Directing Cell-free Expressed Membrane Proteins into Lipid Bilayers: Evaluating the L-CF Mode and the Nanodisc Technology

Conventional protocols for the production of membrane protein (MP) samples inserted into defined lipid bilayers are time consuming and include protein solubilisation, purification procedures and in vitro reconstitution steps. Cell-free expression in presence of supplemented artificial liposomes (L-CF mode) would provide a much more straight-forward approach to shorten workload and time requirements by the direct co-translational integration of the nascent polypeptide-chain.

We have evaluated requirements for the efficient L-CF expression of MPs in E. coli cell-free expression systems. A library of different MP-GFP fusions comprising transporters, channels and GPCRs was used as targets. Parameters analyzed for their effects on the co-translational MP translocation in the L-CF mode included (I) type of supplemented lipids; (II) size and composition of artificial liposomes; (III) lipid/detergent mixtures; (IV) nanodiscs; (V) translation rate and (VI) concentrations of system compounds. The L-CF expressed MPs have been characterized by insertion rates and functional activity. The results will help to define general guidelines for the efficient L-CF expression with regard to MP characteristics (size, number of TMS, topology etc.) in order to allow a time optimized expression and simultaneous lipid bilayer insertion of MPs of different origins.