Makino Shin-ichi, Goren Michael A, Fox Brian G, Markley John L
Department of Biochemistry, Center for Eukaryotic Structural Genomics, College of Agricultural and Life Sciences, University of Wisconsin, Madison, WI, USA.
Methods Mol Biol. 2010;607:127-47. doi: 10.1007/978-1-60327-331-2_12.
This chapter describes the current implementation of the cell-free translation platform developed at the Center for Eukaryotic Structural Genomics (CESG) and practical aspects of the production of stable isotope-labeled eukaryotic proteins for NMR structure determination. Protocols are reported for the use of wheat germ cell-free translation in small-scale screening for the level of total protein expression, the solubility of the expressed protein, and the success in purification as predictive indicators of the likelihood that a protein may be obtained in sufficient quantity and quality to initiate structural studies. In most circumstances, the small-scale reactions also produce sufficient protein to permit bioanalytical and functional characterizations. The protocols incorporate the use of robots specialized for small-scale cell-free translation, large-scale protein production, and automated purification of soluble, His(6)-tagged proteins. The integration of isotopically labeled proteins into the sequence of experiments required for NMR structure determination is outlined, and additional protocols for production of integral membrane proteins in the presence of either detergents or unilamellar liposomes are presented.
本章描述了真核结构基因组学中心（CESG）开发的无细胞翻译平台的当前实施方案，以及用于核磁共振结构测定的稳定同位素标记真核蛋白质生产的实际情况。报告了使用小麦胚无细胞翻译进行小规模筛选的方案，以总蛋白表达水平、表达蛋白的溶解度和纯化成功率作为预测指标，来判断是否有可能获得足够数量和质量的蛋白质以启动结构研究。在大多数情况下，小规模反应也能产生足够的蛋白质用于生物分析和功能表征。这些方案包括使用专门用于小规模无细胞翻译、大规模蛋白质生产以及可溶性His(6)标签蛋白自动纯化的机器人。概述了将同位素标记蛋白整合到核磁共振结构测定所需实验序列中的过程，并介绍了在存在去污剂或单层脂质体的情况下生产整合膜蛋白的其他方案。
