Abstract
This chapter addresses two major bottlenecks in cell-free membrane protein production. Firstly, we describe the optimization of expression templates for obtaining membrane proteins in preparative scales. We present details for a newly established tag variation screen providing high success rates in improving expression efficiencies while having only minimal impacts on the target protein structure. Secondly, we present protocols for the efficient co-translational insertion of membrane proteins into defined lipid bilayers. We describe the production of nanodiscs and their implementation into cell-free expression reactions for the co-translational reconstitution of membrane proteins. In addition we give guidelines for the loading of nanodiscs with different lipids in order to systematically analyze effects of lipids on the translocation, functional folding, and stability of cell-free expressed membrane proteins.
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Acknowledgements
This work was supported by the Collaborative Research Center (SFB) 807 of the German Research Foundation (DFG). We further thank the European Drug Initiative on Channels and Transporters (EDICT), contract number HEALTH-F4-2007-201924; the European initiative on Structural Biology of Membrane Proteins (SBMP), contract number PITN-GA-2008-211800; and the NIH (grant number U54 GM094608) for funding. We further thank Oliver Ernst for helpful advice and Vladimir Shirokov for critical discussions.
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Roos, C. et al. (2014). High-Level Cell-Free Production of Membrane Proteins with Nanodiscs. In: Alexandrov, K., Johnston, W. (eds) Cell-Free Protein Synthesis. Methods in Molecular Biology, vol 1118. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-782-2_7
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DOI: https://doi.org/10.1007/978-1-62703-782-2_7
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Publisher Name: Humana Press, Totowa, NJ
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