| Project/Area Number |
16580083
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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 |
Applied biochemistry
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| Research Institution | University of Tsukuba (2006)
The Institute of Physical and Chemical Research (2004-2005) |
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Principal Investigator |
USUI Takeo University of Tsukuba, Graduate School of Life and Environmental Sciences, Associate Professor, 大学院生命環境科学研究科, 助教授 (60281648)
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| Co-Investigator(Kenkyū-buntansha) |
MUROI Makoto RIKEN, Antibiotics Laboratory, Senor Research Scientist, 長田抗生物質研究室, 先任研究員 (30261168)
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| Project Period (FY) |
2004 – 2006
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| Project Status |
Completed (Fiscal Year 2006)
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| Budget Amount *help |
¥3,300,000 (Direct Cost: ¥3,300,000)
Fiscal Year 2006: ¥500,000 (Direct Cost: ¥500,000)
Fiscal Year 2005: ¥1,100,000 (Direct Cost: ¥1,100,000)
Fiscal Year 2004: ¥1,700,000 (Direct Cost: ¥1,700,000)
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| Keywords | Yeast / Tubulin / Single allele / タキソール / ピロネチン |
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| Research Abstract |
Tubulin is a heterodimer composed of α-and β-polypeptides, each requiring a distinct set of chaperons for proper folding. Apart from this complexity, multiple α-and β-tubulin genes are found in most eukaryotic cells, with each subunit undergoing different post-translational modifications. Consequently, it has been difficult to express and purify isotypically pure tubulin in biochemically useful amounts. In this project, we have used the budding yeast Saccharomyces cerevisiae to conduct mutant analyses on microtubules. S.cerevisiae contains only two α-tubulin genes (TUB1 and TUB3) and one β-tubulin gene (TUB2); since TUB3 is nonessential, it provides a potential source of isotypically pure tubulin. To prepare isotypically pure tubulin, we used TUB3-null yeast cells, having only a single a-and β-tubulin gene, TUB1 and TUB2, respectively. To obtain yeast microtubules that were stable at low concentrations, Taxol-binding ability was introduced into a β-tubulin gene by site-directed mutagenesis at five amino acids, and the gene tub2-A19K-T23V-G26D-N227H-Y270F thus obtained was referred to as TUB2^<tax>. The yeast strain expressing TUB1 and TUB2^<tax> was used as the wild-type. To purify tubulin from the cell lysate, a couple of anion exchange column chromatographies were successively used, and the crude tubulin fraction eluted from the second column was further purified by polymerization and depolymerization. This procedure generated approximately 30 μg of assembly-competent tubulin from 6 liters of culture with purity higher than 95% on SDS gel electrophoresis.
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