Cell-Free Protein Synthesis Technology in NMR High-Throughput Structure Determination
Cell-Free Protein Synthesis Technology in NMR High-Throughput Structure Determination
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DOI：
10.1007/978-1-60327-331-2_12
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发表时间：
2010-01-01
期刊：
影响因子：
--
通讯作者：
Markley, John L.
中科院分区：
文献类型：
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作者：
Makino, Shin-ichi;Goren, Michael A.;Markley, John L.
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情况，以及用于核磁共振结构测定的稳定同位素标记真核蛋白生产的实际方面。据报道，小麦无生殖细胞翻译在小范围筛选中用于总蛋白表达水平、表达蛋白的溶解度和纯化成功率，作为获得足够数量和质量的蛋白质以启动结构研究的可能性的预测指标。在大多数情况下，小规模反应也产生足够的蛋白质，以允许生物分析和功能表征。该方案包括使用专门用于小规模无细胞翻译的机器人，大规模蛋白质生产和可溶性His(6)标记蛋白质的自动纯化。概述了将同位素标记的蛋白质整合到NMR结构测定所需的实验序列中，并提出了在洗涤剂或单层脂质体存在下生产整体膜蛋白的附加方案。
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 arc presented.