Cell-free expression of membrane proteins

Cell-free (CF) expression systems have emerged in recent times as promising tools in order to accelerate and to streamline MP expression approaches. The elimination of a living host environment during protein overexpression in combination with the open accessibility of the reaction offers a variety of valuable advantages. It is evident that problems with toxic or inhibitory effects of the recombinant MPs to the host cell physiology are minimized or even completely eliminated. The expression reaction is not enclosed by membranes and any compound can thus be directly added without considering transport or metabolic conversion problems. Protease inhibitors, ligands, cofactors or substrates can be considered as possible additives that might be helpful in stabilizing the freshly translated MPs. CF extracts are devoid of native membranes and complicated transportation or translocation systems for the synthesized MPs are therefore no longer necessary. In contrast, the MPs can be maintained soluble in artificial hydrophobic environments like detergent micelles. Furthermore, CF reactions are carried out in small volumes of few millilitres or even microlitres and incubation times of few hrs are already sufficient for the production of mg amounts of MPs. Altogether, these properties make CF systems interesting for the preparative scale expression of MPs as well as for throughput screening and proteomics approaches.

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