2002 Fiscal Year Final Research Report Summary
Development of rapid proteomic analysis for effective production of recombinant proteins by secretion
| Project/Area Number |
13555226
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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 | 展開研究 |
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| Research Field |
生物・生体工学
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| Research Institution | Kobe University |
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Principal Investigator |
KATOH Shigeo Kobe University, Graduate School of Science and Technology, Professor, 自然科学研究科, 教授 (20026272)
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| Co-Investigator(Kenkyū-buntansha) |
TANAKA Hisao Kyowa Hakko Kogyo Co., LTD., General Manager, 工務部, 技術開発室長
SHIOMI Naofumi Kobe College, Dept. of Human Science, Associate Professor, 人間科学部, 助教授 (20299077)
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| Project Period (FY) |
2001 – 2002
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| Keywords | production by secretion / α-amylase / proteomic analysis / liposome / membrane blotting / methanol assimilating enzymes |
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| Research Abstract |
Recombinant proteins have been produced by secretion from recombinant cells, since biologically active proteins are obtained through easy separation processes. However, the productivity of target proteins is not always high because of incorrect processing of signal sequences and degradation by proteases. In this work we propose an effective control and production method for secretion of recombinant proteins based on the proteomic analysis with rapid characterization of produced proteins and measurements of the activities of marker enzymes and proteases.
As a model protein α-amylase was secreted from S. cerevisiae and Pichia pastoris, and the effects of fermentation conditions on the productivity, the activities of marker enzymes and the characteristics of secreted protein were studied. The following results were obtained;
* Characteristics of C-terminus of secreted α-amylase were clarified by use of an anti-peptide antibody against the C-terminal sequences.
* Pichia pastoris could effectively secrete α-amylase by induction with methanol, and the feed of methanol could be controlled by the DO-statt for effective production.
* A novel membrane blotting method utilizing immunoliposomes was developed for rapid and highly sensitive measurement of proteins.
* The activities of enzymes in the metabolic pathway of methanol increased by induction with methanol and were related to the specific production rate of α-amylase.
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