Klammt Christian, Schwarz Daniel, Dötsch Volker, Bernhard Frank
Centre for Biomolecular Magnetic Resonance, University of Frankfurt/Main, Institute for Biophysical Chemistry, Frankfurt/Main, Germany.
Methods Mol Biol. 2007;375:57-78. doi: 10.1007/978-1-59745-388-2_3.
The chapter will focus on the high level cell-free production of integral membrane proteins having multiple transmembrane segments by using an individual coupled transcription/translation system based on an Escherichia coli S30-extract. We describe in detail the setup and optimization of the cell-free expression technique to obtain the maximum yield of recombinant proteins. The protocol can be used for the expression of soluble membrane proteins as well as for their production as a precipitate. In addition, we will provide protocols for the efficient solubilization and reconstitution of membrane proteins directly from the cell-free produced precipitates.
本章将重点介绍通过使用基于大肠杆菌S30提取物的单个耦合转录/翻译系统，在无细胞体系中高水平生产具有多个跨膜区段的整合膜蛋白。我们详细描述了无细胞表达技术的设置和优化，以获得重组蛋白的最大产量。该方案可用于可溶性膜蛋白的表达以及将其作为沉淀物进行生产。此外，我们将提供直接从无细胞产生的沉淀物中高效溶解和重组膜蛋白的方案。
