| Project/Area Number |
18560755
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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 |
Biofunction/Bioprocess
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| Research Institution | Sojo University |
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Principal Investigator |
MATSUOKA Masayoshi Sojo University, Faculty of Biotechnology and Life Science, Professor (10121667)
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| Co-Investigator(Kenkyū-buntansha) |
OGAWA Takahira Sojo University, Faculty of Biotechnology and Life Science, Professor (40029244)
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| Project Period (FY) |
2006 – 2007
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| Project Status |
Completed (Fiscal Year 2007)
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| Budget Amount *help |
¥3,760,000 (Direct Cost: ¥3,400,000、Indirect Cost: ¥360,000)
Fiscal Year 2007: ¥1,560,000 (Direct Cost: ¥1,200,000、Indirect Cost: ¥360,000)
Fiscal Year 2006: ¥2,200,000 (Direct Cost: ¥2,200,000)
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| Keywords | photosystem II / cyanobacteria / water splitting / D 1 / D2 heterodimer / gene replacement / histidine tag / affinity chromatography / thermostability / rps12 |
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| Research Abstract |
A minimum unit required for photochemical water-splitting reaction is a heterodimer consisting of D1 and D2 proteins in reaction center of photosystem II (PSII). We investigated possible isolation of D 1/D2 heterodimer from mesophilic cyanobacterium, Synechococcus elongatus PCC 7942 expressing thermostable Dl/D2 proteins from a thermophile Thermosynechococcus vulcanus. To this end, we replaced ORFs of D1 and D2 genes in S elongatus with those from T vulcanus via rps12-mediated gene replacement method, a newly developed technology without limitation of the availability of drug-resistance markers. A C-terminal histidine tag was introduced into CP47 protein encoded by psbB gene to facilitate the purification of PSII complex. The resultant psbB mutant strain was heterogeneous for psbB gene, so that a psbT gene downstream the psbB gene was activated by inserting an extra copy of promoter upstream the psbT gene. In addition, a long stretch of DNA encompassing the psbB psbTregion was cloned to enhance the recombination frequency between plasmid and chromosome. Using psbB mutant strains thus created, Dl/D2 heterodimer was efficiently purified from PSII complex via Ni-affinity chromatography.
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