 2mM Lactitols with or without 0.2mM Umami taste stimuli Pidolidone, sweetener stimulant sucrose and the D-trp and salty taste stimulus sodium chloride taste of IMP Feel threshold value.The Lactitol of millimolar concentration is remarkably improved the detection threshold value of sweet taste feel and umami taste stimuli, feels to saline taste Stimulant is quite different.Figure 16 contains these results.
In short, (i) these find further to support the present inventor's it is assumed that i.e. T1R1/T1R3 is unique delicate flavour feel by Relevant Lactitol binding structural domain on body, and the possible apokoinou construction of (ii) T1R1/T1R3 and T1R2/T1R3 receptors.
Although several embodiments of the present invention have been described in the above detailed descriptionthe, it should be understood that above description is only said Bright and unrestricted the displosure invention.The present invention is limited only by the following claims.
This specification further includes the following contents：
1. by least one T1R1 polypeptides (or variant, segment or chimera of the T1R1 polypeptides) and/or at least one The receptor of T1R3 polypeptides (or variant, segment or chimera of the T1R3 polypeptides) composition, wherein the receptor specific can be tied Close umami taste stimuli and/or by its activation.
2. the receptor of project 1, it includes segment, variant or the chimeras of natural hT1R1 polypeptides.
3. the receptor of project 1, it includes segment, variant or the chimeras of natural hT1R3 polypeptides.
4. the receptor of project 1, wherein the T1R1 and T1R3 derives from same species.
5. the receptor of project 1, wherein the T1R1 and T1R3 derives from different plant species.
6. the receptor of project 1, wherein the T1R1 and/or T1R3 is from mammal, fish, reptile, amphibious Animal or birds.
7. the receptor of project 1, wherein the T1R1 and/or T1R3 from hT1R1, hT1R3, rT1R1, rT1R3, mT1R1, mT1R3；Or its segment, variant or chimera.
8. the composition of the receptor comprising project 1.
9. the composition of the receptor comprising project 2.
10. the composition of the receptor comprising project 3.
11. the composition of the receptor comprising project 4.
12. the composition of the receptor comprising project 5.
13. the composition of the receptor comprising project 6.
14. the composition of the receptor comprising project 7.
15. expression is by least one T1R1 polypeptides (or variant, segment or chimera of the T1R1 polypeptides) and/or extremely A kind of cell of the receptor of few T1R3 polypeptides (or variant, segment or chimera of the T1R3 polypeptides) composition, wherein it is described by Body can specifically bind umami taste stimuli and/or by its activation.
16. the cell of project 15, selected from HEK293, COS and Chinese hamster ovary celI and Xenopus Oocytes.
17. the cell of project 15, wherein the T1R1 and T1R3 come from same species.
18. the cell of project 15, wherein the T1R1 and T1R3 derives from different plant species.
19. the cell of project 15, wherein the T1R1 and/or T1R3 is from mammal, fish, reptile, two Dwell animal or birds.
20. the cell of project 15, wherein the T1R1 and T1R3 be selected from hT1R1, hT1R3, mT1R1, mT1R3, rT1R1 and rT1R3；Or its segment, variant or chimera.
21. by least one T1R2 polypeptides (or variant, segment or chimera of the T1R2 polypeptides) and/or at least one The receptor that kind T1R3 polypeptides (or variant, segment or chimera of the T1R3 polypeptides) form, wherein the receptor can specificity In conjunction with sweet taste stimuli and/or by its activation.
22. the receptor of project 21, it includes segment, variant or the chimeras of natural hT1R2 polypeptides.
23. the receptor of project 21, it includes segment, variant or the chimeras of natural hT1R3 polypeptides.
24. the receptor of project 21, wherein the T1R2 and T1R3 derives from same species.
25. the receptor of project 21, wherein the T1R2 and T1R3 derives from different plant species.
26. the receptor of project 21, wherein the T1R2 and/or T1R3 is from mammal, fish, reptile, two Dwell animal or birds.
27. the receptor of project 21, wherein the T1R2 and/or T1R3 come from hT1R2, hT1R3, rT1R2, rT1R3, mT1R2、mT1R3；Or its segment, variant or chimera.
28. the composition of the receptor comprising project 21.
29. the composition of the receptor comprising project 22.
30. the composition of the receptor comprising project 23.
31. the composition of the receptor comprising project 24.
32. the composition of the receptor comprising project 25.
33. the composition of the receptor comprising project 26.
34. the composition of the receptor comprising project 27.
35. expression is by least one T1R2 polypeptides (or variant, segment or chimera of the T1R2 polypeptides) and/or extremely A kind of cell of the receptor of few T1R3 polypeptides (or variant, segment or chimera of the T1R3 polypeptides) composition, wherein it is described by Body can specifically bind sweet taste stimuli and/or by its activation.
36. the cell of project 35, selected from HEK293, COS and Chinese hamster ovary celI and Xenopus Oocytes.
37. the cell of project 35, wherein the T1R2 and T1R3 come from same species.
38. the cell of project 35, wherein the T1R2 and T1R3 derives from different plant species.
39. the cell of project 35, wherein the T1R2 and/or T1R3 is from mammal, fish, reptile, two Dwell animal or birds.
40. the cell of project 35, wherein the T1R2 and T1R3 be selected from hT1R2, hT1R3, mT1R2, mT1R3, rT1R2 and rT1R3；Or its segment, variant or chimera.
41. being incorporated into the receptor of the project 1-7 or 21-27 of solid phase.
42. the receptor of the project 1-7 or 21-27 in solution.
43. the receptor in lipid bilayer or project 1-7 or 21-27 in vesica.
44. the method that the compound of taste perception is adjusted in identification, by identifying combinable, activation, inhibiting, enhancing And/or one or more receptors of reconciling items 1-7 and/or 21-27 compound and identify.
45. the method for project 44, wherein the receptor is incorporated into solid formation.
46. the method for project 44, wherein the receptor is in solution.
47. the method for project 44, wherein the receptor is in lipid bilayer or vesica.
48. the method for project 44, wherein identifying the compound using receptor-binding assay.
49. the method for project 44, wherein the analysis method based on receptor active is used to identify the compound.
50. the method for project 44, wherein the expression of receptor is in cell.
51. the method for project 50, wherein the cell is HEK-293, COS or Chinese hamster ovary celI.
52. the method for project 50, wherein identifying the compound by its effect to receptor internalisation.
53. the method for project 50, wherein identifying the compound by its effect to receptor phosphorylation.
54. the method for project 50, wherein identifying the compound by its effect to inhibition protein translocation.
55. the method for project 44 uses the analysis method for second messenger.
56. the method for project 55, wherein the second messenger is cAMP or IP3。
57. the method for project 50 uses voltage sensitivity or calcium sensitivity dyestuff.
58. the method for project 50, wherein at least one G-protein of cell expression.
59. the method for project 58, wherein the G-protein is mixed G-protein of dwelling.
60. the method for project 59, wherein the mixed G-protein of dwelling is G α15Or G α16。
61. the method for project 44, wherein expressing the receptor using viral vectors.
62. the method for project 61, wherein the viral vectors are expressed in mammalian cell.
63. the method for project 44, wherein the receptor is generated by In Vitro Translation.
64. the method for project 44, wherein the receptor is detached with film combining form.
65. the method for project 44, wherein identifying the compound by its effect to the receptor activation G-protein.
66. the method for project 44, wherein identifying the compound by its effect to receptor conformation.
67. the method for project 66, wherein detecting the conformation change by Susceptible change to proteolysis.
68. the method for project 66, wherein detecting the conformation change by NMR spectroscopy.
69. the method for project 66, wherein detecting the conformation change by fluorescence spectroscopy.
70. the method for project 44, wherein passing through its work to the ligand binding of the receptor and radioactivity or fluorescent marker With and identify the compound.
71. the method for project 70, wherein measuring the displacement of the labeled compound using fluorescence polarization or FRET analyses.
72. the method for project 44 is high flux screening analysis method.
73. the method for project 44, wherein receptor active are associated with reporter gene.
74. the method for project 73, wherein the reporter gene be luciferase, alkaline phosphatase, beta galactosidase or Beta-lactamase.
75. the method for project 44, wherein the receptor is constitutive activity variant.
76. the method for project 44, wherein the expression of the receptor is located under constitutive promoter control.
77. the method for project 44, wherein the expression of the receptor is located under the control of adjustment type promoter.
78. the method for project 44, wherein the receptor is merged with that can promote the peptide of surface expression.
79. the method for project 78, wherein the peptide is PDZ structural domain interacting peptides.
80. the method for project 44, wherein predicting the compound to this receptor according to the x-ray crystal structure of the receptor Effect.
81. the method for project 44, wherein by the effect of its natural to expression or transgenosis T1R receptors non-human animal Identify the compound.
82. the method for project 81, wherein identifying the compound by its effect to behavior.
83. the method for project 81, wherein identifying the compound by its effect to taste receptor cells.
84. the method for project 81, wherein the non-human animal is mouse, rat, worm, fish or insect.
85. the method for project 44, wherein identifying describedization by its effect to the yeast cells for expressing the receptor Close object.
86. the method for project 44, wherein identifying the compound from compound combination library.
87. the method for project 44, wherein identifying the compound from peptide library.
88. the method for project 44, wherein identifying the compound from small molecule random library.
89. changing the method for animal taste using the compound that project 44 is identified.
90. the method for project 89 is felt wherein the sense of taste is delicate flavour.
91. the method for project 89 is felt wherein the sense of taste is sweet taste.
92. the method for project 89, wherein the animal is people, dog, cat, fish, ox, sheep or pig.
93. the method for project 89, wherein the compound is formulated in food, beverage or combination of oral medication.
94. the method for quantitative each compound or the taste of food or beverage composition for treating dental erosion, using project 1-7 and/or One or more receptors of 21-27.
95. stablize expression by variant, segment or the chimera of at least one T1R1 polypeptides or the T1R1 polypeptides and/or The cell of the receptor of the variant of at least one T1R3 polypeptides or the T1R3 polypeptides, segment or chimera composition, wherein it is described by Body can specifically bind umami taste stimuli and/or by its activation.
96. the cell of project 95, selected from HEK293, COS and Chinese hamster ovary celI and Xenopus Oocytes.
97. the cell of project 95, wherein the T1R1 and T1R3 come from same species.
98. the cell of project 95, wherein the T1R1 and T1R3 derives from different plant species.
99. the cell of project 95, wherein the T1R1 and/or T1R3 is from mammal, fish, reptile, two Dwell animal or birds.
100. the cell of project 95, wherein the T1R1 and T1R3 is selected from hT1R1, hT1R3, mT1R1, mT1R3, rT1R1 And rT1R3；Or its segment, variant or chimera.
101. the cell of project 95 is to stablize expression G α15HEK-293 cells.
102. the composition of the cell comprising project 95.
103. the composition of the cell comprising project 96.
104. the composition of the cell comprising project 97.
105. the composition of the cell comprising project 98.
106. including the composition of the cell of project 99.
107. the composition of the cell comprising project 100.
108. the composition of the cell comprising project 101.
109. stablize expression by variant, segment or the chimera of at least one T1R2 polypeptides or the T1R2 polypeptides and/or The cell of the receptor of the variant of at least one T1R3 polypeptides or the T1R3 polypeptides, segment or chimera composition, wherein it is described by Body can specifically bind sweet taste stimuli and/or by its activation.
110. the cell of project 109, selected from HEK293, COS and Chinese hamster ovary celI and Xenopus Oocytes.
111. the cell of project 109, wherein the T1R2 and T1R3 come from same species.
112. the cell of project 109, wherein the T1R2 and T1R3 derives from different plant species.
113. the cell of project 109, wherein the T1R2 and/or T1R3 from mammal, fish, reptile, Amphibian or birds.
114. the cell of project 109, wherein the T1R2 and T1R3 is selected from hT1R2, hT1R3, mT1R2, mT1R3, rT1R2 And rT1R3；Or its segment, variant or chimera.
115. the cell of project 109 stablizes expression hT1R2, hT1R3 and G α15。
116. identification be adjusted taste perception compound method, by identify it is combinable, activation, inhibit and/or It adjusts the compound of receptor and identifies, wherein expressing the cell expression this receptor of at least one T1R by stablizing.
117. the method for project 116, wherein the cell combination is in solid phase.
118. the method for project 117, wherein the cell is in solution.
119. the method for project 117, wherein identifying the compound using receptor-binding assay.
120. the method for project 116, wherein the cell is the cell of any one of project 95-101 or 109-115.
121. the method for project 116, wherein the analysis method based on receptor active is used to identify the compound.
122. the method for project 116, wherein the T1R expression of receptor is in cell.
123. the method for project 116, wherein the cell is HEK-293, COS or Chinese hamster ovary celI.
124. the method for project 116, wherein identifying the compound by its effect to the cell internalizing receptor.
125. the method for project 116, wherein identifying the compound by its effect to receptor phosphorylation.
126. the method for project 116, wherein identifying the compound by its effect to inhibition protein translocation.
127. the method for project 116 uses the analysis method for second messenger.
128. the method for project 127, wherein the second messenger is cAMP or IP3。
129. the method for project 116 uses voltage sensitivity or calcium sensitivity dyestuff.
130. the method for project 116, wherein at least one G-protein of cell expression.
131. the method for project 130, wherein the G-protein is mixed G-protein of dwelling.
132. the method for project 131, wherein the mixed G-protein of dwelling is G α15Or G α16。
133. the method for project 116 expresses the receptor wherein stablizing using viral promoter.
134. the method for project 116, wherein the cell is mammalian cell.
135. the method for project 116, wherein identifying the chemical combination by its effect to the receptor activation G-protein Object.
136. the method for project 116, wherein identifying the compound by its effect to receptor conformation.
137. the method for project 136, wherein detecting the conformation change by Susceptible change to proteolysis.
138. the method for project 136, wherein detecting the conformation change by NMR spectroscopy.
139. the method for project 136, wherein detecting the conformation change by fluorescence spectroscopy.
140. the method for project 136, wherein by it to the receptor of the stable expression and radioactivity or fluorescent marker The effect of ligand binding and identify the compound.
The method of 141. projects 140, wherein measuring the shifting of the labeled compound using fluorescence polarization or FRET analyses Position.
142. the method for project 116 is high flux screening analysis method.
The method of 143. projects 116, wherein receptor active are associated with reporter gene.
The method of 144. projects 143, wherein the reporter gene is luciferase, alkaline phosphatase, beta galactosidase Or beta-lactamase.
The method of 145. projects 140, wherein the receptor is constitutive activity variant.
The method of 146. projects 140, wherein the expression of the receptor is located under constitutive promoter control.
The method of 147. projects 140, wherein the expression of the receptor is located under the control of adjustment type promoter.
The method of 148. projects 140, wherein the receptor is merged with that can promote the peptide of surface expression.
The method of 149. projects 140, wherein the peptide is PDZ structural domain interacting peptides.
The method of 150. projects 140, wherein according to the x-ray crystal structure of the receptor predict the compound to this by The effect of body.
The method of 151. projects 116, wherein being identified by its effect to the yeast cells for expressing the receptor described Compound.
The method of 152. projects 116, wherein identifying the compound from compound combination library.
The method of 153. projects 116, wherein identifying the compound from peptide library.
The method of 154. projects 116, wherein identifying the compound from small molecule random library.
155. change the method for animal taste using the compound that project 116 is identified.
The method of 156. projects 155 is felt wherein the sense of taste is delicate flavour.
The method of 157. projects 155 is felt wherein the sense of taste is sweet taste.
The method of 158. projects 155, wherein the animal is people, dog, cat, fish, ox, sheep or pig.
The method of 159. projects 155, wherein the compound is formulated in food, beverage or combination of oral medication.
The method of 160. quantitative each compounds or the taste of food or beverage composition for treating dental erosion, is encoded using expression is stablized The cell of the heterologous nucleic acid sequence of at least one T1R of one of project 1-7 and/or 15-21.
The cell line of 161. inducible expression hT1R1s/T1R3 umami taste receptors or T1R2/T1R3 sweet taste receptors.
The cell line of 162. projects 161, the wherein cell line are CHO, COS, HEK or bhk cell system.
The cell line of 163. projects 162 is HEK-293 cell lines.
The cell line of 164. projects 161 expresses G-protein.
The cell line of 165. projects 164, wherein the G-protein is G α15Or G α16。
The cell line of 166. projects 161 is stablized and expresses the T1R1/T1R3 receptors.
The cell line of 167. projects 161, wherein by GeneSwitch protein induced expressions.
168. can excitement or antagonism T1R1/T1R3 receptors or T1R2/T1R3 receptors using the cell line identification of projects 161 The method of compound.
The method of 169. projects 168, is bonding analysis method.
The method of 170. projects 168 is high flux screening analysis method.
The method of 171. projects 168, is fluorescence analysis method.
The method of 172. projects 168, screening can be with Pidolidone or L-Aspartic acid competitive binding T1R1/T1R3 delicate flavours Feel the compound of receptor.
The method of 173. projects 168 is the high flux screening analysis method using automation phosphorimager.
The method of 174. projects 168 can enhance or adjust Pidolidone activation T1R1/ for being screened in library of compounds The active compound of T1R3 umami taste receptors.
The method of 175. projects 168, in library of compounds screening can excitement or antagonism T1R2/T1R3 sweet tastes feel by The compound of body.
The method of 176. projects 168, screening can feel with IMP, GMP or its analog competitive binding T1R1/T1R3 delicate flavour The compound of receptor.
The method of 177. projects 168 can simulate IMP, GMP or the enhancing of its analog for being screened in library of compounds The active compound of T1R1/T1R3 agonist activities.
The method of 178. projects 168 can enhance or adjust sweetener activation T1R2/ for being screened in library of compounds The active compound of T1R3 sweet taste receptors.
179. inhibit the method for T1R1/T1R3 umami taste receptors, inhibit it includes the receptor and simultaneously T1R1/T1R3 sweet teas The sweet-taste inhibitor of taste receptors and T1R2/T1R3 taste receptors is in contact.
The method of 180. projects 179, wherein the inhibitor is Lactitol.
The method that the compound of T1R1/T1R3 umami taste receptors is adjusted in 181. identifications, can be with Lactitol by screening Competitive binding and/or inhibit T1R1/T1R3 umami taste receptors compound and identify.
The method of 182. projects 179 is the analysis method based on cell.
The method of 183. projects 182, wherein cell line of the analysis method using coexpression T1R1 and T1R3.
The method of 184. projects 183, wherein the cell line is HEK-G α15Cell line.
The method of 185. projects 183, wherein the cell line, which is stablized, expresses the receptor.
The method of 186. projects 183, wherein receptor described in the cell line transient expression.
The method of 187. projects 186, wherein the G-protein is G α15Or G α16。
The method of 188. projects 181 is the analysis method based on cell.
The method of 189. projects 188, wherein cell line of the analysis method using coexpression T1R1 and T1R3.
The method of 190. projects 189, wherein the cell line is HEK-G α15The cell line.
The method of 191. projects 189, wherein the cell line, which is stablized, expresses the receptor.
The method of 192. projects 191, wherein receptor described in the cell line transient expression.
The method of 193. projects 192, wherein the G-protein is G α15Or G α16。

Claims (9)

1. separation by least one T1R1 polypeptides or variant, segment or the chimera and at least one T1R3 of the T1R1 polypeptides The variant of polypeptide or the T1R3 polypeptides, the umami taste receptor of segment or chimera composition or the umami taste receptor containing the separation Composition, wherein the receptor-specific combination umami taste stimuli and/or by its activation.
2. the receptor or composition of claim 1, segment, variant or chimera containing natural hT1R1 or hT1R3 polypeptides.
3. expression is more by variant, segment or the chimera and at least one T1R3 of at least one T1R1 polypeptides or the T1R1 polypeptides The recombinant cell of the umami taste receptor of the variant of peptide or the T1R3 polypeptides, segment or chimera composition, wherein the receptor is special The opposite sex combines umami taste stimuli and/or by its activation.
4. the cell of claim 3, wherein segment of the umami taste receptor containing natural hT1R1 or hT1R3 polypeptides, variant or embedding It is fit.
5. the receptor of the separation of claim 1, is incorporated into solid formation, in solution, or it is contained in lipid bilayer or vesica.
6. identification adjust taste perception compound method, by identify combine, activation, inhibit, enhancing claim 1 or 2 umami taste receptor, or block the activation of the umami taste receptor of claims 1 or 2.
7. the method for claim 7, wherein the receptor is expressed in cell or the receptor is incorporated into solid formation, in molten In liquid, or it is contained in lipid bilayer or vesica.
8. the method for claim 7 or 8, using selected from following method：Divide for identifying that the receptor of the compound combines Analysis method, the analysis method based on receptor active for identifying the compound, receptor internalisation analysis method, receptor phosphorylation Analysis method inhibits protein translocation analysis method to detect the analysis method of intracellular calcium levels for the analysis method of second messenger Or detection compound is to the analysis method of the effect of receptor conformation.
9. at least one of compound identified according to claim 8 or 9 is preparing the delicate flavour feel for adjusting people experimenter Composition in purposes.
CN201710563289.1A 2001-06-26 2002-06-26 The taste receptors of T1R oligomerics and the cell line for expressing the receptor and its purposes in identification of taste compounds Pending CN108329393A (en)

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US30043401P 2001-06-26 2001-06-26
US60/300,434 2001-06-26
US09/897,427 US6955887B2 (en) 2001-03-30 2001-07-03 Use of T1R hetero-oligomeric taste receptor to screen for compounds that modulate taste signaling
US09/897,427 2001-07-03
US30474901P 2001-07-13 2001-07-13
US60/304,749 2001-07-13
US31049301P 2001-08-08 2001-08-08
US60/310,493 2001-08-08
US33177101P 2001-11-21 2001-11-21
US60/331,771 2001-11-21
US33947201P 2001-12-14 2001-12-14
US60/339,472 2001-12-14
US10/035,045 2002-01-03
US10/035,045 US7241880B2 (en) 2001-01-03 2002-01-03 T1R taste receptors and genes encoding same
US37209002P 2002-04-15 2002-04-15
US60/372,090 2002-04-15
US37414302P 2002-04-22 2002-04-22
US60/374,143 2002-04-22
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