|["Cell-free expression techniques have been established as efficient tools for the fast production of integral membrane proteins as they generally eliminate most central bottlenecks restricting membrane protein production in conventional cellular expression systems. Systematic protocol development results in success rates of > 80 % independent from size, topology, function or origin of the membrane protein target. The open accessibility of cell-free reactions allows furthermore the direct synthesis and solubilization of membrane proteins into defined artificial hydrophobic environments composed of detergents, surfactants or lipids. Cell-free expression therefore combines high success rates in membrane protein production with rapid access to usually very difficult and complex pharmaceutical targets.
A diverse number of options to modify the cell-free reaction conditions are possible and we demonstrate that this is a valuable tool in order to optimize membrane protein quality by applying throughput strategies. I will discuss how efficient cell-free expression protocols for membrane proteins are established and I will give an overview on our recent results obtained with the preparative scale cell-free production of G-protein coupled receptors, transporters and membrane integrated enzymes involved in peptidoglycan synthesis. Co- and post-translational modulation of the protein environment was systematically used in order to optimize membrane protein activity and stability. An emerging tool is the combination of nanodiscs with cell-free expression in order to produce highly soluble membrane proteins inserted into their natural lipid environments. We demonstrate that enzymatic activity of membrane proteins can strongly be modulated by the type of lipids provided with the nanodiscs.
Many membrane proteins form larger complexes and are not functional as monomers. Even large molecular machines can assemble in cell-free systems as analysed by single particle analysis of the ATP synthase, a 530 kDa complex composed out of 25 protomers. In addition, I will discuss structural approaches of cell-free expressed membrane proteins including subunits of the y-secretase complex, a key player in Alzheimer's disease development."]