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Preparative scale expression of membrane proteins in Escherichia coli-based continuous exchange cell-free systems

Abstract

Cell-free expression is emerging as a prime method for the rapid production of preparative quantities of high-quality membrane protein samples. The technology facilitates easy access to large numbers of proteins that have been extremely difficult to obtain. Most frequently used are cell-free systems based on extracts of Escherichia coli cells, and the reaction procedures are reliable and efficient. This protocol describes the preparation of all essential reaction components such as the E. coli cell extract, T7 RNA polymerase, DNA templates as well as the individual stock solutions. The setups of expression reactions in analytical and preparative scales, including a variety of reaction designs, are illustrated. We provide detailed reaction schemes that allow the preparation of milligram amounts of functionally folded membrane proteins of prokaryotic and eukaryotic origin in less than 24 h.

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Figure 1: Design of CECF expression reaction containers.
Figure 2: DNA template for CF expression.
Figure 3: CF expression modes for high-level MP production.
Figure 4: Flowchart of CF MP expression.
Figure 5: High-level CF production of MPs.

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Correspondence to Frank Bernhard.

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Schwarz, D., Junge, F., Durst, F. et al. Preparative scale expression of membrane proteins in Escherichia coli-based continuous exchange cell-free systems. Nat Protoc 2, 2945–2957 (2007). https://doi.org/10.1038/nprot.2007.426

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