Development of cell-free gene translator as an equipment to support researches in bioscience and biotechnology

开发无细胞基因翻译器作为支持生物科学和生物技术研究的设备

• 批准号: 10555287
• 负责人: YAMANE Tsuneo
• 金额: $ 8.06万
• 依托单位: Nagoya University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 1998
• 资助国家: 日本
• 起止时间: 1998 至 2001
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-10555287/
• 关键词: cell-free protein synthesis; transcription; translation; bioreactor; ATP; stabilization of protein; E. coli S30 extract; Wheat germ extract; たん白質の安定化; DNAの安定化; 無細胞蛋白合成; ミニバイオリアクター; 無細胞遺伝子翻訳装置; ホローファイバー; 中空糸; 転写・翻訳供役反応

Wth aims of developing a laboratory instrument which enables to obtain several mg of foreign active protein in one or two days, and of establishing method of preparing large amount of Escherichia coli S30 extract which is used in the system, the following have been achieved.1. Achievement of Nagoya UniversityA cell-discruption equipment(trade name : beads schocker) was found to be useful to get large amount of S30 extract. Maximally about 50mL of the extract could be obtained. Optimal operational conditions of this equipment was established. A reaction condition to synthesize active protein having correct disulfide bridge was also elucidated.2. Achievement of Tomy Manufacturing Co., Ltd.A small closed dialyzer having semipermiable membrane with working volume of 0. 1〜 0. 3mL was developed. Using this dialyzer, ca. 1. 2mg/mL of chlaramphenicol acetyltransferase could be synthesized by cell-free transcription/ translation system containing E. coli S30 extract in 12 hours.3. Achievement of Nippon Flour Mills Co., Ltd.Long-term stability of S30 extract in its storage in liquid nitrogen was tested. The extract was found to be stably stored for about one year.

为了开发一种能够在一天或两天内获得几毫克外源活性蛋白的实验室仪器，并建立制备用于该系统的大量大肠杆菌S30提取物的方法，进行了以下工作。名古屋大学的成果发现细胞破碎设备（商品名：beads schocker）可用于获得大量的S30提取物。最多可获得约50 mL浸提液。确定了该装置的最佳操作条件。阐明了合成具有正确二硫键的活性蛋白的反应条件. Tomy Manufacturing Co.，一种小型的半渗透膜封闭式透析器，工作容积为0。1块钱。3mL已开发。使用这种透析器，CA。1.用含有E. coli S30提取液12 h后，日本面粉米尔斯公司的成就，测试了S30提取物在液氮中储存的长期稳定性。发现提取物稳定储存约一年。

项目成果

Yugo Iwasaki: "Imporlance of Disulfide Bridge Formation on Folding of Phospholipase D from Streptomyces antibioticus"Journal of Bioscience and Bioengineering. 89・5. 506-508 (2000)

岩崎佑吾：“二硫键形成对抗生素链霉菌磷脂酶 D 折叠的重要性”生物科学与生物工程杂志 89・5（2000）。

Hideo Nakano: "Efficient coupled transcription/translation for PCR template by hollow fiber membrane bioreactor" Biotechnology and Bioengineering. 62巻(印刷中). (1999)

Hideo Nakano：“通过中空纤维膜生物反应器进行 PCR 模板的高效偶联转录/翻译”，《生物技术与生物工程》第 62 卷（出版中）。

Hideo Nakano: "Single-Step Single-Molecule PCR of DNA with a Home-Priming Sequence Using a Single Primer and Hot-Startable DNA Polymerase"Journal of Bioscience and Bioengineering. 90・4. 456-458 (2000)

Hideo Nakano：“使用单引物和热启动 DNA 聚合酶进行 DNA 的单步单分子 PCR”《生物科学与生物工程杂志》90・4（2000 年）。

山根恒夫: "組換えタンパク質生産法、第4章、pp 169-180"学会出版センター. 12 (2001)

Tsuneo Yamane：“重组蛋白质生产方法，第4章，第169-180页”协会出版中心12（2001）。

Suang Rungpragayphan: "High-throughput, cloning-independent protein library construction by combining single-molecule DNA amplivication with in vitro expression"Journal of Molecular Biology. (印刷中). (2002)

Suang Rungpragayphan：“通过结合单分子 DNA 扩增与体外表达构建高通量、不依赖于克隆的蛋白质文库”《分子生物学杂志》（2002 年出版）。