JP6924590B2 - Fusion protein for bitterness evaluation and bitterness evaluation method - Google Patents
Fusion protein for bitterness evaluation and bitterness evaluation method Download PDFInfo
- Publication number
- JP6924590B2 JP6924590B2 JP2017048197A JP2017048197A JP6924590B2 JP 6924590 B2 JP6924590 B2 JP 6924590B2 JP 2017048197 A JP2017048197 A JP 2017048197A JP 2017048197 A JP2017048197 A JP 2017048197A JP 6924590 B2 JP6924590 B2 JP 6924590B2
- Authority
- JP
- Japan
- Prior art keywords
- fusion protein
- bitterness
- bitter taste
- amino acid
- cells
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 235000019658 bitter taste Nutrition 0.000 title claims description 133
- 102000037865 fusion proteins Human genes 0.000 title claims description 109
- 108020001507 fusion proteins Proteins 0.000 title claims description 109
- 238000011156 evaluation Methods 0.000 title claims description 35
- 239000000126 substance Substances 0.000 claims description 72
- 125000003275 alpha amino acid group Chemical group 0.000 claims description 63
- 238000012360 testing method Methods 0.000 claims description 42
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 claims description 39
- 229910052791 calcium Inorganic materials 0.000 claims description 39
- 239000011575 calcium Substances 0.000 claims description 39
- 238000000034 method Methods 0.000 claims description 37
- 108091005708 gustatory receptors Proteins 0.000 claims description 28
- 239000013604 expression vector Substances 0.000 claims description 26
- 102100039215 Guanine nucleotide-binding protein G(t) subunit alpha-3 Human genes 0.000 claims description 24
- 108010005995 gustducin Proteins 0.000 claims description 24
- 108091033319 polynucleotide Proteins 0.000 claims description 23
- 102000040430 polynucleotide Human genes 0.000 claims description 23
- 239000002157 polynucleotide Substances 0.000 claims description 23
- 108091006099 G alpha subunit Proteins 0.000 claims description 15
- 102000034353 G alpha subunit Human genes 0.000 claims description 15
- 238000012216 screening Methods 0.000 claims description 9
- 230000007423 decrease Effects 0.000 claims description 3
- 210000004027 cell Anatomy 0.000 description 137
- 108090000623 proteins and genes Proteins 0.000 description 33
- 230000003834 intracellular effect Effects 0.000 description 27
- 230000036327 taste response Effects 0.000 description 21
- 230000004044 response Effects 0.000 description 20
- 241000282414 Homo sapiens Species 0.000 description 18
- 230000008859 change Effects 0.000 description 17
- NGFMICBWJRZIBI-JZRPKSSGSA-N Salicin Natural products O([C@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@H](CO)O1)c1c(CO)cccc1 NGFMICBWJRZIBI-JZRPKSSGSA-N 0.000 description 15
- NGFMICBWJRZIBI-UHFFFAOYSA-N alpha-salicin Natural products OC1C(O)C(O)C(CO)OC1OC1=CC=CC=C1CO NGFMICBWJRZIBI-UHFFFAOYSA-N 0.000 description 15
- NGFMICBWJRZIBI-UJPOAAIJSA-N salicin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1OC1=CC=CC=C1CO NGFMICBWJRZIBI-UJPOAAIJSA-N 0.000 description 15
- 229940120668 salicin Drugs 0.000 description 15
- 102000005962 receptors Human genes 0.000 description 14
- 108020003175 receptors Proteins 0.000 description 14
- 108090000045 G-Protein-Coupled Receptors Proteins 0.000 description 13
- 102000003688 G-Protein-Coupled Receptors Human genes 0.000 description 12
- 150000001413 amino acids Chemical class 0.000 description 12
- 108091006027 G proteins Proteins 0.000 description 11
- 102000030782 GTP binding Human genes 0.000 description 11
- 108091000058 GTP-Binding Proteins 0.000 description 11
- FULZLIGZKMKICU-UHFFFAOYSA-N N-phenylthiourea Chemical compound NC(=S)NC1=CC=CC=C1 FULZLIGZKMKICU-UHFFFAOYSA-N 0.000 description 11
- BBFQZRXNYIEMAW-UHFFFAOYSA-N aristolochic acid I Chemical compound C1=C([N+]([O-])=O)C2=C(C(O)=O)C=C3OCOC3=C2C2=C1C(OC)=CC=C2 BBFQZRXNYIEMAW-UHFFFAOYSA-N 0.000 description 11
- 241000282412 Homo Species 0.000 description 10
- RXWNCPJZOCPEPQ-NVWDDTSBSA-N puromycin Chemical compound C1=CC(OC)=CC=C1C[C@H](N)C(=O)N[C@H]1[C@@H](O)[C@H](N2C3=NC=NC(=C3N=C2)N(C)C)O[C@@H]1CO RXWNCPJZOCPEPQ-NVWDDTSBSA-N 0.000 description 10
- 239000013598 vector Substances 0.000 description 10
- 239000002609 medium Substances 0.000 description 9
- 108020004414 DNA Proteins 0.000 description 8
- 210000004899 c-terminal region Anatomy 0.000 description 8
- 239000013612 plasmid Substances 0.000 description 8
- 108010063331 type 2 taste receptors Proteins 0.000 description 8
- IXQKXEUSCPEQRD-NRNCYQGDSA-N (2S,4R,23E)-2,16beta,20-trihydroxy-9beta,10,14-trimethyl-1,11,22-trioxo-4,9-cyclo-9,10-secocholesta-5,23-dien-25-yl acetate Chemical compound C([C@H]1[C@]2(C)C[C@@H](O)[C@@H]([C@]2(CC(=O)[C@]11C)C)[C@@](C)(O)C(=O)C=CC(C)(C)OC(=O)C)C=C2[C@H]1C[C@H](O)C(=O)C2(C)C IXQKXEUSCPEQRD-NRNCYQGDSA-N 0.000 description 7
- IHTCCHVMPGDDSL-ZJNDIJRCSA-N Cucurbitacin A Natural products O=C([C@@](O)(C)[C@H]1[C@@H](O)C[C@]2(C)[C@]1(C)CC(=O)[C@]1(CO)[C@H]2CC=C2C(C)(C)C(=O)[C@H](O)C[C@@H]12)/C=C/C(OC(=O)C)(C)C IHTCCHVMPGDDSL-ZJNDIJRCSA-N 0.000 description 7
- CVKKIVYBGGDJCR-SXDZHWHFSA-N Cucurbitacin B Natural products CC(=O)OC(C)(C)C=CC(=O)[C@@](C)(O)[C@@H]1[C@@H](O)C[C@]2(C)C3=CC[C@@H]4C(C)(C)C(=O)[C@H](O)C[C@@]4(C)[C@@H]3CC(=O)[C@@]12C CVKKIVYBGGDJCR-SXDZHWHFSA-N 0.000 description 7
- QZJJDOYZVRUEDY-UHFFFAOYSA-N Dihydrocucurbitacin B Natural products CC12C(=O)CC3(C)C(C(C)(O)C(=O)CCC(C)(C)OC(=O)C)C(O)CC3(C)C1CC=C1C2CC(O)C(=O)C1(C)C QZJJDOYZVRUEDY-UHFFFAOYSA-N 0.000 description 7
- 101000674775 Homo sapiens Taste receptor type 2 member 16 Proteins 0.000 description 7
- 101000787850 Homo sapiens Taste receptor type 2 member 38 Proteins 0.000 description 7
- 102100021234 Taste receptor type 2 member 16 Human genes 0.000 description 7
- 102100025867 Taste receptor type 2 member 38 Human genes 0.000 description 7
- 230000001419 dependent effect Effects 0.000 description 7
- 239000012895 dilution Substances 0.000 description 7
- 238000010790 dilution Methods 0.000 description 7
- 235000019640 taste Nutrition 0.000 description 7
- 230000036962 time dependent Effects 0.000 description 7
- 101000892439 Homo sapiens Taste receptor type 2 member 10 Proteins 0.000 description 6
- 101000787816 Homo sapiens Taste receptor type 2 member 39 Proteins 0.000 description 6
- LHNKBXRFNPMIBR-UHFFFAOYSA-N Picrotoxin Natural products CC(C)(O)C1(O)C2OC(=O)C1C3(O)C4OC4C5C(=O)OC2C35C LHNKBXRFNPMIBR-UHFFFAOYSA-N 0.000 description 6
- 239000000872 buffer Substances 0.000 description 6
- 238000001514 detection method Methods 0.000 description 6
- 230000004927 fusion Effects 0.000 description 6
- 239000003550 marker Substances 0.000 description 6
- 238000005259 measurement Methods 0.000 description 6
- VJKUPQSHOVKBCO-AHMKVGDJSA-N picrotoxin Chemical compound O=C([C@@]12O[C@@H]1C[C@]1(O)[C@@]32C)O[C@@H]3[C@H]2[C@@H](C(=C)C)[C@@H]1C(=O)O2.O=C([C@@]12O[C@@H]1C[C@]1(O)[C@@]32C)O[C@@H]3[C@H]2[C@@H](C(C)(O)C)[C@@H]1C(=O)O2 VJKUPQSHOVKBCO-AHMKVGDJSA-N 0.000 description 6
- LSHVYAFMTMFKBA-TZIWHRDSSA-N (-)-epicatechin-3-O-gallate Chemical compound O([C@@H]1CC2=C(O)C=C(C=C2O[C@@H]1C=1C=C(O)C(O)=CC=1)O)C(=O)C1=CC(O)=C(O)C(O)=C1 LSHVYAFMTMFKBA-TZIWHRDSSA-N 0.000 description 5
- LSHVYAFMTMFKBA-UHFFFAOYSA-N ECG Natural products C=1C=C(O)C(O)=CC=1C1OC2=CC(O)=CC(O)=C2CC1OC(=O)C1=CC(O)=C(O)C(O)=C1 LSHVYAFMTMFKBA-UHFFFAOYSA-N 0.000 description 5
- 101000714926 Homo sapiens Taste receptor type 2 member 14 Proteins 0.000 description 5
- 101000838287 Homo sapiens Taste receptor type 2 member 46 Proteins 0.000 description 5
- 102100040649 Taste receptor type 2 member 10 Human genes 0.000 description 5
- 102100036720 Taste receptor type 2 member 14 Human genes 0.000 description 5
- 102100025866 Taste receptor type 2 member 31 Human genes 0.000 description 5
- 102100025861 Taste receptor type 2 member 39 Human genes 0.000 description 5
- 102100029029 Taste receptor type 2 member 46 Human genes 0.000 description 5
- 229950010131 puromycin Drugs 0.000 description 5
- 230000035945 sensitivity Effects 0.000 description 5
- 230000001953 sensory effect Effects 0.000 description 5
- 241000283690 Bos taurus Species 0.000 description 4
- 241000588724 Escherichia coli Species 0.000 description 4
- 101000787841 Homo sapiens Taste receptor type 2 member 31 Proteins 0.000 description 4
- 241001465754 Metazoa Species 0.000 description 4
- 108091028043 Nucleic acid sequence Proteins 0.000 description 4
- 238000010367 cloning Methods 0.000 description 4
- 239000002299 complementary DNA Substances 0.000 description 4
- 238000012258 culturing Methods 0.000 description 4
- 230000000694 effects Effects 0.000 description 4
- 102000004169 proteins and genes Human genes 0.000 description 4
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 3
- QGWNDRXFNXRZMB-UUOKFMHZSA-K GDP(3-) Chemical group C1=NC=2C(=O)NC(N)=NC=2N1[C@@H]1O[C@H](COP([O-])(=O)OP([O-])([O-])=O)[C@@H](O)[C@H]1O QGWNDRXFNXRZMB-UUOKFMHZSA-K 0.000 description 3
- 108010043121 Green Fluorescent Proteins Proteins 0.000 description 3
- 102000004144 Green Fluorescent Proteins Human genes 0.000 description 3
- 101000838273 Homo sapiens Taste receptor type 2 member 43 Proteins 0.000 description 3
- 241000700159 Rattus Species 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 102100029031 Taste receptor type 2 member 43 Human genes 0.000 description 3
- 238000003556 assay Methods 0.000 description 3
- 230000003247 decreasing effect Effects 0.000 description 3
- 229940079593 drug Drugs 0.000 description 3
- 239000003814 drug Substances 0.000 description 3
- 235000013305 food Nutrition 0.000 description 3
- 230000006870 function Effects 0.000 description 3
- 239000005090 green fluorescent protein Substances 0.000 description 3
- QGWNDRXFNXRZMB-UHFFFAOYSA-N guanidine diphosphate Natural products C1=2NC(N)=NC(=O)C=2N=CN1C1OC(COP(O)(=O)OP(O)(O)=O)C(O)C1O QGWNDRXFNXRZMB-UHFFFAOYSA-N 0.000 description 3
- 210000004962 mammalian cell Anatomy 0.000 description 3
- 238000004519 manufacturing process Methods 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 238000002360 preparation method Methods 0.000 description 3
- 239000002904 solvent Substances 0.000 description 3
- JKMHFZQWWAIEOD-UHFFFAOYSA-N 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-UHFFFAOYSA-N 0.000 description 2
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 description 2
- 241000894006 Bacteria Species 0.000 description 2
- 238000003650 Calcium Assay Kit Methods 0.000 description 2
- 241000282472 Canis lupus familiaris Species 0.000 description 2
- 108091033380 Coding strand Proteins 0.000 description 2
- 102000053602 DNA Human genes 0.000 description 2
- 241000283086 Equidae Species 0.000 description 2
- 241000282326 Felis catus Species 0.000 description 2
- 241000282560 Macaca mulatta Species 0.000 description 2
- 241000699666 Mus <mouse, genus> Species 0.000 description 2
- 102000016978 Orphan receptors Human genes 0.000 description 2
- 108070000031 Orphan receptors Proteins 0.000 description 2
- 241000288906 Primates Species 0.000 description 2
- 241000283984 Rodentia Species 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 241000282887 Suidae Species 0.000 description 2
- 102000014384 Type C Phospholipases Human genes 0.000 description 2
- 108010079194 Type C Phospholipases Proteins 0.000 description 2
- 108091023045 Untranslated Region Proteins 0.000 description 2
- 108010084455 Zeocin Proteins 0.000 description 2
- 230000004913 activation Effects 0.000 description 2
- 239000000556 agonist Substances 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 238000007796 conventional method Methods 0.000 description 2
- 239000000539 dimer Substances 0.000 description 2
- 239000000796 flavoring agent Substances 0.000 description 2
- 235000019634 flavors Nutrition 0.000 description 2
- 230000001965 increasing effect Effects 0.000 description 2
- 244000144972 livestock Species 0.000 description 2
- 230000035772 mutation Effects 0.000 description 2
- 239000002773 nucleotide Substances 0.000 description 2
- 125000003729 nucleotide group Chemical group 0.000 description 2
- CWCMIVBLVUHDHK-ZSNHEYEWSA-N phleomycin D1 Chemical compound N([C@H](C(=O)N[C@H](C)[C@@H](O)[C@H](C)C(=O)N[C@@H]([C@H](O)C)C(=O)NCCC=1SC[C@@H](N=1)C=1SC=C(N=1)C(=O)NCCCCNC(N)=N)[C@@H](O[C@H]1[C@H]([C@@H](O)[C@H](O)[C@H](CO)O1)O[C@@H]1[C@H]([C@@H](OC(N)=O)[C@H](O)[C@@H](CO)O1)O)C=1N=CNC=1)C(=O)C1=NC([C@H](CC(N)=O)NC[C@H](N)C(N)=O)=NC(N)=C1C CWCMIVBLVUHDHK-ZSNHEYEWSA-N 0.000 description 2
- 108090000765 processed proteins & peptides Proteins 0.000 description 2
- 230000019491 signal transduction Effects 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- 230000014616 translation Effects 0.000 description 2
- 102000040650 (ribonucleotides)n+m Human genes 0.000 description 1
- NCYCYZXNIZJOKI-IOUUIBBYSA-N 11-cis-retinal Chemical compound O=C/C=C(\C)/C=C\C=C(/C)\C=C\C1=C(C)CCCC1(C)C NCYCYZXNIZJOKI-IOUUIBBYSA-N 0.000 description 1
- BFSVOASYOCHEOV-UHFFFAOYSA-N 2-diethylaminoethanol Chemical compound CCN(CC)CCO BFSVOASYOCHEOV-UHFFFAOYSA-N 0.000 description 1
- 241000243290 Aequorea Species 0.000 description 1
- BHPQYMZQTOCNFJ-UHFFFAOYSA-N Calcium cation Chemical compound [Ca+2] BHPQYMZQTOCNFJ-UHFFFAOYSA-N 0.000 description 1
- 241000208365 Celastraceae Species 0.000 description 1
- 108091026890 Coding region Proteins 0.000 description 1
- 230000006820 DNA synthesis Effects 0.000 description 1
- 102000004163 DNA-directed RNA polymerases Human genes 0.000 description 1
- 108090000626 DNA-directed RNA polymerases Proteins 0.000 description 1
- 229920002307 Dextran Polymers 0.000 description 1
- 102000005915 GABA Receptors Human genes 0.000 description 1
- 108010005551 GABA Receptors Proteins 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- 102100023954 Guanine nucleotide-binding protein subunit alpha-15 Human genes 0.000 description 1
- 239000007995 HEPES buffer Substances 0.000 description 1
- 241000238631 Hexapoda Species 0.000 description 1
- 101000888145 Homo sapiens Guanine nucleotide-binding protein G(t) subunit alpha-3 Proteins 0.000 description 1
- 101000904080 Homo sapiens Guanine nucleotide-binding protein subunit alpha-15 Proteins 0.000 description 1
- 101000829138 Homo sapiens Somatostatin receptor type 3 Proteins 0.000 description 1
- 101100152261 Homo sapiens TAS2R16 gene Proteins 0.000 description 1
- 101000626659 Homo sapiens Taste receptor type 2 member 1 Proteins 0.000 description 1
- 101000714920 Homo sapiens Taste receptor type 2 member 13 Proteins 0.000 description 1
- 101000674770 Homo sapiens Taste receptor type 2 member 19 Proteins 0.000 description 1
- 101000674784 Homo sapiens Taste receptor type 2 member 20 Proteins 0.000 description 1
- 101000626662 Homo sapiens Taste receptor type 2 member 3 Proteins 0.000 description 1
- 101000674777 Homo sapiens Taste receptor type 2 member 30 Proteins 0.000 description 1
- 101000626629 Homo sapiens Taste receptor type 2 member 4 Proteins 0.000 description 1
- 101000596268 Homo sapiens Taste receptor type 2 member 40 Proteins 0.000 description 1
- 101000596174 Homo sapiens Taste receptor type 2 member 41 Proteins 0.000 description 1
- 101000838279 Homo sapiens Taste receptor type 2 member 42 Proteins 0.000 description 1
- 101000838288 Homo sapiens Taste receptor type 2 member 45 Proteins 0.000 description 1
- 101000652662 Homo sapiens Taste receptor type 2 member 5 Proteins 0.000 description 1
- 101000836166 Homo sapiens Taste receptor type 2 member 50 Proteins 0.000 description 1
- 101000836159 Homo sapiens Taste receptor type 2 member 60 Proteins 0.000 description 1
- 101000652668 Homo sapiens Taste receptor type 2 member 7 Proteins 0.000 description 1
- 101000652677 Homo sapiens Taste receptor type 2 member 8 Proteins 0.000 description 1
- 101000715025 Homo sapiens Taste receptor type 2 member 9 Proteins 0.000 description 1
- 239000012097 Lipofectamine 2000 Substances 0.000 description 1
- 241000699670 Mus sp. Species 0.000 description 1
- 102000008300 Mutant Proteins Human genes 0.000 description 1
- 108010021466 Mutant Proteins Proteins 0.000 description 1
- 229930193140 Neomycin Natural products 0.000 description 1
- AYRXSINWFIIFAE-UHFFFAOYSA-N O6-alpha-D-Galactopyranosyl-D-galactose Natural products OCC1OC(OCC(O)C(O)C(O)C(O)C=O)C(O)C(O)C1O AYRXSINWFIIFAE-UHFFFAOYSA-N 0.000 description 1
- 102000012547 Olfactory receptors Human genes 0.000 description 1
- 108050002069 Olfactory receptors Proteins 0.000 description 1
- 241000283973 Oryctolagus cuniculus Species 0.000 description 1
- 241000282579 Pan Species 0.000 description 1
- 241000282577 Pan troglodytes Species 0.000 description 1
- 102000007074 Phospholipase C beta Human genes 0.000 description 1
- 108010047834 Phospholipase C beta Proteins 0.000 description 1
- 101000633010 Rattus norvegicus Somatostatin Proteins 0.000 description 1
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 1
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 1
- 102100040756 Rhodopsin Human genes 0.000 description 1
- 108090000820 Rhodopsin Proteins 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- 108091081021 Sense strand Proteins 0.000 description 1
- 235000000336 Solanum dulcamara Nutrition 0.000 description 1
- 102100023803 Somatostatin receptor type 3 Human genes 0.000 description 1
- 102100024845 Taste receptor type 2 member 1 Human genes 0.000 description 1
- 102100036737 Taste receptor type 2 member 13 Human genes 0.000 description 1
- 102100021233 Taste receptor type 2 member 19 Human genes 0.000 description 1
- 102100021214 Taste receptor type 2 member 20 Human genes 0.000 description 1
- 102100024842 Taste receptor type 2 member 3 Human genes 0.000 description 1
- 102100021235 Taste receptor type 2 member 30 Human genes 0.000 description 1
- 102100024854 Taste receptor type 2 member 4 Human genes 0.000 description 1
- 102100035240 Taste receptor type 2 member 40 Human genes 0.000 description 1
- 102100035216 Taste receptor type 2 member 41 Human genes 0.000 description 1
- 102100029030 Taste receptor type 2 member 42 Human genes 0.000 description 1
- 102100028980 Taste receptor type 2 member 45 Human genes 0.000 description 1
- 102100030827 Taste receptor type 2 member 5 Human genes 0.000 description 1
- 102100027220 Taste receptor type 2 member 50 Human genes 0.000 description 1
- 102100027216 Taste receptor type 2 member 60 Human genes 0.000 description 1
- 102100030842 Taste receptor type 2 member 7 Human genes 0.000 description 1
- 102100030839 Taste receptor type 2 member 8 Human genes 0.000 description 1
- 102100036711 Taste receptor type 2 member 9 Human genes 0.000 description 1
- 239000004098 Tetracycline Substances 0.000 description 1
- 108010022394 Threonine synthase Proteins 0.000 description 1
- 241000209140 Triticum Species 0.000 description 1
- 235000021307 Triticum Nutrition 0.000 description 1
- 229960000723 ampicillin Drugs 0.000 description 1
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 210000004102 animal cell Anatomy 0.000 description 1
- 230000000692 anti-sense effect Effects 0.000 description 1
- 239000012131 assay buffer Substances 0.000 description 1
- 102000016966 beta-2 Adrenergic Receptors Human genes 0.000 description 1
- 108010014499 beta-2 Adrenergic Receptors Proteins 0.000 description 1
- 230000005540 biological transmission Effects 0.000 description 1
- 238000009395 breeding Methods 0.000 description 1
- 230000001488 breeding effect Effects 0.000 description 1
- 229910001424 calcium ion Inorganic materials 0.000 description 1
- 239000001506 calcium phosphate Substances 0.000 description 1
- 229910000389 calcium phosphate Inorganic materials 0.000 description 1
- 235000011010 calcium phosphates Nutrition 0.000 description 1
- 238000000423 cell based assay Methods 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 230000005754 cellular signaling Effects 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 230000001268 conjugating effect Effects 0.000 description 1
- 230000021615 conjugation Effects 0.000 description 1
- 210000004748 cultured cell Anatomy 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 102000004419 dihydrofolate reductase Human genes 0.000 description 1
- 239000012636 effector Substances 0.000 description 1
- 238000004520 electroporation Methods 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 210000003527 eukaryotic cell Anatomy 0.000 description 1
- 230000005284 excitation Effects 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 239000012737 fresh medium Substances 0.000 description 1
- 238000001641 gel filtration chromatography Methods 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- DLRVVLDZNNYCBX-CQUJWQHSSA-N gentiobiose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1OC[C@@H]1[C@@H](O)[C@H](O)[C@@H](O)C(O)O1 DLRVVLDZNNYCBX-CQUJWQHSSA-N 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 239000005556 hormone Substances 0.000 description 1
- 229940088597 hormone Drugs 0.000 description 1
- 102000056291 human TAS2R10 Human genes 0.000 description 1
- 102000056415 human TAS2R39 Human genes 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 238000007852 inverse PCR Methods 0.000 description 1
- 238000005342 ion exchange Methods 0.000 description 1
- 239000003446 ligand Substances 0.000 description 1
- 239000002502 liposome Substances 0.000 description 1
- 108020004999 messenger RNA Proteins 0.000 description 1
- 239000011259 mixed solution Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 230000036651 mood Effects 0.000 description 1
- 229960004927 neomycin Drugs 0.000 description 1
- 239000002858 neurotransmitter agent Substances 0.000 description 1
- 235000019645 odor Nutrition 0.000 description 1
- 239000000825 pharmaceutical preparation Substances 0.000 description 1
- 229940127557 pharmaceutical product Drugs 0.000 description 1
- 239000013600 plasmid vector Substances 0.000 description 1
- 229920001184 polypeptide Polymers 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 238000001243 protein synthesis Methods 0.000 description 1
- 238000003259 recombinant expression Methods 0.000 description 1
- 230000006798 recombination Effects 0.000 description 1
- 238000005215 recombination Methods 0.000 description 1
- 230000004043 responsiveness Effects 0.000 description 1
- 108091008146 restriction endonucleases Proteins 0.000 description 1
- 210000001995 reticulocyte Anatomy 0.000 description 1
- 238000007423 screening assay Methods 0.000 description 1
- 239000006152 selective media Substances 0.000 description 1
- 238000002741 site-directed mutagenesis Methods 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 210000001584 soft palate Anatomy 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 238000001308 synthesis method Methods 0.000 description 1
- 210000001779 taste bud Anatomy 0.000 description 1
- 229960002180 tetracycline Drugs 0.000 description 1
- 229930101283 tetracycline Natural products 0.000 description 1
- 235000019364 tetracycline Nutrition 0.000 description 1
- 150000003522 tetracyclines Chemical class 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 238000013518 transcription Methods 0.000 description 1
- 230000035897 transcription Effects 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 238000011426 transformation method Methods 0.000 description 1
- 238000013519 translation Methods 0.000 description 1
- QORWJWZARLRLPR-UHFFFAOYSA-H tricalcium bis(phosphate) Chemical compound [Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O QORWJWZARLRLPR-UHFFFAOYSA-H 0.000 description 1
- 239000013638 trimer Substances 0.000 description 1
- 235000019583 umami taste Nutrition 0.000 description 1
- 238000010200 validation analysis Methods 0.000 description 1
Images
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Peptides Or Proteins (AREA)
Description
本発明は、苦味評価のための融合タンパク質および苦味評価方法に関する。 The present invention relates to a fusion protein for bitterness evaluation and a bitterness evaluation method.
飲食物の風味の評価方法としては、専らヒトの感覚に頼った官能評価が利用されている。官能評価は、官能検査、官能試験とも呼ばれ、ヒトの感覚（視覚、聴覚、味覚、臭覚、触覚）を使って対象物を評価するものであり、総合的な風味の評価には適しているが、経験、年齢、個人差、感覚疲労、体調・気分等の生理的変化などの主観的要素が評価に影響する欠点がある。そのため、より客観的な評価が可能であり、再現性が高い評価系の開発が望まれている。 As a method for evaluating the flavor of food and drink, sensory evaluation that relies exclusively on human senses is used. Sensory evaluation, also called sensory test or sensory test, evaluates an object using human senses (sight, hearing, taste, smell, touch), and is suitable for comprehensive flavor evaluation. However, there is a drawback that subjective factors such as experience, age, individual differences, sensory fatigue, and physiological changes such as physical condition and mood affect the evaluation. Therefore, it is desired to develop an evaluation system that enables more objective evaluation and has high reproducibility.
甘み、うま味および苦味の伝達には、軟口蓋や舌に存在している味蕾にある味細胞に発現しているGタンパク質共役受容体（GPCR）が関与することが知られている。GPCRは、ヒトでは約750種のGPCR遺伝子があり、ヒトゲノム中での最大のスーパーファミリーである。750種の約半分は匂いを感ずる嗅覚受容体である。残り半分は、光や味物質、ホルモン、神経伝達物質などを認識する受容体である。ヒトGPCRのうち約150種類は、働きが分かっていないオーファン受容体であるとされている。またGPCRは、アミノ酸配列や機能の類似性に基づいて6つのサブグループに分類されている。GPCRは、Gタンパク質と共役して、カルシウムイオンなどのセカンドメッセンジャーを介し、情報を伝達する。Gタンパク質はGs、Gi、Gq、Gtなどのファミリーに分類され、それぞれ役割が異なっている。また、Gタンパク質は、Gα、Gβ、Gγのサブユニットから構成されている。例えば、Gqファミリーのαサブユニット（Gqα）はホスホリパーゼC-βの活性化に関わっている。 G protein-coupled receptors (GPCRs) expressed on taste cells in the taste buds present on the soft palate and tongue are known to be involved in the transmission of sweetness, umami and bitterness. GPCRs are the largest superfamily in the human genome, with approximately 750 GPCR genes in humans. About half of the 750 species are olfactory receptors that sense odors. The other half are receptors that recognize light, taste substances, hormones, neurotransmitters, and so on. About 150 types of human GPCRs are said to be orphan receptors of unknown function. GPCRs are also divided into 6 subgroups based on their amino acid sequence and functional similarity. GPCRs couple with G proteins to transmit information via second messengers such as calcium ions. G proteins are classified into families such as Gs, Gi, Gq, and Gt, each of which has a different role. In addition, G protein is composed of subunits of Gα, Gβ, and Gγ. For example, the α subunit of the Gq family (Gqα) is involved in the activation of phospholipase C-β.
ヒトにおける苦味の認識は、苦味受容体、Taste type 2 receptor（TAS2R, T2R）で受容されていることが知られている。TAS2RはGPCRの一種であり、光受容を担うロドプシンに代表されるクラスAに属している。ヒトではおよそ25種類の苦味受容体が機能しているとされている。他のGPCRと同様に、TAS2Rに共役するGタンパク質は、Gα、Gβ、Gγのサブユニットから構成されており、Gαにグアノシン５’二リン酸（GDP）が結合していると、Gタンパク質は不活性型として三量体構造Gαβγを取ってTAS2Rと結合している。苦味物質がTAS2Rに結合すると、Gαに結合していたGDPがGTPに置換されて、GαとGβγ二量体、TAS2Rに解離する。解離したGα、Gβγ二量体はそれぞれが別のエフェクター分子に作用してシグナルを伝達すると考えられている。味細胞におけるGαは主にガストデューシン（gustducin）とされており、苦味受容体はガストデューシンと共役し、細胞内のセカンドメッセンジャーを介する情報伝達過程を経て、細胞内のカルシウム濃度が上昇し苦味シグナルを伝達することが知られている。 It is known that the recognition of bitter taste in humans is accepted by the bitter taste receptor, Taste type 2 receptor (TAS2R, T2R). TAS2R is a type of GPCR and belongs to class A represented by rhodopsin, which is responsible for photoreception. Approximately 25 types of bitter taste receptors are said to be functioning in humans. Like other GPCRs, TAS2R-coupled G proteins are composed of subunits of Gα, Gβ, and Gγ, and when Gα is bound to guanosine 5'diphosphate (GDP), the G protein is It takes the trimer structure Gαβγ as an inactive form and binds to TAS2R. When a bitter substance binds to TAS2R, GDP bound to Gα is replaced with GTP and dissociated into Gα and Gβγ dimer, TAS2R. The dissociated Gα and Gβγ dimers are thought to act on different effector molecules to transmit signals. Gα in taste cells is mainly considered to be gustducin, and the bitter taste receptor is coupled to gustducin, and the intracellular calcium concentration rises through the signal transduction process via the intracellular second messenger. It is known to transmit bitterness signals.
苦味受容体の活性化を検出する方法としては、他の一般的なGPCRと同様、細胞内のカルシウム動態測定がよく用いられている。このような細胞ベースのアッセイは、苦味物質や苦味調節物質の発見および検証に好ましい。培養細胞に一過性にTAS2Rおよびそれに共役するGαを発現させる方法もあるが、より安定した再現性のある結果を得るためには、TAS2RとGαの両者を安定発現させた細胞を用いた方法が望ましい。 As a method for detecting the activation of bitter taste receptors, intracellular calcium kinetics measurement is often used as in other general GPCRs. Such cell-based assays are preferred for the discovery and validation of bitter and bitter taste regulators. There is also a method of transiently expressing TAS2R and Gα conjugated to it in cultured cells, but in order to obtain more stable and reproducible results, a method using cells in which both TAS2R and Gα are stably expressed. Is desirable.
Gαの中でもGα16は、HEK293細胞など哺乳類細胞において内在性のβ/γサブユニットと複合体を形成することができ、HEK293細胞を用いた評価系において感度のよい測定を可能とするが、味覚受容体との共役には適していない。一方、苦味受容体はガストデューシンと共役する。このことから、Gα15またはGα16の最後の44個のアミノ酸をガストデューシンの44個のアミノ酸に置換したキメラGαタンパク質を用いて、味覚シグナルを測定した例がある（特許文献１）。 Among Gα, Gα16 can form a complex with endogenous β / γ subunits in mammalian cells such as HEK293 cells, and enables sensitive measurement in an evaluation system using HEK293 cells, but taste receptor. Not suitable for conjugation with the body. On the other hand, the bitter taste receptor is conjugate with gustducin. From this, there is an example in which the taste signal was measured using a chimeric Gα protein in which the last 44 amino acids of Gα15 or Gα16 were replaced with 44 amino acids of gustducin (Patent Document 1).
GPCRとGαを融合タンパク質として発現させる方法を用いることによって、その受容体のリガンドの同定や機能の解明を実践できることが知られている（特許文献２、非特許文献１）。しかし、これらは、GABA受容体やβ２アドレナリン受容体の例であり、アミノ酸配列も機能も異なる味覚受容体について苦味物質や苦味調節物質の苦味応答性を安定して測定できる系は確立されていない。さらに、苦味受容体の中には、オーファン受容体の中に含まれ未同定の受容体があり、実際、苦味受容体との対応が未だ明らかでない苦味成分が多数残されている。 It is known that the identification of the ligand of the receptor and the elucidation of the function can be practiced by using the method of expressing GPCR and Gα as a fusion protein (Patent Document 2 and Non-Patent Document 1). However, these are examples of GABA receptors and β2 adrenergic receptors, and a system that can stably measure the bitterness responsiveness of bitterness substances and bitterness regulators for taste receptors having different amino acid sequences and functions has not been established. .. Furthermore, among the bitter taste receptors, there are unidentified receptors contained in the orphan receptor, and in fact, many bitter taste components whose correspondence with the bitter taste receptor has not yet been clarified remain.
本発明は、苦味受容体とＧタンパク質の融合タンパク質を安定発現する形質転換細胞を用いて新たな苦味の評価系を構築し、客観性および再現性の高い苦味評価方法を提供することを課題とする。また、本発明は、当該苦味評価方法に用いる新たな苦味の評価系を構築するための融合タンパク質、ポリヌクレオチド、発現ベクター、形質転換細胞を提供することを課題とする。 An object of the present invention is to construct a new bitterness evaluation system using transformed cells that stably express a fusion protein of a bitterness receptor and a G protein, and to provide a bitterness evaluation method with high objectivity and reproducibility. do. Another object of the present invention is to provide a fusion protein, a polynucleotide, an expression vector, and transformed cells for constructing a new bitterness evaluation system used in the bitterness evaluation method.
本発明は、上記課題を解決するために、以下の各発明を包含する。
［１］苦味受容体ＴＡＳ２Ｒと、該苦味受容体に共役するＧタンパク質αサブユニットとを含む融合タンパク質。
［２］前記Ｇタンパク質αサブユニットが、Ｇｑファミリーのαサブユニットとガストデューシンのキメラタンパク質である前記［１］に記載の融合タンパク質。
［３］前記ＧｑファミリーのαサブユニットがＧα１５またはＧα１６である前記［２］に記載の融合タンパク質。
［４］配列番号１、３、５、７、９、２５または２７に示されるアミノ酸配列と同一または実質的に同一のアミノ酸配列を有する前記［３］に記載の融合タンパク質。
［５］前記［１］〜［４］のいずれかに記載の融合タンパク質をコードするポリヌクレオチド。
［６］前記［５］に記載のポリヌクレオチドを含む発現ベクター。
［７］前記［６］に記載の発現ベクターが導入されている形質転換細胞。
［８］前記［１］〜［４］のいずれかに記載の融合タンパク質を安定発現する前記［７］に記載の形質転換細胞。
［９］前記［７］または［８］に記載の形質転換細胞に被験物質を接触させる工程と、該形質転換細胞内のカルシウム濃度を測定する工程を含むことを特徴とする被験物質の苦味評価方法。
［１０］前記［７］または［８］に記載の形質転換細胞に苦味物質と被験物質を接触させる工程と、該形質転換細胞内のカルシウム濃度を測定する工程と、得られたカルシウム濃度を苦味物質のみを接触させた形質転換細胞内のカルシウム濃度と比較し、カルシウム濃度を増加または低下させる被験物質を選択する工程を含む苦味調節物質のスクリーニング方法。
The present invention includes the following inventions in order to solve the above problems.
[1] A fusion protein containing the bitter taste receptor TAS2R and a G protein α subunit conjugated to the bitter taste receptor.
[2] The fusion protein according to the above [1], wherein the G protein α subunit is a chimeric protein of the Gq family α subunit and gustducin.
[3] The fusion protein according to the above [2], wherein the α subunit of the Gq family is Gα15 or Gα16.
[4] The fusion protein according to the above [3], which has the same or substantially the same amino acid sequence as the amino acid sequence shown in SEQ ID NO: 1, 3, 5, 7, 9, 25 or 27.
[5] A polynucleotide encoding the fusion protein according to any one of the above [1] to [4].
[6] An expression vector containing the polynucleotide according to the above [5].
[7] Transformed cells into which the expression vector according to [6] has been introduced.
[8] The transformed cell according to the above [7], which stably expresses the fusion protein according to any one of the above [1] to [4].
[9] Evaluation of bitterness of the test substance, which comprises a step of bringing the test substance into contact with the transformed cell according to the above [7] or [8] and a step of measuring the calcium concentration in the transformed cell. Method.
[10] The step of bringing the bitter taste substance and the test substance into contact with the transformed cell according to the above [7] or [8], the step of measuring the calcium concentration in the transformed cell, and the obtained calcium concentration being bitter taste. A method for screening a bitterness-regulating substance, which comprises a step of selecting a test substance for increasing or decreasing the calcium concentration as compared with the calcium concentration in the transformed cells to which only the substance is contacted.
本発明によれば、客観性および再現性の高い苦味評価方法を提供することができる。また、本発明によれば、当該苦味評価方法に用いる新たな苦味の評価系を構築するための融合タンパク質、ポリヌクレオチド、発現ベクター、形質転換細胞を提供することができる。さらに、本発明の形質転換細胞を用いた苦味調節物質のスクリーニング方法を提供することができる。 According to the present invention, it is possible to provide a bitterness evaluation method having high objectivity and reproducibility. Further, according to the present invention, it is possible to provide a fusion protein, a polynucleotide, an expression vector, and a transformed cell for constructing a new bitterness evaluation system used in the bitterness evaluation method. Furthermore, it is possible to provide a method for screening a bitterness regulator using the transformed cells of the present invention.
〔融合タンパク質〕
本発明は、苦味受容体ＴＡＳ２Ｒと、該苦味受容体に共役するＧタンパク質αサブユニットとを含む融合タンパク質を提供する。本発明の融合タンパク質に含まれる苦味受容体は、ＴＡＳ２Ｒファミリーに属するものであればどのような動物由来のＴＡＳ２Ｒでもよい。なかでも、ヒト、霊長類、げっ歯類、家畜（ウシ、ウマ、ブタ等）、ペット（イヌ、ネコ等）由来のＴＡＳ２Ｒが好ましい。特にヒトを対象とする飲食品の開発に利用する場合は、ヒトのＴＡＳ２Ｒを用いることが好ましい。
[Fusion protein]
The present invention provides a fusion protein comprising the bitter taste receptor TAS2R and a G protein α subunit conjugated to the bitter taste receptor. The bitter taste receptor contained in the fusion protein of the present invention may be TAS2R derived from any animal as long as it belongs to the TAS2R family. Of these, TAS2R derived from humans, primates, rodents, livestock (cattle, horses, pigs, etc.) and pets (dogs, cats, etc.) is preferable. In particular, when it is used for the development of foods and drinks for humans, it is preferable to use human TAS2R.
ＴＡＳ２Ｒファミリーには多くの種類があり、ヒトでは２５種類（TAS2R1、TAS2R3、TAS2R4、TAS2R5、TAS2R7、TAS2R8、TAS2R9、TAS2R10、TAS2R13、TAS2R14、TAS2R16、TAS2R38、TAS2R39、TAS2R40、TAS2R41、TAS2R42、TAS2R43、TAS2R44、TAS2R45、TAS2R46、TAS2R47、TAS2R48、TAS2R49、TAS2R50、TAS2R60）のＴＡＳ２Ｒが知られている（Chandrashekar J et al., The receptors and cells for mammalian taste. Nature, 2006; 444; 288-294）。本発明者らは、TAS2R10、TAS2R14、TAS2R16、TAS2R38、TAS2R39、TAS2R43、TAS2R44およびTAS2R46を用いて融合タンパク質を作製し、目的の効果を奏することを確認している。それゆえ、ヒトの他のＴＡＳ２Ｒや他の動物のＴＡＳ２Ｒを用いて作製した融合タンパク質も目的の効果を奏することが容易に推認できる。公知のＴＡＳ２Ｒのアミノ酸配列およびそれをコードする遺伝子の塩基配列は、公知のデータベース（DDBJ／GenBank／EMBL）等から取得することができる。 There are many types in the TAS2R family, and 25 types in humans (TAS2R1, TAS2R3, TAS2R4, TAS2R5, TAS2R7, TAS2R8, TAS2R9, TAS2R10, TAS2R13, TAS2R14, TAS2R16, TAS2R38, TAS2R39, TAS2R40, TAS2R41, TAS2R42. , TAS2R45, TAS2R46, TAS2R47, TAS2R48, TAS2R49, TAS2R50, TAS2R60) are known (Chandrashekar J et al., The receptors and cells for mammalian taste. Nature, 2006; 444; 288-294). The present inventors have made fusion proteins using TAS2R10, TAS2R14, TAS2R16, TAS2R38, TAS2R39, TAS2R43, TAS2R44 and TAS2R46, and confirmed that they achieve the desired effects. Therefore, it can be easily inferred that fusion proteins prepared by using TAS2R of other humans or TAS2R of other animals also exert the desired effect. The known amino acid sequence of TAS2R and the base sequence of the gene encoding the same can be obtained from a known database (DDBJ / GenBank / EMBL) or the like.
本発明の融合タンパク質に含まれるＧタンパク質αサブユニットは、融合される苦味受容体に共役することができ、かつホスホリパーゼＣを活性化するαサブユニットであることが好ましい。このようなＧタンパク質αサブユニットとして、Ｇｑファミリーのαサブユニットとガストデューシンのキメラタンパク質を好適に用いることができる。Ｇｑファミリーのαサブユニットは、Ｇタンパク質共役型受容体（GPCR）と共役してホスホリパーゼＣを活性化するαサブユニットであり、ガストデューシンは、ＴＡＳ２Ｒが苦味物質と結合すると共役するＧタンパク質αサブユニットであることが知られている。それゆえ、Ｇｑファミリーのαサブユニットとガストデューシンのキメラタンパク質は、本発明の融合タンパク質に含まれるＧタンパク質αサブユニットとして好適である。 The G protein α subunit contained in the fusion protein of the present invention is preferably an α subunit that can be conjugated to the bitter taste receptor to be fused and activates phospholipase C. As such a G protein α subunit, a chimeric protein of the Gq family α subunit and gustducin can be preferably used. The α subunit of the Gq family is an α subunit that binds to a G protein-coupled receptor (GPCR) to activate phospholipase C, and gustducin is a G protein α that binds to TAS2R when it binds to a bitter substance. It is known to be a subunit. Therefore, the chimeric protein of the α subunit of the Gq family and gustducin is suitable as the G protein α subunit contained in the fusion protein of the present invention.
Ｇｑファミリーのαサブユニットには、Ｇαｑ、Ｇα１１、Ｇα１４、Ｇα１５およびＧα１６が含まれ、これらはいずれも本発明の融合タンパク質に含まれるＧタンパク質αサブユニットとして好適に用いることができる。好ましくは、Ｇα１５またはＧα１６である。一般に、ヒトの遺伝子はＧα１６と称され、マウスやらラットのオルソログはＧα１５と称される。Ｇｑファミリーのαサブユニットおよびガストデューシンはどのような動物由来のものでもよいが、ヒト、霊長類、げっ歯類、家畜（ウシ、ウマ、ブタ等）、ペット（イヌ、ネコ等）由来のものを好適に用いることができる。公知のＧｑファミリーのαサブユニットおよび公知のガストデューシンのアミノ酸配列およびそれをコードする遺伝子の塩基配列は、公知のデータベース（DDBJ／GenBank／EMBL）等から取得することができる。表１にヒト、マウス、ラット、ウシ、チンパンジー、アカゲザルのＧα１５またはＧα１６の塩基配列およびアミノ酸配列のアクセッション番号を示す。表２に、ヒト、マウス、ラット、ウシ、チンパンジー、アカゲザルのガストデューシンの塩基配列およびアミノ酸配列のアクセッション番号を示す。 The α subunit of the Gq family includes Gαq, Gα11, Gα14, Gα15 and Gα16, all of which can be suitably used as the G protein α subunit contained in the fusion protein of the present invention. Preferably, it is Gα15 or Gα16. Generally, the human gene is called Gα16, and the ortholog of mouse or rat is called Gα15. The α subunit and gustducin of the Gq family can be derived from any animal, but are derived from humans, primates, rodents, livestock (cattle, horses, pigs, etc.) and pets (dogs, cats, etc.). Can be preferably used. The α-subunit of the known Gq family, the amino acid sequence of the known gustducin, and the base sequence of the gene encoding the same can be obtained from a known database (DDBJ / GenBank / EMBL) or the like. Table 1 shows the accession numbers of the nucleotide sequences and amino acid sequences of Gα15 or Gα16 of human, mouse, rat, bovine, chimpanzee, and rhesus monkey. Table 2 shows the accession numbers of the nucleotide sequences and amino acid sequences of gastoducin in humans, mice, rats, cattle, chimpanzees, and rhesus monkeys.
Ｇｑファミリーのαサブユニットとガストデューシンのキメラタンパク質は、ＧｑファミリーのαサブユニットのＣ末端側の約３６〜約４４アミノ酸がガストデューシンのＣ末端側の約３６〜約４４アミノ酸に置換されたものを好適に用いることができる。より好ましくは、ＧｑファミリーのαサブユニットのＣ末端側の約３８〜約４２アミノ酸がガストデューシンのＣ末端側の約３８〜約４２アミノ酸に置換されたものである。このようなＧｑファミリーのαサブユニットとガストデューシンのキメラタンパク質として、例えば特表２００６−５２４４８３やＵｅｄａらの論文（Ueda T，et al., J. Neurosci，23，7376-7380（2003））に記載のキメラタンパク質を好適に用いることができる。 In the chimeric protein of the α subunit of the Gq family and gustducin, about 36 to about 44 amino acids on the C-terminal side of the α subunit of the Gq family are replaced with about 36 to about 44 amino acids on the C-terminal side of gustducin. Can be preferably used. More preferably, about 38 to about 42 amino acids on the C-terminal side of the α subunit of the Gq family are replaced with about 38 to about 42 amino acids on the C-terminal side of gustducin. As chimeric proteins of such Gq family α subunit and gustducin, for example, the papers of Special Table 2006-524483 and Ueda et al. (Ueda T, et al., J. Neurosci, 23, 7376-7380 (2003)). The chimeric protein described in 1 can be preferably used.
本発明の融合タンパク質において、ＴＡＳ２ＲはＮ末端側に配置され、Ｇタンパク質αサブユニットはＣ末端側に配置される。本発明の融合タンパク質はＴＡＳ２ＲおよびＧタンパク質αサブユニット以外のアミノ酸配列を含んでいてもよく、このようなアミノ酸配列としては、例えばタグ配列、リンカー配列、マーカー配列などが挙げられる。 In the fusion protein of the present invention, the TAS2R is located on the N-terminal side and the G protein α subunit is located on the C-terminal side. The fusion protein of the present invention may contain an amino acid sequence other than TAS2R and G protein α subunit, and examples of such an amino acid sequence include a tag sequence, a linker sequence, a marker sequence and the like.
本発明の融合タンパク質は、配列番号１、３、５、７、９、２５または２７に示されるアミノ酸配列と同一または実質的に同一のアミノ酸配列を有することが好ましい。配列番号１に示されるアミノ酸配列はTAS2R14-改変型Gα16gust40融合タンパク質のアミノ酸配列、配列番号３に示されるアミノ酸配列はTAS2R16-改変型Gα16gust40融合タンパク質のアミノ酸配列、配列番号５に示されるアミノ酸配列はTAS2R38-改変型Gα16gust40融合タンパク質のアミノ酸配列、配列番号７に示されるアミノ酸配列はTAS2R43-改変型Gα16gust40融合タンパク質のアミノ酸配列、配列番号９に示されるアミノ酸配列はTAS2R14-改変型Gα16gust40融合タンパク質のアミノ酸配列、配列番号２５に示されるアミノ酸配列はTAS2R10-改変型Gα16gust40融合タンパク質のアミノ酸配列、配列番号２７に示されるアミノ酸配列はTAS2R39-改変型Gα16gust40融合タンパク質のアミノ酸配列である（実施例参照）。本発明の融合タンパク質は、配列番号１、３、５、７、９、２５または２７に示されるアミノ酸配列と同一または実質的に同一のアミノ酸配列以外の配列を含んでいてもよい。例えば、マーカー配列、タグ配列、リンカー配列などを含んでいてもよい。また、本発明の融合タンパク質は、配列番号１、３、５、７、９、２５または２７に示されるアミノ酸配列と同一または実質的に同一のアミノ酸配列のみからなるものであってもよい。 The fusion protein of the present invention preferably has the same or substantially the same amino acid sequence as the amino acid sequence shown in SEQ ID NO: 1, 3, 5, 7, 9, 25 or 27. The amino acid sequence shown in SEQ ID NO: 1 is the amino acid sequence of the TAS2R14-modified Gα16gust40 fusion protein, the amino acid sequence shown in SEQ ID NO: 3 is the amino acid sequence of the TAS2R16-modified Gα16gust40 fusion protein, and the amino acid sequence shown in SEQ ID NO: 5 is TAS2R38. -The amino acid sequence of the modified Gα16gust40 fusion protein, the amino acid sequence shown in SEQ ID NO: 7 is TAS2R43-The amino acid sequence of the modified Gα16gust40 fusion protein, the amino acid sequence shown in SEQ ID NO: 9 is the amino acid sequence of TAS2R14-modified Gα16gust40 fusion protein, The amino acid sequence shown in SEQ ID NO: 25 is the amino acid sequence of the TAS2R10-modified Gα16gust40 fusion protein, and the amino acid sequence shown in SEQ ID NO: 27 is the amino acid sequence of the TAS2R39-modified Gα16gust40 fusion protein (see Examples). The fusion protein of the present invention may contain a sequence other than the amino acid sequence that is the same as or substantially the same as the amino acid sequence shown in SEQ ID NO: 1, 3, 5, 7, 9, 25 or 27. For example, it may include a marker sequence, a tag sequence, a linker sequence, and the like. Further, the fusion protein of the present invention may consist only of an amino acid sequence that is the same as or substantially the same as the amino acid sequence shown in SEQ ID NO: 1, 3, 5, 7, 9, 25 or 27.
配列番号１に示されるアミノ酸配列と実質的に同一のアミノ酸配列としては、例えば、配列番号１に示されるアミノ酸配列において、１〜数個のアミノ酸が欠失、置換もしくは付加されたアミノ酸配列が挙げられる。「１〜数個のアミノ酸が欠失、置換もしくは付加された」とは、部位特異的突然変異誘発法等の公知の変異ペプチド作製法により欠失、置換もしくは付加できる程度の数（好ましくは１０個以下、より好ましくは７個以下、さらに好ましくは５個以下）のアミノ酸が欠失、置換もしくは付加されることを意味する。このような変異タンパク質は、公知の変異ポリペプチド作製法により人為的に導入された変異を有するタンパク質に限定されるものではなく、天然に存在するタンパク質を単離精製したものであってもよい。また、実質的に同一のアミノ酸配列は、配列番号１に示されるアミノ酸配列と少なくとも８０％同一、より好ましくは少なくとも８５％、９０％、９２％、９５％、９６％、９７％、９８％または９９％同一であるアミノ酸配列が挙げられる。配列番号３、５、７、９、２５または２７に示されるアミノ酸配列と実質的に同一のアミノ酸配列も同様である。 Examples of the amino acid sequence substantially the same as the amino acid sequence shown in SEQ ID NO: 1 include an amino acid sequence in which one to several amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 1. Be done. "One to several amino acids have been deleted, substituted or added" is a number (preferably 10) that can be deleted, substituted or added by a known mutant peptide preparation method such as a site-specific mutagenesis method. It means that less than or equal to, more preferably 7 or less, still more preferably 5 or less) amino acids are deleted, substituted or added. Such a mutant protein is not limited to a protein having a mutation artificially introduced by a known mutant polypeptide production method, and may be a naturally occurring protein isolated and purified. Further, the substantially identical amino acid sequence is at least 80% identical to the amino acid sequence shown in SEQ ID NO: 1, more preferably at least 85%, 90%, 92%, 95%, 96%, 97%, 98% or Amino acid sequences that are 99% identical can be mentioned. The same applies to the amino acid sequence substantially the same as the amino acid sequence shown in SEQ ID NO: 3, 5, 7, 9, 25 or 27.
配列番号１に示されるアミノ酸配列と実質的に同一のアミノ酸配列を有する融合タンパク質としては、配列番号１に示されるアミノ酸配列を有する融合タンパク質と実質的に同質の活性を有する融合タンパク質が好ましい。具体的には、融合タンパク質のＴＡＳ２Ｒ部分が苦味物質と結合した際の細胞内カルシウム濃度の変化量が、配列番号１に示されるアミノ酸配列からなる融合タンパク質と同等（例えば、約０．５〜２倍）であることが挙げられる。配列番号３、５、７、９、２５または２７に示されるアミノ酸配列と実質的に同一のアミノ酸配列を有する融合タンパク質も同様である。 As the fusion protein having substantially the same amino acid sequence as the amino acid sequence shown in SEQ ID NO: 1, a fusion protein having substantially the same activity as the fusion protein having the amino acid sequence shown in SEQ ID NO: 1 is preferable. Specifically, the amount of change in intracellular calcium concentration when the TAS2R portion of the fusion protein binds to the bitterness substance is equivalent to that of the fusion protein consisting of the amino acid sequence shown in SEQ ID NO: 1 (for example, about 0.5 to 2). Double). The same applies to a fusion protein having an amino acid sequence substantially the same as the amino acid sequence shown in SEQ ID NO: 3, 5, 7, 9, 25 or 27.
本発明の融合タンパク質は、公知の遺伝子工学的手法により、本発明の融合タンパク質をコードする遺伝子を発現可能に挿入した組み換え発現ベクターを構築し、これを適当な宿主細胞に導入して組み換えタンパク質として発現させ、精製することにより製造することができる。また、本発明の融合タンパク質は、本発明の融合タンパク質をコードする遺伝子と公知のIn vitro転写・翻訳系（例えば、ウサギ網状赤血球、コムギ胚芽または大腸菌由来の無細胞タンパク質合成系等）を用いて、製造することができる。 For the fusion protein of the present invention, a recombinant expression vector in which a gene encoding the fusion protein of the present invention is inserted in an expressible manner is constructed by a known genetic engineering technique, and this is introduced into an appropriate host cell to obtain a recombinant protein. It can be produced by expressing and purifying. Further, the fusion protein of the present invention uses a gene encoding the fusion protein of the present invention and a known in vitro transcription / translation system (for example, rabbit reticulocyte, wheat germ, cell-free protein synthesis system derived from Escherichia coli, etc.). , Can be manufactured.
〔ポリヌクレオチド〕
本発明のポリヌクレオチドは、上記本発明の融合タンパク質をコードするポリヌクレオチドであればよい。ポリヌクレオチドは、ＲＮＡ（例えば、ｍＲＮＡ）の形態、またはＤＮＡの形態（例えば、ｃＤＮＡまたはゲノムＤＮＡ）で存在することができる。ポリヌクレオチドは、二本鎖でもよく一本鎖でもよい。二本鎖の場合は、二本鎖ＤＮＡ、二本鎖ＲＮＡまたはＤＮＡと ＲＮＡとのハイブリッドのいずれであってもよい。一本鎖の場合は、コード鎖（センス鎖）または非コード鎖（アンチセンス鎖）のいずれであってもよい。また、本発明のポリヌクレオチドは、その５’側または３’側でタグ標識（タグ配列またはマーカー配列）をコードするポリヌクレオチドに融合されていてもよい。さらに、非翻訳領域（ＵＴＲ）の配列やベクター配列（発現ベクター配列を含む）などの配列を含むものであってもよい。
[Polynucleotide]
The polynucleotide of the present invention may be any polynucleotide encoding the fusion protein of the present invention. Polynucleotides can be present in the form of RNA (eg, mRNA), or in the form of DNA (eg, cDNA or genomic DNA). The polynucleotide may be double-stranded or single-stranded. In the case of double-stranded DNA, it may be either double-stranded DNA, double-stranded RNA, or a hybrid of DNA and RNA. In the case of a single strand, it may be either a coding strand (sense strand) or a non-coding strand (antisense strand). Further, the polynucleotide of the present invention may be fused to a polynucleotide encoding a tag label (tag sequence or marker sequence) on the 5'side or 3'side thereof. Furthermore, it may contain a sequence such as an untranslated region (UTR) sequence or a vector sequence (including an expression vector sequence).
本発明のポリヌクレオチドは、公知のＤＮＡ合成法やＰＣＲ法等によって取得することができる。具体的には、例えば、ＴＡＳ２Ｒをコードする遺伝子、Ｇｑファミリーのαサブユニットをコードする遺伝子、ガストデューシンをコードする遺伝子の塩基配列情報を公知のデータベース（DDBJ／GenBank／EMBL）等から取得し、目的の遺伝子領域を増幅するためのプライマーを設計し、対応する動物のゲノムＤＮＡまたはｃＤＮＡ等を鋳型にしてＰＣＲ等を行い、その後に公知の遺伝子組換え技術を用いて各ＤＮＡを融合することにより、取得することができる。 The polynucleotide of the present invention can be obtained by a known DNA synthesis method, PCR method, or the like. Specifically, for example, the base sequence information of the gene encoding TAS2R, the gene encoding the α subunit of the Gq family, and the gene encoding gustducin is obtained from a known database (DDBJ / GenBank / EMBL) or the like. , Design a primer for amplifying the target gene region, perform PCR etc. using the corresponding animal genomic DNA or cDNA as a template, and then fuse each DNA using a known gene recombination technique. Can be obtained by
配列番号１、３、５、７、９、２５または２７に示されるアミノ酸配列からなる融合タンパク質をコードするポリヌクレオチドとしては、例えば、それぞれ、配列番号２、４、６、８、１０、２６または２８に示される塩基配列からなるポリヌクレオチドが挙げられるがこれに限定されない。配列番号１、３、５、７、９、２５または２７に示されるアミノ酸配列と同一または実質的に同一のアミノ酸配列をコードするポリヌクレオチドであれば塩基配列は限定されない。好ましいポリヌクレオチドとしては、例えば、配列番号２、４、６、８、１０、２６または２８に示される塩基配列のいずれかと８０％同一、より好ましくは少なくとも８５％、９０％、９２％、９５％、９６％、９７％、９８％または９９％同一である塩基配列からなるポリヌクレオチドが挙げられる。 Examples of the polynucleotide encoding the fusion protein consisting of the amino acid sequence shown in SEQ ID NO: 1, 3, 5, 7, 9, 25 or 27 include, for example, SEQ ID NO: 2, 4, 6, 8, 10, 26 or, respectively. Examples include, but are not limited to, polynucleotides consisting of the nucleotide sequence shown in 28. The base sequence is not limited as long as it is a polynucleotide encoding the same or substantially the same amino acid sequence as the amino acid sequence shown in SEQ ID NO: 1, 3, 5, 7, 9, 25 or 27. Preferred polynucleotides are, for example, 80% identical to any of the nucleotide sequences set forth in SEQ ID NO: 2, 4, 6, 8, 10, 26 or 28, more preferably at least 85%, 90%, 92%, 95%. , 96%, 97%, 98% or 99% identical polynucleotides.
〔発現ベクター〕
本発明は、上記本発明の融合タンパク質を製造するため、または宿主に発現させるために使用される発現ベクターを提供する。本発明の発現ベクターは、本発明の融合タンパク質をコードするポリヌクレオチドを含むものであれば特に限定されないが、ＲＮＡポリメラーゼの認識配列を有するプラスミドベクターが好ましい。発現ベクターの作製方法としては、プラスミド、ファージ、またはコスミドなどを用いる方法が挙げられるが特に限定されない。ベクターの具体的な種類は限定されず、宿主細胞中で発現可能なベクターを適宜選択することができる。すなわち、宿主細胞の種類に応じて、確実に本発明のポリヌクレオチドを発現させるために適宜プロモーター配列を選択し、これと本発明のポリヌクレオチドを各種プラスミド等に組み込んだベクターを発現ベクターとして用いればよい。本発明の発現ベクターを用いて形質転換された宿主を、培養、栽培または飼育した後、培養物などから慣用的な手法（例えば、濾過、遠心分離、細胞の破砕、ゲル濾過クロマトグラフィー、イオン交換クロマトグラフィーなど）に従って、本発明の融合タンパク質を回収、精製することができる。
[Expression vector]
The present invention provides an expression vector used to produce the fusion protein of the present invention or to express it in a host. The expression vector of the present invention is not particularly limited as long as it contains a polynucleotide encoding the fusion protein of the present invention, but a plasmid vector having a recognition sequence of RNA polymerase is preferable. Examples of the method for producing an expression vector include, but are not limited to, a method using a plasmid, phage, cosmid, or the like. The specific type of vector is not limited, and a vector that can be expressed in a host cell can be appropriately selected. That is, if a promoter sequence is appropriately selected in order to reliably express the polynucleotide of the present invention according to the type of host cell, and a vector in which this and the polynucleotide of the present invention are incorporated into various plasmids or the like is used as an expression vector. good. After culturing, cultivating or breeding a host transformed using the expression vector of the present invention, conventional methods (for example, filtration, centrifugation, cell disruption, gel filtration chromatography, ion exchange) are performed from the culture or the like. The fusion protein of the present invention can be recovered and purified according to (chromatography, etc.).
発現ベクターは、少なくとも１つの選択マーカーを含むことが好ましい。このようなマーカーとしては、真核生物細胞培養についてはジヒドロ葉酸レダクターゼまたはネオマイシン耐性遺伝子、大腸菌および他の細菌における培養についてはテトラサイクリン耐性遺伝子またはアンピシリン耐性遺伝子が挙げられる。上記選択マーカーを用いれば、本発明のポリヌクレオチドが宿主細胞に導入されたか否か、さらには宿主細胞中で確実に発現しているか否かを確認することができる。あるいは、例えばオワンクラゲ由来の緑色蛍光タンパク質ＧＦＰ（Green Fluorescent Protein）をマーカーとして用い、本発明の融合タンパク質をＧＦＰ融合タンパク質として発現させてもよい。 The expression vector preferably contains at least one selectable marker. Such markers include dihydrofolate reductase or neomycin resistance genes for eukaryotic cell cultures and tetracycline or ampicillin resistance genes for cultures in E. coli and other bacteria. By using the above-mentioned selectable marker, it is possible to confirm whether or not the polynucleotide of the present invention has been introduced into the host cell and whether or not it is reliably expressed in the host cell. Alternatively, for example, a green fluorescent protein (GFP) derived from Aequorea coeruleus may be used as a marker, and the fusion protein of the present invention may be expressed as a GFP fusion protein.
〔形質転換細胞〕
本発明は、上記本発明の発現ベクターが導入された形質転換細胞を提供する。本発明の形質転換細胞（以下、「本発明の細胞」という）は、本発明の融合タンパク質を一過性に発現するものでもよいが、本発明の融合タンパク質を安定発現するものが好ましい。宿主となる細胞は特に限定されず、本発明の発現ベクターが導入されて本発明の融合タンパク質を発現できる細胞であればどのような細胞も好適に用いることができる。例えば、大腸菌等の細菌、酵母、昆虫細胞、動物細胞などが挙げられる。本発明の細胞を後述する苦味物質の評価に用いる場合は、哺乳動物細胞を用いることが好ましい。本発明の発現ベクターを宿主細胞に導入する方法、すなわち形質転換法も特に限定されるものではなく、電気穿孔法、リン酸カルシウム法、リポソーム法、ＤＥＡＥデキストラン法等の従来公知の方法を好適に用いることができる。
[Transformed cells]
The present invention provides transformed cells into which the expression vector of the present invention has been introduced. The transformed cell of the present invention (hereinafter, referred to as “cell of the present invention”) may transiently express the fusion protein of the present invention, but a cell that stably expresses the fusion protein of the present invention is preferable. The host cell is not particularly limited, and any cell into which the expression vector of the present invention can be introduced and capable of expressing the fusion protein of the present invention can be preferably used. For example, bacteria such as Escherichia coli, yeast, insect cells, animal cells and the like can be mentioned. When the cells of the present invention are used for the evaluation of bitterness substances described later, it is preferable to use mammalian cells. The method for introducing the expression vector of the present invention into a host cell, that is, the transformation method is not particularly limited, and conventionally known methods such as electroporation method, calcium phosphate method, liposome method, and DEAE dextran method are preferably used. Can be done.
本発明の細胞は、ＴＡＳ２ＲとＧタンパク質αサブユニットとの融合タンパク質を発現する細胞であるので、ＴＡＳ２ＲとＧタンパク質αサブユニットを別々の発現ベクターを用いて導入した共発現細胞と比較して、作製のステップを大幅に削減できる点で非常に有利である。すなわち、共発現細胞を作製する場合、１つ目の発現ベクターを導入して高発現細胞を選択した後、２つ目の発現ベクターを導入して再び高発現細胞を選択するという２段階のステップを行う必要がある。通常、発現ベクターを導入して安定性の良い高発現細胞を選択し取得するために１か月以上を要するため、導入された２つの遺伝子について、安定性の高い高発現細胞を選択し取得するためには２〜３か月を要する。また、共発現細胞では、細胞のゲノム上にランダムに二つの遺伝子が入るので、その遺伝子の挿入数や安定性、発現量が揃わない可能性が高い。実際に共発現細胞は、継代を繰り返すと導入した遺伝子が欠落し、苦味物質の検出感度が大幅に低下することを本発明者らは経験している（実施例６参照）。 Since the cell of the present invention is a cell that expresses a fusion protein of TAS2R and G protein α-subunit, it is compared with a co-expressing cell in which TAS2R and G protein α-subunit are introduced using separate expression vectors. It is very advantageous in that the number of manufacturing steps can be significantly reduced. That is, when producing co-expressing cells, a two-step step of introducing a first expression vector to select a highly expressing cell and then introducing a second expression vector to select a highly expressing cell again. Need to be done. Normally, it takes one month or more to introduce an expression vector and select and obtain highly stable highly expressed cells. Therefore, for the two introduced genes, highly stable highly expressed cells are selected and obtained. It takes 2-3 months for this. In addition, in co-expressing cells, two genes are randomly placed on the cell genome, so there is a high possibility that the number of inserted genes, stability, and expression level will not be the same. In fact, the present inventors have experienced that the introduced gene is deleted from the co-expressing cells when the passage is repeated, and the detection sensitivity of the bitter substance is significantly reduced (see Example 6).
これに対して本発明の細胞は、１つの発現ベクターを導入して高発現細胞を選択する１つのステップのみで作製することができると共に、ＴＡＳ２ＲとＧタンパク質αサブユニットの発現量は常に均等である。さらに、ＴＡＳ２ＲとＧタンパク質αサブユニットが繋がった１つのタンパク質として接近した場で発現しているので、互いに共役する効率が高いと考えられる。それゆえ、本発明の細胞は、以下の苦味の評価方法および苦味調節物質のスクリーニング方法における評価系として、非常に有用である。 On the other hand, the cells of the present invention can be prepared by introducing one expression vector and selecting highly expressed cells in only one step, and the expression levels of TAS2R and G protein α subunit are always equal. be. Furthermore, since TAS2R and G protein α subunit are expressed in close proximity as one protein linked to each other, it is considered that the efficiency of conjugating with each other is high. Therefore, the cells of the present invention are very useful as an evaluation system in the following methods for evaluating bitterness and screening methods for bitterness regulators.
〔苦味評価方法〕
本発明は、上記本発明の細胞を用いる苦味評価方法を提供する。本発明の苦味評価方法は、本発明の細胞に被験物質を接触させる工程および被験物質を接触させた細胞における細胞内カルシウム濃度を測定する工程を含むものであればよい。本発明の苦味評価方法により、被験物質が苦味を感じさせる物質であるか否かを評価することができる。また、本発明の苦味評価方法により、被験物質により生体が感知する苦味の程度（強さ）を評価することができる。
[Bitter taste evaluation method]
The present invention provides a method for evaluating bitterness using the cells of the present invention. The bitterness evaluation method of the present invention may include a step of contacting the cells of the present invention with the test substance and a step of measuring the intracellular calcium concentration in the cells contacted with the test substance. According to the bitterness evaluation method of the present invention, it is possible to evaluate whether or not the test substance is a substance that makes the test substance feel bitter. In addition, the bitterness evaluation method of the present invention can evaluate the degree (strength) of bitterness perceived by a living body by a test substance.
被験物質は特に限定されないが、ヒトに経口摂取される可能性のある物質であることが好ましい。具体的には、例えば飲食品に含まれる成分、医薬品に含まれる成分などが挙げられる。本発明の細胞に被験物質を接触させる方法としては、例えば本発明の細胞を培養している培地に被験物質を添加する方法が挙げられる。被験物質は培地に直接添加してもよく、適当な溶媒に溶解または懸濁して添加してもよい。また、被験物質を接触させない対照群を設けることが好ましい。 The test substance is not particularly limited, but is preferably a substance that can be orally ingested by humans. Specific examples thereof include components contained in foods and drinks, components contained in pharmaceutical products, and the like. Examples of the method of bringing the test substance into contact with the cells of the present invention include a method of adding the test substance to the medium in which the cells of the present invention are cultured. The test substance may be added directly to the medium, or may be added by dissolving or suspending in a suitable solvent. In addition, it is preferable to provide a control group that does not come into contact with the test substance.
細胞内カルシウム濃度を測定する方法は特に限定されず、市販の試薬やキットを好適に用いることができる。被験物質との接触後に細胞内カルシウム濃度が上昇すれば、当該被験物質は苦味物質であると判断することができる。また、細胞内カルシウム濃度の上昇レベルが高い被験物質は苦味の程度が強いと判断することができる。 The method for measuring the intracellular calcium concentration is not particularly limited, and commercially available reagents and kits can be preferably used. If the intracellular calcium concentration increases after contact with the test substance, it can be determined that the test substance is a bitter taste substance. In addition, it can be determined that the test substance having a high level of increase in intracellular calcium concentration has a strong degree of bitterness.
〔苦味調節物質のスクリーニング方法〕
本発明は、上記本発明の細胞を用いる苦味調節物質のスクリーニング方法を提供する。本発明のスクリーニング方法は、本発明の細胞に苦味物質および被験物質を接触させる工程、苦味物質および被験物質を接触させた細胞における細胞内カルシウム濃度を測定する工程、および得られたカルシウム濃度を苦味物質のみを接触させた本発明の細胞における細胞内カルシウム濃度と比較し、カルシウム濃度を増加または低下させる被験物質を選択する工程を含むものであればよい。
[Screening method for bitterness regulators]
The present invention provides a method for screening a bitterness regulator using the cells of the present invention. The screening method of the present invention includes a step of contacting the cells of the present invention with a bitter taste substance and a test substance, a step of measuring the intracellular calcium concentration in the cells contacted with the bitter taste substance and the test substance, and a bitter taste of the obtained calcium concentration. Anything may include a step of selecting a test substance that increases or decreases the calcium concentration as compared with the intracellular calcium concentration in the cells of the present invention that have been contacted with only the substance.
被験物質は特に限定されないが、経口摂取した際に毒性を発現しない物質が好ましい。本発明の細胞に苦味物質および被験物質を接触させる方法としては、例えば本発明の細胞を培養している培地に苦味物質および被験物質を添加する方法が挙げられる。苦味物質および被験物質は培地に直接添加してもよく、適当な溶媒に溶解または懸濁して添加してもよい。溶媒に溶解または懸濁する場合、両者の混合溶液を用いてもよく、それぞれ別の溶液にして用いてもよい。本発明のスクリーニング方法では、別途苦味物質のみを接触させる対照群を設ける。 The test substance is not particularly limited, but a substance that does not develop toxicity when taken orally is preferable. Examples of the method of bringing the bitterness substance and the test substance into contact with the cells of the present invention include a method of adding the bitterness substance and the test substance to the medium in which the cells of the present invention are cultured. The bitterness substance and the test substance may be added directly to the medium, or may be added by dissolving or suspending in a suitable solvent. When dissolved or suspended in a solvent, a mixed solution of both may be used, or different solutions may be used. In the screening method of the present invention, a control group is separately provided in which only bitter substances are brought into contact with each other.
細胞内カルシウム濃度を測定する方法は特に限定されず、市販の試薬やキットを好適に用いることができる。苦味物質のみを接触させた細胞の細胞内カルシウム濃度と比較して、被験物質を同時に接触させた細胞の細胞内カルシウム濃度が低下していれば、当該被験物質は苦味抑制作用を有すると判断できる。被験物質が細胞内カルシウム濃度を低下させる程度は特に限定されないが、例えば、苦味物質のみを接触させた細胞の細胞内カルシウム濃度を５０％以下に低下させる被験物質が好ましく、２５％以下に低下させる被験物質がより好ましい。 The method for measuring the intracellular calcium concentration is not particularly limited, and commercially available reagents and kits can be preferably used. If the intracellular calcium concentration of the cells to which the test substance is simultaneously contacted is lower than the intracellular calcium concentration of the cells to which only the bitterness substance is contacted, it can be determined that the test substance has a bitterness suppressing effect. .. The degree to which the test substance reduces the intracellular calcium concentration is not particularly limited, but for example, a test substance that reduces the intracellular calcium concentration of cells in contact with only a bittersweet substance to 50% or less is preferable, and the intracellular calcium concentration is reduced to 25% or less. The test substance is more preferred.
一方、苦味物質のみを接触させた細胞の細胞内カルシウム濃度と比較して、被験物質を同時に接触させた細胞の細胞内カルシウム濃度が増加していれば、当該被験物質は苦味増強作用を有すると判断できる。被験物質が細胞内カルシウム濃度を増加させる程度は特に限定されないが、例えば、苦味物質のみを接触させた細胞の細胞内カルシウム濃度を２５％以上増加させる被験物質が好ましく、５０％以上増加させる被験物質がより好ましい。 On the other hand, if the intracellular calcium concentration of the cells to which the test substance is simultaneously contacted is increased as compared with the intracellular calcium concentration of the cells to which only the bitterness substance is contacted, the test substance has a bitterness enhancing effect. I can judge. The degree to which the test substance increases the intracellular calcium concentration is not particularly limited, but for example, a test substance that increases the intracellular calcium concentration of cells in contact with only a bitter substance by 25% or more is preferable, and a test substance that increases the intracellular calcium concentration by 50% or more is preferable. Is more preferable.
本発明のスクリーニング方法において、被験物質自身が苦味を有するか否かを確認する工程を設けてもよい。被験物質自身の苦味の有無を確認する工程は、上記本発明の苦味評価方法と同じ工程により確認することができる。 In the screening method of the present invention, a step of confirming whether or not the test substance itself has a bitter taste may be provided. The step of confirming the presence or absence of the bitterness of the test substance itself can be confirmed by the same step as the bitterness evaluation method of the present invention.
以下、実施例により本発明を詳細に説明するが、本発明はこれらに限定されるものではない。 Hereinafter, the present invention will be described in detail with reference to Examples, but the present invention is not limited thereto.
〔実施例１：ヒト苦味受容体遺伝子のクローニング〕
TAS2R14（塩基配列：配列番号11、アミノ酸配列：配列番号12）、TAS2R16（塩基配列：配列番号13、アミノ酸配列：配列番号14）、TAS2R38（塩基配列：配列番号15、アミノ酸配列：配列番号16）、TAS2R43（塩基配列：配列番号17、アミノ酸配列：配列番号18）、TAS2R44（塩基配列：配列番号19、アミノ酸配列：配列番号20）、TAS2R10（塩基配列：配列番号29、アミノ酸配列：配列番号30）、TAS2R39（塩基配列：配列番号31、アミノ酸配列：配列番号32）およびTAS2R46は、GenBankに登録されている配列情報を基に、ヒトゲノムDNA (BD Clontech)を鋳型として、PCR法により各遺伝子を増幅した。各受容体のコード領域のN末端側に、ラットソマトスタチン タイプ３の最初の45アミノ酸をタグ配列（塩基配列：配列番号21、アミノ酸配列：配列番号22）として付加し、pEAK10ベクター（Edge Biosystems）のAsc I-Not Iサイトに組込んだ。
[Example 1: Cloning of human bitter taste receptor gene]
TAS2R14 (base sequence: SEQ ID NO: 11, amino acid sequence: SEQ ID NO: 12), TAS2R16 (base sequence: SEQ ID NO: 13, amino acid sequence: SEQ ID NO: 14), TAS2R38 (base sequence: SEQ ID NO: 15, amino acid sequence: SEQ ID NO: 16) , TAS2R43 (base sequence: SEQ ID NO: 17, amino acid sequence: SEQ ID NO: 18), TAS2R44 (base sequence: SEQ ID NO: 19, amino acid sequence: SEQ ID NO: 20), TAS2R10 (base sequence: SEQ ID NO: 29, amino acid sequence: SEQ ID NO: 30) ), TAS2R39 (base sequence: SEQ ID NO: 31, amino acid sequence: SEQ ID NO: 32) and TAS2R46 are based on the sequence information registered in GenBank, and each gene is selected by PCR using human genomic DNA (BD Clontech) as a template. Amplified. The first 45 amino acids of rat somatostatin type 3 were added as a tag sequence (base sequence: SEQ ID NO: 21, amino acid sequence: SEQ ID NO: 22) to the N-terminal side of the coding region of each receptor, and the pEAK10 vector (Edge Biosystems) was added. Incorporated into the Asc I-Not I site.
〔実施例２：改変型Gα16gust40遺伝子のクローニング〕
苦味受容体は生体内では、ガストデューシンのようなGi型のGタンパク質と共役するが、Gα15やGα16のようなGq型のGタンパク質とは共役しないことが知られている。一方で、Gq型のGタンパク質はHEK293細胞のような哺乳類細胞において内在性のβ/γと複合体を形成することができ、ガストデューシンのようなGi型のGタンパク質よりも、より感度の高いシグナル伝達を可能とする。したがって、宿主細胞において、Gα16のようなGq型のαサブユニットのC末端側のアミノ酸配列をガストデューシンのようなGi型のαサブユニットのアミノ酸配列と置換したキメラタンパク質を苦味受容体と共に強制発現させることにより、細胞内のカルシウム濃度動態が観察できる。
ヒトGα16とヒトガストデューシンは、ともにORIGENEより購入した[GNA15 (NM_002068) Human cDNA Clone, GNAT3 (NM_001102386) Human cDNA Clone]。まず初めに、文献［Ueda T，et al., J. Neurosci，23，7376-7380（2003）］に従い、Gα16のアミノ酸配列のC末端側の40アミノ酸をガストデューシンのC末端側の40アミノ酸に置換させたキメラαサブユニットをコードする遺伝子を作製するため、購入したプラスミドを鋳型として各遺伝子をPCR法にて増幅し、pEAK10ベクター（Edge Biosystems）のEco RI-Not Iサイトに組込んだ。さらにGα16とガストデューシンの繋ぎ目のアミノ酸配列に対して部位特異的変異を導入して改変型Gα16gust40であるGα16(1-331)-AlaGluThr-Gustducin(317-354)を作製した（塩基配列：配列番号23、アミノ酸配列：配列番号24）。
[Example 2: Cloning of modified Gα16gust40 gene]
It is known that the bitter taste receptor is conjugated to a Gi-type G protein such as gustducin in vivo, but not to a Gq-type G protein such as Gα15 or Gα16. On the other hand, Gq-type G proteins can form a complex with endogenous β / γ in mammalian cells such as HEK293 cells and are more sensitive than Gi-type G proteins such as gustducin. Enables high signal transduction. Therefore, in the host cell, a chimeric protein in which the amino acid sequence on the C-terminal side of the Gq-type α subunit such as Gα16 is replaced with the amino acid sequence of the Gi-type α subunit such as gustducin is forced together with the bitter taste receptor. By expressing it, intracellular calcium concentration dynamics can be observed.
Both human Gα16 and human gustducin were purchased from ORIGENE [GNA15 (NM_002068) Human cDNA Clone, GNAT3 (NM_001102386) Human cDNA Clone]. First, according to the literature [Ueda T, et al., J. Neurosci, 23, 7376-7380 (2003)], the 40 amino acids on the C-terminal side of the amino acid sequence of Gα16 are replaced with the 40 amino acids on the C-terminal side of gustducin. In order to prepare a gene encoding the chimeric α-subunit substituted with, each gene was amplified by PCR using the purchased plasmid as a template and incorporated into the Eco RI-Not I site of the pEAK10 vector (Edge Biosystems). .. Furthermore, a site-specific mutation was introduced into the amino acid sequence at the joint between Gα16 and gustducin to prepare a modified Gα16 gust40, Gα16 (1-331) -AlaGluThr-Gustducin (317-354) (base sequence: SEQ ID NO: 23, amino acid sequence: SEQ ID NO: 24).
〔実施例３：ヒト苦味受容体と改変型Gα16gust40との融合遺伝子の作製〕
融合遺伝子はClontechのIn-FusionTM Advantage PCR Cloning Kitを用いて作製した。最初にマニュアルに従って、実施例２で作成した改変型Gα16gust40/pEAK10ベクターをinverse PCRにより線状化した。次に作製した線状ベクターの末端と相同な配列を5’末端側に付加させた目的遺伝子を増幅するためのPrimerを設計し、SSTR3タグ-TAS2Rの遺伝子を増幅した。In-Fusion反応により、これらの遺伝子断片を融合させて、大腸菌に形質転換を行った。得られたコロニーを培養してプラスミドの精製を行い、そのDNA配列を確認して、目的の融合遺伝子を含むプラスミドを得た（図１参照）。
[Example 3: Preparation of a fusion gene of a human bitter taste receptor and a modified Gα16gust40]
The fusion gene was prepared using Clontech's In-Fusion TM Advantage PCR Cloning Kit. First, according to the manual, the modified Gα16gust40 / pEAK10 vector prepared in Example 2 was linearized by inverse PCR. Next, we designed a Primer for amplifying the target gene with a sequence homologous to the end of the prepared linear vector added to the 5'end side, and amplified the SSTR3 tag-TAS2R gene. These gene fragments were fused by an In-Fusion reaction to transform Escherichia coli. The obtained colonies were cultured to purify the plasmid, and the DNA sequence thereof was confirmed to obtain a plasmid containing the desired fusion gene (see FIG. 1).
〔実施例４：苦味受容体と改変型Gα16gust40の融合遺伝子発現細胞の作製〕
TAS2R14、TAS2R16、TAS2R38、TAS2R43、TAS2R44、TAS2R10、TAS2R39およびTAS2R46の融合タンパク質をそれぞれ発現させたHEK293T細胞は次のように作製した。まず初めに各融合遺伝子を含んだプラスミドを制限酵素（Bgl IIまたはPme I）にて切断し、線状化した。HEK293T細胞（GE Healthcare等より入手可能）は60mm dishに4〜7 x 105 細胞数になるよう播いて、37℃、5％CO2を保持したインキュベーター内で培養しておいた。翌日、線状化プラスミド（6 μg）とLipofectamine 2000（15 μl）を別々のチューブ内にて各500 μlのOPTI-MEM（Invitrogen Corporation）に加えておき、室温にて5分間静置後、両者を混合した。20分後にこの混合液を静かにHEK293T細胞に添加し、CO2インキュベーター内で培養した。24時間後、細胞を常法に従って、dishより剥がし、培地で適宜希釈を行い、一部を12枚の100mm dishに播き直した。24時間の培養後、puromycinが最終濃度 10 μg/mlになるよう添加し、薬剤選抜を開始した。数日おきにpuromycinを加えた新鮮な培地に交換しながら、約3週間程度培養を継続した。薬剤耐性の細胞がそれぞれコロニーとして生育してくるので、順次ピッキングし、それぞれを個別に24穴もしくは6穴のプレート内に移し、培養を継続して増殖させ、25 cm2もしくは75 cm2のフラスコで培養後、ストックを作製した。計20〜30個のコロニーから取得した各セルラインのストックは、順次解凍して培養を行い、各受容体に応じたアゴニストである苦味成分を添加するスクリーニングアッセイを実施し、最も応答強度が高いセルラインを選んだ。
[Example 4: Preparation of fusion gene-expressing cells of bitter taste receptor and modified Gα16gust40]
HEK293T cells expressing the fusion proteins of TAS2R14, TAS2R16, TAS2R38, TAS2R43, TAS2R44, TAS2R10, TAS2R39 and TAS2R46 were prepared as follows. First, the plasmid containing each fusion gene was cut with a restriction enzyme (Bgl II or Pme I) and linearized. HEK293T cells (available from GE Healthcare, etc.) were sown in a 60 mm dish to a number of 4 to 7 x 10 5 cells and cultured in an incubator holding 5% CO 2 at 37 ° C. The next day, linearized plasmid (6 μg) and Lipofectamine 2000 (15 μl) were added to each 500 μl OPTI-MEM (Invitrogen Corporation) in separate tubes, allowed to stand at room temperature for 5 minutes, and then both. Was mixed. After 20 minutes, the mixture was gently added to HEK293T cells and cultured in a CO 2 incubator. After 24 hours, the cells were detached from the dish according to a conventional method, diluted appropriately with medium, and a part was re-sown on 12 100 mm dishes. After culturing for 24 hours, puromycin was added to a final concentration of 10 μg / ml, and drug selection was started. The culture was continued for about 3 weeks while exchanging with a fresh medium containing puromycin every few days. As each drug-resistant cell grows as a colony, pick it in sequence, transfer each individually into a 24-well or 6-well plate, continue culturing, and grow in a 25 cm 2 or 75 cm 2 flask. After culturing in, a stock was prepared. The stock of each cell line obtained from a total of 20 to 30 colonies is sequentially thawed and cultured, and a screening assay in which a bitterness component that is an agonist corresponding to each receptor is added is performed, and the response intensity is the highest. I chose the cell line.
〔実施例５：苦味評価〕
（１）細胞内カルシウム濃度変化の測定
苦味受容体と改変型Gα16gust40の融合タンパク質を発現しているHEK293T細胞に苦味物質を投与すると、苦味を感知した苦味受容体は融合している改変型Gα16gust40と共役しPLCβ2を活性化させて、細胞内カルシウム量を増加させる。上記で作製した各細胞をアッセイの前日に96穴プレートに播種し、培養を開始した。24時間後に培養物から培地を取り除き、アッセイ用のバッファーに交換した（50 μL）。バッファーの組成は、10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 130 mM NaCl, 10 mM glucose, 5 mM KCl, 2 mM CaCl2, 1.2 mM MgCl2, pH7.4(NaOHにて調整)である。
[Example 5: Evaluation of bitterness]
(1) Measurement of intracellular calcium concentration change When a bitter taste substance is administered to HEK293T cells expressing a fusion protein of bitter taste receptor and modified Gα16gust40, the bitter taste receptor that senses bitterness is fused with modified Gα16gust40. It conjugates and activates PLCβ2 to increase the amount of intracellular calcium. Each cell prepared above was seeded on a 96-well plate the day before the assay and culture was initiated. After 24 hours, the medium was removed from the culture and replaced with assay buffer (50 μL). The composition of the buffer is 10 mM 4- (2-hydroxyethyl) -1-piperazineethanesulfonic acid (HEPES), 130 mM NaCl, 10 mM glucose, 5 mM KCl, 2 mM CaCl 2 , 1.2 mM MgCl 2 , pH 7.4 (NaOH). (Adjusted by).
次にFLIPR Calcium Assay Kit（Molecular Devices）の細胞内カルシウム蛍光指示薬Calcium 5を同バッファーで希釈し、各ウェルへ50 μLずつ添加後、CO2インキュベーター内で静置した。50分後に、37℃に設定しておいたFlexStation（Molecular Devices）装置内にプレートを移し、励起波長485nmにおける検出波長525nmの蛍光強度測定を開始した。測定開始から18秒後に、苦味物質溶液100 μLを添加し、細胞内カルシウム濃度変化を検出した。解析は、測定開始時から終了までの90秒間における蛍光強度の変化量の最大値（ΔＦ）を細胞の応答強度とした。各物質の応答強度はバッファーのみを添加した時の（ΔＦ）を差し引いて算出した。
Next, the intracellular calcium
（２）TAS2R16-改変型Gα16gust40融合タンパク質発現細胞による苦味評価
ヒトTAS2R16はサリシンやゲンチオビオースなどのβグルコピラノシドを特異的に認識する苦味受容体であることが知られている。そこで、TAS2R16-改変型Gα16gust40融合タンパク質発現細胞を用いて、サリシンのTAS2R16に対する苦味応答を検出した。サリシンは最終濃度が最大10 mMの濃度から3倍希釈のシリーズで添加した。
結果を図２に示した。図２において縦軸はサリシンを添加した際に生じる蛍光強度の変化量を示し、横軸は時間（秒）を示す。また、図２のデータから、サリシンの濃度とその投与により生じた蛍光強度の変化量をバッファーのみで生じた蛍光強度の変化量で差し引いたものを応答強度としてその関係を図３に示した。図３において縦軸はサリシンを添加した際に生じる蛍光強度の最大変化量（ΔＦ）−バッファーのみで生じる蛍光強度の最大変化量（ΔＦ）を示し、横軸はサリシンの最終濃度を示す。サリシンは濃度依存性の応答を示したことから、TAS2R16-改変型Gα16gust40融合タンパク質発現細胞は苦味の評価に使用できることが示された。
(2) Evaluation of bitterness by TAS2R16-modified Gα16gust40 fusion protein-expressing cells Human TAS2R16 is known to be a bitterness receptor that specifically recognizes β-glucopyranoside such as salicin and gentiobiose. Therefore, using TAS2R16-modified Gα16gust40 fusion protein-expressing cells, the bitter taste response of salicin to TAS2R16 was detected. Salicin was added in a series of 3-fold dilutions from concentrations with a final concentration of up to 10 mM.
The results are shown in FIG. In FIG. 2, the vertical axis indicates the amount of change in fluorescence intensity that occurs when salicin is added, and the horizontal axis indicates time (seconds). Further, the relationship is shown in FIG. 3 as the response intensity obtained by subtracting the amount of change in fluorescence intensity caused by the concentration of salicin and its administration from the data in FIG. 2 by the amount of change in fluorescence intensity generated only in the buffer. In FIG. 3, the vertical axis represents the maximum amount of change in fluorescence intensity (ΔF) generated when salicin is added-the maximum amount of change in fluorescence intensity generated only by the buffer (ΔF), and the horizontal axis represents the final concentration of salicin. Salicin showed a concentration-dependent response, indicating that TAS2R16-modified Gα16gust40 fusion protein-expressing cells could be used to assess bitterness.
（３）TAS2R14-改変型Gα16gust40融合タンパク質発現細胞による苦味評価
ヒトTAS2R14は様々な苦味に応答する比較的特異性の低い苦味受容体であることが知られている。そこで、TAS2R14-改変型Gα16gust40融合タンパク質発現細胞を用いて、アリストロキア酸のTAS2R14に対する苦味応答を検出した。アリストロキア酸は最終濃度が最大10 μMの濃度から3倍希釈のシリーズで添加した。
結果を図４および図５に示した。アリストロキア酸は濃度依存性の応答を示したことから、TAS2R14-改変型Gα16gust40融合タンパク質発現細胞は苦味の評価に使用できることが示された。
(3) Evaluation of bitterness by TAS2R14-modified Gα16gust40 fusion protein-expressing cells Human TAS2R14 is known to be a relatively low-specific bitterness receptor that responds to various bitternesses. Therefore, using TAS2R14-modified Gα16gust40 fusion protein-expressing cells, the bitter taste response of aristolochic acid to TAS2R14 was detected. Aristolochic acid was added in a series of 3-fold dilutions from concentrations with a final concentration of up to 10 μM.
The results are shown in FIGS. 4 and 5. Aristolochic acid showed a concentration-dependent response, indicating that TAS2R14-modified Gα16gust40 fusion protein-expressing cells could be used to assess bitterness.
（４）TAS2R38-改変型Gα16gust40融合タンパク質発現細胞による苦味評価
ヒトTAS2R38はフェニルチオカルバミド（PTC）の苦味受容体であることが知られている。またTAS2R38は一塩基多型により苦味応答性が全く異なることが報告されている（Curr Biol. 2005 Feb 22;15(4):322-7）。そこで、TAS2R38-改変型Gα16gust40融合タンパク質発現細胞を用いて、PTCのTAS2R38に対する苦味応答を検出した。PTCは最終濃度が最大100 μMの濃度から3倍希釈のシリーズで添加した。
結果を図６および図７に示した。PTCは濃度依存性の応答を示したことから、TAS2R38-改変型Gα16gust40融合タンパク質発現細胞は苦味の評価に使用できることが示された。
(4) Evaluation of bitterness by TAS2R38-modified Gα16gust40 fusion protein-expressing cells Human TAS2R38 is known to be a bitter taste receptor for phenylthiocarbamide (PTC). It has also been reported that TAS2R38 has a completely different bitterness response depending on the single nucleotide polymorphism (Curr Biol. 2005 Feb 22; 15 (4): 322-7). Therefore, we detected the bitter taste response of PTC to TAS2R38 using TAS2R38-modified Gα16gust40 fusion protein-expressing cells. PTC was added in a series of 3-fold dilutions from concentrations with a final concentration of up to 100 μM.
The results are shown in FIGS. 6 and 7. PTC showed a concentration-dependent response, indicating that TAS2R38-modified Gα16gust40 fusion protein-expressing cells could be used to assess bitterness.
（５）TAS2R43-改変型Gα16gust40融合タンパク質発現細胞およびTAS2R44-改変型Gα16gust40融合タンパク質発現細胞による苦味評価
ヒトTAS2R43とTAS2R44はヒト苦味受容体の中で最も相同性が高く、また応答する苦味物質も共通しているものが多い。そこで、TAS2R44-改変型Gα16gust40融合タンパク質発現細胞を用いて、アリストロキア酸のTAS2R44に対する苦味応答を検出した。アリストロキア酸は最終濃度が最大10 μMの濃度から3倍希釈のシリーズで添加した。
結果を図８に示した。アリストロキア酸は濃度依存性の応答を示したことから、TAS2R44-改変型Gα16gust40融合タンパク質発現細胞は苦味の評価に使用できることが示された。結果を示していないが、アリストロキア酸はTAS2R43-改変型Gα16gust40融合タンパク質発現細胞に対しても同様の反応を示した。
(5) Evaluation of bitterness by TAS2R43-modified Gα16gust40 fusion protein-expressing cells and TAS2R44-modified Gα16gust40 fusion protein-expressing cells Human TAS2R43 and TAS2R44 have the highest homology among human bitter taste receptors, and also have a common bitterness substance that responds. There are many things that are done. Therefore, using TAS2R44-modified Gα16gust40 fusion protein-expressing cells, the bitter taste response of aristolochic acid to TAS2R44 was detected. Aristolochic acid was added in a series of 3-fold dilutions from concentrations with a final concentration of up to 10 μM.
The results are shown in FIG. Aristolochic acid showed a concentration-dependent response, indicating that TAS2R44-modified Gα16gust40 fusion protein-expressing cells could be used to assess bitterness. Although no results have been shown, aristolochic acid showed a similar response to TAS2R43-modified Gα16gust40 fusion protein-expressing cells.
（６）TAS2R10-改変型Gα16gust40融合タンパク質発現細胞による苦味評価
ヒトTAS2R10は様々な苦味に応答する比較的特異性の低い苦味受容体であることが知られている。そこで、TAS2R10-改変型Gα16gust40融合タンパク質発現細胞を用いて、ククルビタシンBのTAS2R10に対する苦味応答を検出した。ククルビタシンBは最終濃度が最大66.5μMの濃度から3倍希釈のシリーズで添加した。
結果を図９および図１０に示した。ククルビタシンBは濃度依存性の応答を示したことから、TAS2R10-改変型Gα16gust40融合タンパク質発現細胞は苦味の評価に使用できることが示された。
(6) Evaluation of bitterness by TAS2R10-modified Gα16gust40 fusion protein-expressing cells Human TAS2R10 is known to be a relatively low-specific bitterness receptor that responds to various bitternesses. Therefore, TAS2R10-modified Gα16gust40 fusion protein-expressing cells were used to detect the bitter taste response of cucurbitacin B to TAS2R10. Cucurbitacin B was added in a series of 3-fold dilutions from concentrations with a final concentration of up to 66.5 μM.
The results are shown in FIGS. 9 and 10. Cucurbitacin B showed a concentration-dependent response, indicating that TAS2R10-modified Gα16gust40 fusion protein-expressing cells could be used to assess bitterness.
（７）TAS2R39-改変型Gα16gust40融合タンパク質発現細胞による苦味評価
ヒトTAS2R39は様々な苦味に応答する比較的特異性の低い苦味受容体であることが知られている。そこで、TAS2R39-改変型Gα16gust40融合タンパク質発現細胞を用いて、エピカテキンガレートのTAS2R39に対する苦味応答を検出した。エピカテキンガレートは最終濃度が最大150 μMの濃度から2倍希釈のシリーズで添加した。
結果を図１１および図１２に示した。ククルビタシンBは濃度依存性の応答を示したことから、TAS2R39-改変型Gα16gust40融合タンパク質発現細胞は苦味の評価に使用できることが示された。
(7) Evaluation of bitterness by TAS2R39-modified Gα16gust40 fusion protein-expressing cells Human TAS2R39 is known to be a relatively low-specific bitterness receptor that responds to various bitternesses. Therefore, using TAS2R39-modified Gα16gust40 fusion protein-expressing cells, the bitter taste response of epicatechin gallate to TAS2R39 was detected. Epicatechin gallate was added in a series of 2-fold dilutions from concentrations with a final concentration of up to 150 μM.
The results are shown in FIGS. 11 and 12. Cucurbitacin B showed a concentration-dependent response, indicating that TAS2R39-modified Gα16gust40 fusion protein-expressing cells could be used to assess bitterness.
（８）TAS2R46-改変型Gα16gust40融合タンパク質発現細胞による苦味評価
ヒトTAS2R46は様々な苦味に応答する比較的特異性の低い苦味受容体であることが知られている。そこで、TAS2R46-改変型Gα16gust40融合タンパク質発現細胞を用いて、ピクロトキシニンのTAS2R46に対する苦味応答を検出した。ピクロトキシニンは最終濃度が最大5 mMの濃度から3倍希釈のシリーズで添加した。
結果を図１３および図１４に示した。ピクロトキシニンは濃度依存性の応答を示したことから、TAS2R46-改変型Gα16gust40融合タンパク質発現細胞は苦味の評価に使用できることが示された。
(8) Evaluation of bitterness by TAS2R46-modified Gα16gust40 fusion protein-expressing cells Human TAS2R46 is known to be a relatively low-specific bitterness receptor that responds to various bitternesses. Therefore, using TAS2R46-modified Gα16gust40 fusion protein-expressing cells, the bitter taste response of picrotoxin to TAS2R46 was detected. Picrotoxin was added in a series of 3-fold dilutions from concentrations with a final concentration of up to 5 mM.
The results are shown in FIGS. 13 and 14. Picrotoxin showed a concentration-dependent response, indicating that TAS2R46-modified Gα16gust40 fusion protein-expressing cells could be used to assess bitterness.
〔実施例６：苦味受容体およびガストデューシンの共発現細胞との比較〕
ヒトTAS2R16遺伝子を挿入した発現ベクターと、ヒトガストデューシン遺伝子を挿入した発現ベクターを別々にHEK293T細胞に挿入し、ヒトTAS2R16とヒトガストデューシンを安定発現するセルラインを選択した。具体的には、先ずpEAK10ベクターに組込んだソマトスタチンタグ付加ヒトTAS2R16（実施例１に記載）を、実施例４で示した方法と同様の手法によりHEK293T細胞に導入した。ピューロマイシン添加培地で生育し得られた各セルラインに対して、改変型Gα16gust40を一過性に発現させて、サリシンに対する最も応答強度の高いセルラインを選択した。次にその選抜したTAS2R16発現細胞セルラインに、改変型Gα16gust40/pcDNA4/TOを導入し安定発現させた。導入方法は実施例４とほぼ同様であるが、プラスミドはNru Iにて線状化を行い、また選択培地はピューロマイシンとともにゼオシンを最終濃度が400 μg/mlになるよう添加したものを使用した。Gα16gust40/pcDNA4/TOは実施例２に記載した改変型Gα16gust40/pEAK10からIn-FusionTM Advantage PCR Cloning Kitを用いてpcDNA4/TOベクター(invitrogen)のEco RI-Xho Iサイトにサブクローニングして作製した。得られた薬剤耐性の細胞セルラインについて、サリシンを投与し、応答性の高いセルラインをTAS2R16と改変型Gα16gust40の共発現細胞として選択した。
[Example 6: Comparison with co-expressing cells of bitter taste receptor and gastoducin]
An expression vector into which the human TAS2R16 gene was inserted and an expression vector into which the human gustducin gene was inserted were separately inserted into HEK293T cells, and a cell line capable of stably expressing human TAS2R16 and human gustducin was selected. Specifically, first, a somatostatin-tagged human TAS2R16 (described in Example 1) incorporated into a pEAK10 vector was introduced into HEK293T cells by the same method as that shown in Example 4. Modified Gα16gust40 was transiently expressed in each cell line obtained by growing in a puromycin-added medium, and the cell line having the highest response intensity to salicin was selected. Next, the modified Gα16gust40 / pcDNA4 / TO was introduced into the selected TAS2R16-expressing cell cell line and stably expressed. The introduction method was almost the same as in Example 4, but the plasmid was linearized with Nru I, and the selective medium used was puromycin and zeocin added to a final concentration of 400 μg / ml. .. Gα16gust40 / pcDNA4 / TO was prepared by subcloning the modified Gα16gust40 / pEAK10 described in Example 2 into the Eco RI-Xho I site of the pcDNA4 / TO vector (invitrogen) using the In-Fusion TM Advantage PCR Cloning Kit. Salicin was administered to the obtained drug-resistant cell lines, and highly responsive cell lines were selected as co-expressing cells of TAS2R16 and modified Gα16gust40.
共発現細胞の2セルラインと、TAS2R16-改変型Gα16gust40融合タンパク質発現細胞の1セルラインを長期継代培養し、定期的にサリシンの苦味応答を検出し、検出感度の変化を調べた。具体的には、共発現細胞のセルラインは、ピューロマイシンとゼオシンを添加した培地で、融合タンパク質発現細胞はピューロマイシン添加培地にてそれぞれ培養を継続した。実施例５に記載したように、適時アッセイの前日に各セルラインを96穴プレートに播種し、24時間後にアッセイ用のバッファーに交換後、FLIPR Calcium Assay Kitを用いてサリシン投与による細胞内カルシウムの濃度変化を測定した。
結果を表３に示した。共発現細胞のセルライン#3は遅くとも20世代までに検出感度が低下し、セルライン#13は遅くとも35世代までに検出感度が低下した。一方、融合タンパク質発現細胞（セルライン#2）は、32世代まで検出感度の低下は認められていない。
Two cell lines of co-expressing cells and one cell line of TAS2R16-modified Gα16gust40 fusion protein-expressing cells were subcultured for a long period of time, and the bitter taste response of salicin was detected regularly to examine changes in detection sensitivity. Specifically, the cell lines of the co-expressing cells were continuously cultured in the medium supplemented with puromycin and zeocin, and the fusion protein-expressing cells were continuously cultured in the medium supplemented with puromycin. As described in Example 5, each cell line was seeded on a 96-well plate the day before the timely assay, replaced with a buffer for the assay 24 hours later, and then the intracellular calcium by salicin administration using the FLIPR Calcium Assay Kit. The change in concentration was measured.
The results are shown in Table 3. The detection sensitivity of cell line # 3 of co-expressing cells decreased by the 20th generation at the latest, and the detection sensitivity of cell line # 13 decreased by the 35th generation at the latest. On the other hand, fusion protein-expressing cells (cell line # 2) did not show any decrease in detection sensitivity until the 32nd generation.
なお本発明は上述した各実施形態および実施例に限定されるものではなく、請求項に示した範囲で種々の変更が可能であり、異なる実施形態にそれぞれ開示された技術的手段を適宜組み合わせて得られる実施形態についても本発明の技術的範囲に含まれる。また、本明細書中に記載された学術文献および特許文献の全てが、本明細書中において参考として援用される。 The present invention is not limited to the above-described embodiments and examples, and various modifications can be made within the scope of the claims, and the technical means disclosed in the different embodiments may be appropriately combined. The obtained embodiments are also included in the technical scope of the present invention. In addition, all the academic documents and patent documents described in the present specification are incorporated herein by reference.
Claims (9)
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2016050320 | 2016-03-14 | ||
| JP2016050320 | 2016-03-14 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JP2017163984A JP2017163984A (en) | 2017-09-21 |
| JP6924590B2 true JP6924590B2 (en) | 2021-08-25 |
Family
ID=59909337
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP2017048197A Active JP6924590B2 (en) | 2016-03-14 | 2017-03-14 | Fusion protein for bitterness evaluation and bitterness evaluation method |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JP6924590B2 (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2023243495A1 (en) | 2022-06-16 | 2023-12-21 | 花王株式会社 | Method for analyzing g-protein conjugated receptor |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP7321493B2 (en) * | 2019-01-31 | 2023-08-07 | 学校法人昭和大学 | Bitterness evaluation method and bitterness evaluation device |
Family Cites Families (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US7883856B2 (en) * | 2001-04-05 | 2011-02-08 | Senomyx Inc. | Identification of bitter ligands that specifically activate human T2R receptors and related assays for identifying human bitter taste modulators |
| JP2003210192A (en) * | 2002-01-18 | 2003-07-29 | Sumitomo Pharmaceut Co Ltd | Method for screening ligand for orphan GPCR |
| JP2003250551A (en) * | 2002-02-25 | 2003-09-09 | Japan Science & Technology Corp | Ligand search method using fusion protein of G protein-coupled receptor and G16α |
| ATE393778T1 (en) * | 2002-12-18 | 2008-05-15 | Givaudan Sa | CHIMERA ALPHA Q-GUSTDUCIN G PROTEINS |
| JP2006340717A (en) * | 2005-05-11 | 2006-12-21 | Sekisui Chem Co Ltd | Method for evaluating bondability to g protein-coupled receptor and composition for evaluation, fused protein, and gene |
-
2017
- 2017-03-14 JP JP2017048197A patent/JP6924590B2/en active Active
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2023243495A1 (en) | 2022-06-16 | 2023-12-21 | 花王株式会社 | Method for analyzing g-protein conjugated receptor |
Also Published As
| Publication number | Publication date |
|---|---|
| JP2017163984A (en) | 2017-09-21 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Leinders-Zufall et al. | A family of nonclassical class I MHC genes contributes to ultrasensitive chemodetection by mouse vomeronasal sensory neurons | |
| JP2009148278A (en) | Natural ligand of G protein-coupled receptor ChemR23 and use thereof | |
| Peng et al. | cGMP-dependent protein kinase encoded by foraging regulates motor axon guidance in Drosophila by suppressing Lola function | |
| JP6924590B2 (en) | Fusion protein for bitterness evaluation and bitterness evaluation method | |
| JP5307404B2 (en) | Natural ligand of G protein coupled receptor RCC356 and use thereof | |
| JP2019074379A (en) | Screening method of raw garlic-like flavor material | |
| JP6522597B2 (en) | Fusion protein suitable for measurement of autophagy, nucleic acid encoding said fusion protein, and use thereof | |
| JP2005501548A5 (en) | ||
| JP2019129772A (en) | Methods for screening compounds that suppress odor of 2-heptanone | |
| JP2019037197A (en) | Roast-like perfume material screening method | |
| JP5328190B2 (en) | Method for evaluating TRPA1 activator | |
| Pajot‐Augy et al. | Engineered yeasts as reporter systems for odorant detection | |
| JP2019010058A (en) | Selecting method of phenolic perfume material | |
| JP6093946B2 (en) | Method for detecting signal transduction of G protein-coupled receptor | |
| JP2019129773A (en) | Methods for screening compounds that suppress odor of 2-nonanone | |
| JP2015505464A (en) | Cell-based sensor | |
| JP2011067190A (en) | Single molecular format probe and utilization of the same | |
| US7951595B2 (en) | Methods for screening modulators of SLC17-type anion transport activity | |
| JP2019039711A (en) | Olfactory receptor response measurement method | |
| US7713713B2 (en) | Polypeptide having intracellular calcium ion indicator function | |
| JP4351952B2 (en) | Cells and methods for assessing effects on neurodevelopmental disorders | |
| KR101599686B1 (en) | Screening Methods of a Ion Channel Modulator Using a Mutated BKCa Channel | |
| Davies et al. | The physiological impact of an N-terminal Halo-tag on GIPR function in mice | |
| Edwards | Identifying Neurotensin Receptor 1-Expressing Cells and Determining the Molecular Requirements of Neurotensin Receptor 1 Signaling | |
| JP2025154616A (en) | Cells and methods for assessing the binding response of β-arrestin 2 to opioid μ receptors |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| A621 | Written request for application examination |
Free format text: JAPANESE INTERMEDIATE CODE: A621 Effective date: 20190807 |
|
| A977 | Report on retrieval |
Free format text: JAPANESE INTERMEDIATE CODE: A971007 Effective date: 20200728 |
|
| A131 | Notification of reasons for refusal |
Free format text: JAPANESE INTERMEDIATE CODE: A131 Effective date: 20200818 |
|
| A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A523 Effective date: 20201014 |
|
| A02 | Decision of refusal |
Free format text: JAPANESE INTERMEDIATE CODE: A02 Effective date: 20210302 |
|
| A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A523 Effective date: 20210602 |
|
| C60 | Trial request (containing other claim documents, opposition documents) |
Free format text: JAPANESE INTERMEDIATE CODE: C60 Effective date: 20210602 |
|
| A911 | Transfer to examiner for re-examination before appeal (zenchi) |
Free format text: JAPANESE INTERMEDIATE CODE: A911 Effective date: 20210609 |
|
| C21 | Notice of transfer of a case for reconsideration by examiners before appeal proceedings |
Free format text: JAPANESE INTERMEDIATE CODE: C21 Effective date: 20210615 |
|
| TRDD | Decision of grant or rejection written | ||
| A01 | Written decision to grant a patent or to grant a registration (utility model) |
Free format text: JAPANESE INTERMEDIATE CODE: A01 Effective date: 20210706 |
|
| A61 | First payment of annual fees (during grant procedure) |
Free format text: JAPANESE INTERMEDIATE CODE: A61 Effective date: 20210802 |
|
| R150 | Certificate of patent or registration of utility model |
Ref document number: 6924590 Country of ref document: JP Free format text: JAPANESE INTERMEDIATE CODE: R150 |
|
| R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |