| Project/Area Number |
11557181
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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 |
Biological pharmacy
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| Research Institution | TOKYO INSTITUTE OF TECHNOLOGY |
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Principal Investigator |
MIYAZAWA Keiji Tokyo Institute of Technology, Graduate School of Bioscience and Biotechnology Associate professor, 大学院・生命理工学研究科, 助教授 (40209896)
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| Co-Investigator(Kenkyū-buntansha) |
SHIMOMURA Takeshi Mitsubishi-Tokyo Pharmaceuticals Inc, Pharmaceuticals Discovery Laboratory Senior Research Scientist, 創薬基盤研究所, 主管研究員
KITAMURA Naomi Tokyo Institute of Technology, Graduate School of Bioscience and Biotechnology Professor, 大学院・生命理工学研究科, 教授 (80107424)
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| Project Period (FY) |
1999 – 2000
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| Project Status |
Completed (Fiscal Year 2000)
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| Budget Amount *help |
¥13,000,000 (Direct Cost: ¥13,000,000)
Fiscal Year 2000: ¥3,500,000 (Direct Cost: ¥3,500,000)
Fiscal Year 1999: ¥9,500,000 (Direct Cost: ¥9,500,000)
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| Keywords | protease / fusion protein / limited digestion / recombinant protein |
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| Research Abstract |
Proteases with high substrate specificity as well as high efficiency are desirable for limited digestion of tagged fusion proteins. In this project, we examined the use of hepatocyte growth factor activator (HGFA) for this purpose.
First, we introduced an HGFA-substrate peptide sequence into GST fusion protein. Anoligonucleotide coding HGFA cleavage sequence (AKTKQLRVVNG) was inserted downstream of thrombin cleavage site of pGEX4T-3 vector. For assesing the utility of this system, JNK interacting protein-1 (JIP-1) was used as a model protein for expression and cleavage. The expressed fusion protein (GST-JIP1) was treated with HGFA as well as thrombin at substrate/enzyme ratio of 50/1 at 37℃ for 4 hours. Thrombin treatment caused extensive degradation of the JIP-1 moiety of the fusion protein, whereas HGFA treatment caused cleavage of the linker peptide without extra digestion. HGFA treatment was successful for other fusion proteins, GST-NK1 (a variant of hepatocyte growth factor) and GST-Met kinase domain. This system worked in the presence of glutathione, indicating that column eluate from glutathione-Sepharose can be processed for digestion without pretreatment. The system, however, failed to work in the presence of 0.5-2M urea, indicating that urea-solubilized proteins should be devoid of urea before digestion.
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