| Project/Area Number |
05680554
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| Research Category |
Grant-in-Aid for General Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Research Field |
Functional biochemistry
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| Research Institution | HIROSIMA UNIVERSITY |
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Principal Investigator |
KOMINAMAI Shiro Fac. Integrated Arts and Sciences, Hiroshima Univ., Professor, 総合科学部, 教授 (10106776)
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| Co-Investigator(Kenkyū-buntansha) |
SAKAKI Toshiyuki Research Institute of Smitomo Pharma ceutical Company, Chief Researcher, 主任研究員
YAMAZAKI Takeshi Fac. Integrated Arts and Sciences Hiroshima Univ., Assisrant Professor, 総合科学部, 助手 (30192397)
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| Project Period (FY) |
1993 – 1994
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| Project Status |
Completed (Fiscal Year 1994)
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| Budget Amount *help |
¥1,900,000 (Direct Cost: ¥1,900,000)
Fiscal Year 1994: ¥400,000 (Direct Cost: ¥400,000)
Fiscal Year 1993: ¥1,500,000 (Direct Cost: ¥1,500,000)
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| Keywords | Gene Expression / P-450XVIIA1 / Membrane Proteins / PCR Mutagenesis / E.coli / Steroidogenesis / Rapid Quenching Method / Successive Reaction / チトクロムP-450 / P-450X VII A1 / 大量発現 / P-4501A1 / リポソーム / P-45011B1 / P-450(C21) |
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| Research Abstract |
The aims of this research project were a large scale expression of bovine adrenal cytochrome P-450XVIIA1 in E.coli and the elucidation of the reaction mechanism in the membrane reconstitude system. In order to express genes of eucaryotic membrane proteins in E.coli, about 20 base pairs of the nucleotide sequences at 5'-terminal of the DNA were converted into the sequences which are generally used for the membrane proteins of E.coli. PCR mutagenesis was performed for this conversion using a primer containing A and T rich sequence at the 5'-terminal end. The mutated DNA was inserted to plasmids, pKSN2 and pCW both of which have tac promoter. The amount of expressed protein was assayd by Western blotting and the concentration of native P-450 was determined by reduced-CO difference spectra. In the case of the constitution with pKSN2, E.coli expressed the P-450 to the range of several % of the total proteins but most of the proteins did not contain heme. On the contrary, 60 nmol of the P-450 was expressed per one liter of the cultivated E.coli as the holo-form by constitution with pCW.This expressed P-450XVIIA1 in E.coli could catalyzed the metabolism of progesterone to 17alpha-hydroxyprogesterone.
The reaction mechanism of P-450XVIIA1 catalyzing metabolism from progesterone to androstenedione was studied with a rapid quenching devie in the membrane reconstituted system using P-450XVIIA1 purified from guinea pig adrenal microsomes. Androstenedione was proved to be produced from progesterone via 17alpha-hydroxyprogesterone by a successive reaction mechanism in which the reaction intermediate does not leave the P-450.
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