| Project/Area Number |
08458174
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| Research Category |
Grant-in-Aid for Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Bioorganic chemistry
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| Research Institution | YOKOHAMA NATIONAL UNIVERSITY |
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Principal Investigator |
KURIHARA Yoshie YOKOHAMA NATIONAL UNIVERSITY,FACULTY OF EDUCATION AND HUMAN SCIENCES,PROFFESOR, 教育人間科学部, 教授 (90017715)
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| Co-Investigator(Kenkyū-buntansha) |
HARADA Shigeharu UNIVERSITY OF TOKYO,FACULTY OF PHARMACEUTICAL SCIENCES,ASSISTANT PROFESSOR, 薬学部, 助教授 (80156504)
OHTANI Hiroyuki YOKOHAMA NATIONAL UNIVERSITY,FACULTY OF EDUCATION AND HUMAN SCIENCES,LECTURER, 教育人間科学部, 講師 (30213763)
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| Project Period (FY) |
1996 – 1997
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| Project Status |
Completed (Fiscal Year 1997)
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| Budget Amount *help |
¥6,200,000 (Direct Cost: ¥6,200,000)
Fiscal Year 1997: ¥2,200,000 (Direct Cost: ¥2,200,000)
Fiscal Year 1996: ¥4,000,000 (Direct Cost: ¥4,000,000)
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| Keywords | miraculin cDNA / expression in yeast / curculin dimer / expression in E.coli / クルクリンcDNA / マビンリンcDNA |
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| Research Abstract |
Miraculin, which exists in the pulp of Richadella dulcifica (miracle fruit), had the unusual property of modifying a sour taste into a sweet taste. cDNA clone for miraculin was isolated and sequenced. The DNA encoding matured miraculin was cloned into the expression vector, pGEX-4T-1 and pET-32A,respectively. The presence of expressed miraculin in the supernatant of cell extract was confirmed by SDS-PAGE and immunoblotting using anti-miraculin serum. The expressed miraculin had no sweetness-inducing activity. The DNA encoding pre-miraculin (MIR) and matured miraculin (MIRPCR) were cloned into the expression vector, pYPR3831 and pYERNAP-Rh. The presence of miraculin in the yeast EH13-15/pYMI-1 extract was confirmed by immunoblotting using anti-miraculin serum. The yeast extracts which were purified by ammonium sulfate fractionation showed no sweetness-inducing activity. Curculin, which exists in the pulp of Curculigo latifolia, has a sweet taste and the ability to induce sweetness. Expression of curculin in E.coli and yeast was performed by the same procedure as that of miraculin Expressed protein showed no activity at present. cDNA for mabinlin, a heat-stable sweet protein, was cloned and the structure of its prepro-protein was determined. Mabinlin expressed in E.coli showed a sweet activity.
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