Preparation of Escherichia coli cell extract for highly productive cell-free protein expression
Preparation of Escherichia coli cell extract for highly productive cell-free protein expression
复制标题
DOI：
10.1023/b:jsfg.0000029204.57846.7d
复制
发表时间：
2004-01-01
期刊：
影响因子：
--
通讯作者：
Yokoyama, Shigeyuki
中科院分区：
文献类型：
--
作者：
Kigawa, Takanori;Yabuki, Takashi;Yokoyama, Shigeyuki
随着结构基因组学和蛋白质组学研究的普及，人们认识到无细胞蛋白质合成系统对于高通量表达的重要性。我们的团队通过使用无细胞蛋白质合成建立了用于结构基因组学和蛋白质组学的蛋白质样品制备的高通量管道。在无细胞蛋白质合成的许多程序中，细胞提取物的制备是建立高效和可重复工作流程的关键步骤。在这篇文章中，我们描述了一个详细的协议，E。大肠杆菌细胞提取物制备无细胞蛋白质合成，我们已经开发和常规使用。根据该方案制备的细胞提取物用于我们的许多无细胞合成应用，包括使用PCR扩增模板的高通量蛋白质表达和用于结构测定的大规模蛋白质生产。
As structural genomics and proteomics research has become popular, the importance of cell-free protein synthesis systems has been realized for high-throughput expression. Our group has established a high-throughput pipeline for protein sample preparation for structural genomics and proteomics by using cell-free protein synthesis. Among the many procedures for cell-free protein synthesis, the preparation of the cell extract is a crucial step to establish a highly efficient and reproducible workflow. In this article, we describe a detailed protocol for E. coli cell extract preparation for cell-free protein synthesis, which we have developed and routinely use. The cell extract prepared according to this protocol is used for many of our cell-free synthesis applications, including high-throughput protein expression using PCR-amplified templates and large-scale protein production for structure determinations.