| Project/Area Number |
60880018
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| Research Category |
Grant-in-Aid for Developmental Scientific Research
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| Allocation Type | Single-year Grants |
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| Research Field |
物質生物化学
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| Research Institution | The University of Tokyo |
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Principal Investigator |
INOUE Keizo The University of Tokyo, 薬学部, 教授 (30072937)
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| Co-Investigator(Kenkyū-buntansha) |
岸本 文貴 住友化学生命工学研究所, 主任研究員
荻野 重男 住友化学生命工学研究所, 所長
KOBAYASHI Tetsuyuki The University of Tokyo, 薬学部, 助手 (50178323)
UMEDA Masato The university of Tokyo, 薬学部, 助手 (10185069)
KUDO Ichiro The University of Tokyo, 薬学部, 助教授 (30134612)
OGINO Shigeo Sumitomo chemical Co. Ltd.
KISHIMOTO Fumitaka Sumitomo chemical Co. Ltd.
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| Project Period (FY) |
1985 – 1987
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| Project Status |
Completed (Fiscal Year 1987)
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| Budget Amount *help |
¥29,400,000 (Direct Cost: ¥29,400,000)
Fiscal Year 1987: ¥4,100,000 (Direct Cost: ¥4,100,000)
Fiscal Year 1986: ¥10,300,000 (Direct Cost: ¥10,300,000)
Fiscal Year 1985: ¥15,000,000 (Direct Cost: ¥15,000,000)
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| Keywords | Signal sequence / Outer membrance of E.coli / Secreetion vector / Processing / Urogastron / プロセシング / 大腸菌 / 組換えDNA / ホスホリパーゼ / 細胞外分泌 / ウロガストロン遺伝子 / ホスホリパーゼ分泌ベクター |
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| Research Abstract |
The aim of this project was to clarify a molecular mechanism of location and processing of one of E. coli outer membrance protein, detergent resistant phospholipase A (DR-phospholipase A). We also attempted to construct a cloning vector which utilized the signal sequence of DR- phospholipase A.
1. Biochemical and genetical analysis of DR-phospholipase A precursor molecule:
The structure gene of DR-phosphclipase A, pldA, was cloned. The in vitro product of pldA was analyzed and was found to bear a molecular weight slightly higher than that generated in vivo< In other experiment, a site-directed mutagenesis, which resulted in one amino acid insertgion, was introduced into the DNA region coding for the signal sequence of the DR-phospholipase A. The mutated pldA gene was found to produce less amount of enzyme. These results strongly suggested the existence of a precursor molecule of DR-phospholipase A. Less amount of production of DR-phospholipase A from mutated pldA gene might be due to the inefficient processing step.
2. Construction of cloning vector:
A synthetic DNA fragment containing multi-cloning sites was introduced immediately after the DNA region which coded for the signal sequence of DR- phospholipase A. The promoter of pldA gene was then replaced by three well-characterized promoters (tac, consensus, and lambda PL), respectively.
3. Application of vectors:
Synthetic genes coding for fuman urogastron of lysozyme were cloned onto these novel cloning vectors. E. coli strains bearing recombinant plasmids were examined for the expression of human proteins. So far, production and accumulation of a mature form of human lysozyme was observed, however, the yield was low. Further improvement in the expression must be performed.
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