CONSTRUCTION OF IN VITRO EVOLUTION SYSTEM OF PROTEINS

蛋白质体外进化体系的构建

• 批准号: 09650872
• 负责人: NAKANO Hideo
• 金额: $ 0.7万
• 依托单位: Nagoya University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1997
• 资助国家: 日本
• 起止时间: 1997 至 1998
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09650872/
• 关键词: single molecule; PCR; cell-free system; transcription; translation; protein synthesis; molecular evolution; library; 1分子; Polymerase Chain Reaction; 無細胞蛋白合成反応; 蛋白質ライブラリー

A novel in vitro method for the generation of a protein library has been developed using the polymerase chain reaction (PCR) amplification of a single DNA molecule followed by in vitro coupled transcription/translation. DNA template encoding green fluorescent protein (GFP) of a jellyfish Aequorea victoria was extensively diluted to one molecule per tube, and then amplified by a total of 80 cycles of PCR with nested primers. The exact number of origins in the amplified DNA fragment was then estimated by directly sequencing a part of the fragment, at which an individual template molecule was marked by PCR with a primer containing three randomized bases. Since the sequehces obtained in 91 independent amplifications were diversified statistically, each amplified fragment was likely originated from a single DNA molecule. In addition, the amplified fragments served as a template for in vitro coupled transcription/translation using T7 RNA polymerase and E.ccli S30 extract. These results suggest that the library obtained by the PCR amplification of a single DNA molecule diluted from a variety of DNA pools is potentially useful in high-throughput generation of protein libraries.

一种新的体外生成蛋白质文库的方法已经被开发出来，该方法使用了单个DNA分子的聚合酶链反应（PCR）扩增，然后在体外偶联转录/翻译。将维多利亚Aequorea victoria水母编码绿色荧光蛋白（GFP）的DNA模板广泛稀释至每管1分子，然后用巢式引物进行共80次PCR扩增。然后通过直接对片段的一部分进行测序来估计扩增DNA片段的确切起源数量，其中单个模板分子通过PCR标记包含三个随机碱基的引物。由于91次独立扩增获得的序列在统计上是多样化的，因此每个扩增片段可能来自单个DNA分子。此外，扩增的片段可作为T7 RNA聚合酶和E.ccli S30提取物体外偶联转录/翻译的模板。这些结果表明，通过PCR扩增从多种DNA池中稀释的单个DNA分子获得的文库可能用于高通量蛋白质文库的生成。

项目成果

Hideo Nakano et al,: "Efficient coupled transcription/translation from PCR template by a hollow fiber membrane bioreactor." Biotechnology & Bioengineering. 印刷中.

Hideo Nakano 等人：“通过中空纤维膜生物反应器进行 PCR 模板的高效耦合转录/翻译。”正在出版。

Hideo Nakano, Tomoya Shinbata, Reiko Okumura, Satoshi Sekiguchi, Masatoshi Fujishiro, and Tsuneo Yamane.: "Efficient coupled transcription/translation from PCR template by a hollow fiber membrane bioreactor." Biotechnology & Bioengineering. (in press.).

Hideo Nakano、Tomoya Shinbata、Reiko Okumura、Satoshi Sekiguchi、Masatoshi Fujishiro 和 Tsuneo Yamane：“通过中空纤维膜生物反应器从 PCR 模板进行高效耦合转录/翻译。”

Hideo Nakano: "Cell-free protein synthesis systems." Biotechnology Advances. 16. 367-384 (1998)

Hideo Nakano：“无细胞蛋白质合成系统。”

Shoji Ohuchi et al,: "In vitro method for the generation of protein libraries using PCR amplification of a single DNA molecule and coupled transcription/translation" Nucleic Acids Research. 26. 4339-4346 (1998)

Shoji Ohuchi 等人：“使用单个 DNA 分子的 PCR 扩增和耦合转录/翻译来生成蛋白质文库的体外方法”核酸研究。

Ohuchi, S., Nakano, H.and Yamane, T.: "In vitro method for the generation of protein libraries using PCR amplification of a single DNA molecule and coupled transcription/translation" Nucleic Acids Research. 26. 4339-4346 (1998)

Ohuchi, S.、Nakano, H. 和 Yamane, T.：“使用单个 DNA 分子的 PCR 扩增和偶联转录/翻译来生成蛋白质文库的体外方法”核酸研究。