1999 Fiscal Year Final Research Report Summary
Search for structural and functional units of a protein by random fragmentation
| Project/Area Number |
09480154
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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 |
Structural biochemistry
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| Research Institution | Tokyo University of Pharmacy & Life Science |
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Principal Investigator |
OSHIMA Tairo School of Life Science, Tokyo University of Pharmacy & Life Science Professor, 生命科学部, 教授 (60167301)
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| Project Period (FY) |
1997 – 1999
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| Keywords | isopropylmalate dehydrogenase / tryptophan synthetase / nicking / structural unit / TIM barrel / fragmented enzyme |
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| Research Abstract |
The investigator has tried to identify structural and functional units of a protein by random fragmentation using genetic manipulation techniques. A new method has been deviced to insert a linker which contains a stop codon, a ribosome binding sequence, and an initiation codon, into any position on a gene coding an enzyme. This method was applied to the E.coli tryptophan synthetase. The gene was cut randomly and the linker bove mentioned was inserted. The constructed fragmented library was expressed in E.coli and the active fragments were screened in a medium lacking tryptophan. Many fragmented genes were isolated indicating that the enzyme can be fragmented without the loss of the activity. Most of the isolated active gene contained overlapping sequences. The nicking sites are mostly located in loop regions. Another attempt was done in 1999 to fragment isopropylmalate dehydrogenase from an extreme thermophile, Thermus thermophilus, by inserting a similar linker at the domain-domain interface. This experiment is still continuing.
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