Cell-free expression techniques have emerged in recent times as powerful tools for the fast and efficient production of membrane proteins. Most central problems associated with conventional cellular expression systems are eliminated and the direct synthesis into defined artificial hydrophobic environments like detergents or liposomes enables completely new strategies for membrane protein production (Fig. 1). In addition, reaction protocols of cell-free expression systems can be individualized according to the specific requirements of particular target proteins. This exceptional versatility guarantees high success rates for the production of even complex membrane proteins.

We have established throughput strategies for the optimization of cell-free reaction protocols for the expression of membrane proteins as integrated processes with robotic platforms (Fig. 2). The potentials of cell-free expression for the synthesis and characterization of a selection of electrochemical potential-dependent transporters involved in transport, efflux, signalling, metabolism or biosynthesis will be explored. Individual cell-free expression protocols for medically relevant transporters at high quality will be optimized or developed. The function of selected transporters solubilized in micelles and inserted into liposomes will be characterized in vitro by an array of complementary techniques. Structural approaches based on unique synergic effects of cell-free expression systems in combination with NMR spectroscopy and X-ray crystallography will be explored (Fig. 3). The project will provide a comprehensive pilot study of new applications generated by the cell-free production of membrane proteins.

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