Cell-Free Production of Membrane Proteins in Escherichia coli Lysates for Functional and Structural Studies
The complexity of membrane protein synthesis is largely reduced in cell-free systems and it results into high success rates of target expression. Protocols for the preparation of bacterial lysates have been optimized in order to ensure reliable efficiencies in membrane protein production that are even sufficient for structural applications. The open accessibility of the semisynthetic cell-free expression reactions allows to adjust membrane protein solubilization conditions according to the optimal folding requirements of individual targets. Two basic strategies will be exemplified. The post-translational solubilization of membrane proteins in detergent micelles is most straightforward for crystallization approaches. The co-translational integration of membrane proteins into preformed nanodiscs will enable their functional characterization in a variety of natural lipid environments.
Key wordsG-protein-coupled receptors Nanodiscs Synthetic biology Membranes Membrane protein crystallization Lipid screening
This work was funded by the Collaborative Research Center (SFB) 807 of the German Research Foundation (DFG) and by the German Ministry of Education and Science (BMBF). Support was further obtained by Instruct, part of the European Strategy Forum on Research Infrastructures (ESFRI).
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