| Project/Area Number |
12450332
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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 |
生物・生体工学
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| Research Institution | Nagoya University |
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Principal Investigator |
NAKANO Hideo Grad.Sch.Bioagri.Sci.,Nagoya Univ.Associate Professor, 大学院・生命農学研究科, 助教授 (00237348)
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| Co-Investigator(Kenkyū-buntansha) |
TAMIYA Eiichi Grad.Sch.Material Sci.,JAIST Professor, 材料科学研究所, 教授 (60179893)
YAMANE Tsuneo Grad.Sch.Bioagri.Sci.,Nagoya Univ.Professor, 大学院・生命農学研究科, 教授 (70026102)
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| Project Period (FY) |
2000 – 2002
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| Project Status |
Completed (Fiscal Year 2002)
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| Budget Amount *help |
¥14,700,000 (Direct Cost: ¥14,700,000)
Fiscal Year 2002: ¥2,600,000 (Direct Cost: ¥2,600,000)
Fiscal Year 2001: ¥2,700,000 (Direct Cost: ¥2,700,000)
Fiscal Year 2000: ¥9,400,000 (Direct Cost: ¥9,400,000)
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| Keywords | cell-free protein synthesis / protein library / single-molecule PCR / micro-chamber array / lipase / GFP / high-throughput screening / antibody / ライブラリー / ビーズ / 均一性 / 蛍光特性 / マイクロアレイ / 蛍光蛋白質 / PCR / 一本鎖抗体 / ハイスループット |
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| Research Abstract |
Herein we have developed a novel protein library system termed SIMPLEX : single-molecule PCR linked in vitro expression combining PCR amplification of single DNA molecule diluted in a well and cell-free protein synthesis. Use of single primer amplification and hot-startable DNA polymerase enabled a single step single-molecule PCR. The obtained protein library by SIMPLEX has been demonstrated to be highly uniform. The uniformity was actually much higher than that obtained by a conventional in vivo system. Also multi-molecule PCR in a well has been shown to be possible. These results have demonstrated that SIMPLEX can provide a high-throughput method for the construction and screening of protein library. Moreover, protein could be synthesized on a micro-chamber array with the concentration of 10,000/cm^2, which will lead the construction of highly condensed protein library.
In addition, in vitro combinatorial mutagenesis was also developed to demonstrate that combinatorial mutation in a specific region of protein selected based on its 3D structure is effective to modulate or improve some properties of proteins.
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