| Project/Area Number |
11640649
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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 |
植物生理
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| Research Institution | Okayama University |
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Principal Investigator |
TAKAHASHI Yuichiro Okayama University, Fac. Of Science, Assoc. Prof., 理学部, 助教授 (50183447)
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| Project Period (FY) |
1999 – 2000
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| Project Status |
Completed (Fiscal Year 2001)
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| Budget Amount *help |
¥3,500,000 (Direct Cost: ¥3,500,000)
Fiscal Year 2000: ¥1,800,000 (Direct Cost: ¥1,800,000)
Fiscal Year 1999: ¥1,700,000 (Direct Cost: ¥1,700,000)
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| Keywords | photosynthesis / photosystem / assembly / tagged protein / affinity chromatography / subunit compositon / chloroplast transformation / Chlamydomonas reinahrdtii / 光化学系複合体 / 生合成 / 葉緑体遺伝子 / Ycf4タンパク / タンパクの精製 / 葉緑体 / 光化学系1 / 形質転換 / タンパク質工学 / ヒスチジンタグ |
|---|
| Research Abstract |
Photosystem I (PSI) is a multiprotein complex consisting of more than ten subunits and one hundred cofactors. Function and structure of this complex have been extensively studied while the assembly of the constituent components into a functional complex is largely unknown. We have already reported that the chloropalst-encoded Ycf4 protein is essential for the assembly of PSI complex in the green alga Chlamydomonas reinhardtii and that Ycf4 is a component of a large complex that is tentatively designated as Ycf4-complex. We have developed a purification procedure of Ycf4-comple after solubilization of thylakoids with dodecyl-maltoside using sucrose density gradient centrifugation, DEAE-chromatography and gel-filtration chromatography. In addition to this classical biochemical approach, we have generated TAP (tandem affinity purification)-tagged Ycf4 complex by chloroplast transformation of Chlamyodmonas. This TAP-technology enabled us to purify Ycf4-complex by two-step affinity chromatography. Comparative study of polypeptide compositions of Ycf4-complexes purified independently, revealed that more than ten subunits are present. It was also estimated that the size of Ycf4-complex is as large as 1800 kDa. We are currently purifying Ycf4-complex on a much larger scale and will determine amino acid sequence of the constituent polypeptides. These sequence data will be used for searching cDNA clones.
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