2001 Fiscal Year Final Research Report Summary
Signal transaction between the functional domains of type II restriction endonuclease
| Project/Area Number |
11660092
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
応用微生物学・応用生物化学
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| Research Institution | TOTTORI UNIVERSITY |
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Principal Investigator |
KITA Keiko Tottori University, Department of Biotechnology, Associate professor, 工学部, 助教授 (70234226)
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| Project Period (FY) |
1999 – 2001
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| Keywords | Restriction endonuclease / Functional domain / Overexpression / Fusion protein / DNA-binding protein / Chimeric enzyme / Crystallization / Folding |
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| Research Abstract |
StsI is a member of unusual class of restriction enzymes that recognize a specific DNA sequence and cleave nonspecifically a short distance away from that sequence. The present study describes mutants of the restriction enzyme StsI that lose DNA cleavage activity. We constructed StsI variants, S233I, W241R, K300E, L365R, E442K, L453S, and I478T, which are unable to cleave DNA specifically. The variants, S233I and E442K, were purified homogeneously from the soluble fraction of recombinant E. coli cell, but the other mutant enzymes produced inclusion bodies. In order to purify these proteins from inclusion bodies, the aggregates were suspended in 6 M guanidine-HCl, and refolded by dilution with the appropriate buffer. The mutant proteins were purified homogeneously with subsequent chromatography using Heparin-Sepharose. The StsI variant, L365R, which defective for both DNA-binding and DNA-cleavage activities, stimulated the rate of DNA cleavage catalyzed by wild-type StsI. These results suggested that the StsI catalytic domains dimerize, as reported by FokI, but a second StsI molecule does not necessarily bind to the recognition site.
In order to solve the crystal structure of StsI, an overexpression system was constructed and the enzyme was purified homogeneously. However, we have not been succeeded in obtaining the StsI crystal yet. Improvements have to be made in purification process of StsI.
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