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.

The preparation of sufficient amounts of high-quality samples is still the major bottleneck for the characterization of membrane proteins by in vitro approaches. The hydrophobic nature, the requirement for complicated transport and modification pathways, and the often observed negative effects on membrane properties are intrinsic features of membrane proteins that frequently cause significant problems in overexpression studies. Establishing efficient protocols for the production of functionally folded membrane proteins is therefore a challenging task, and numerous specific characteristics have to be considered. In addition, a variety of expression systems have been developed, and choice of appropriate techniques could strongly depend on the desired target membrane proteins as well as on their intended applications. The production of membrane proteins is a highly dynamic field and new or modified approaches are frequently emerging. The review will give an overview of currently established processes for the production of functionally folded membrane proteins.

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Cell-free expression has emerged as a promising tool for the fast and efficient production of membrane proteins. The rapidly growing number of successfully produced targets in combination with the continuous development of new applications significantly promotes the distribution of this technology. Membrane protein synthesis by cell-free expression does not appear to be restricted by origin, size or topology of the target, and its global application is therefore a highly valuable characteristic. The technology is relatively fast to establish in standard biochemical labs, and it does not require expensive equipment. Moreover, it enables the production of membrane proteins in completely new modes, like the direct translation into detergent micelles, which is not possible with any other expression system. In this protocol, we focus on the currently most efficient cell-free expression system for membrane proteins based on Escherichia coli extracts.

The functional and structural characterization of G-protein-coupled receptors (GPCRs) still suffers from tremendous difficulties during sample preparation. Cell-free expression has recently emerged as a promising alternative approach for the synthesis of polytopic integral membrane proteins and, in particular, for the production of G-protein-coupled receptors. We have now analyzed the quality and functional folding of cell-free produced human endothelin type B receptor samples as an example of the rhodopsin-type family of G-protein-coupled receptors in correlation with different cell-free expression modes. Human endothelin B receptor was cell-free produced as a precipitate and subsequently solubilized in detergent, or was directly synthesized in micelles of various supplied mild detergents. Purified cell-free-produced human endothelin B receptor samples were evaluated by single-particle analysis and by ligand-binding assays. The soluble human endothelin B receptor produced is predominantly present as dimeric complexes without detectable aggregation, and the quality of the sample is very similar to that of the related rhodopsin isolated from natural sources. The binding of human endothelin B receptor to its natural peptide ligand endothelin-1 is demonstrated by coelution, pull-down assays, and surface plasmon resonance assays. Systematic functional analysis of truncated human endothelin B receptor derivatives confined two key receptor functions to the membrane-localized part of human endothelin B receptor. A 39 amino acid fragment spanning residues 93-131 and including the proposed transmembrane segment 1 was identified as a central area involved in endothelin-1 binding as well as in human endothelin B receptor homo-oligomer formation. Our approach represents an efficient expression technique for G-protein-coupled receptors such as human endothelin B receptor, and might provide a valuable tool for fast structural and functional characterizations.