Functional Cell-free Synthesis of a Seven Helix Membrane Protein: In situ Insertion of Bacteriorhodopsin into Liposomes

Abstract

The expression of membrane proteins for functional and structural studies or medicinal applications is still not very well established. Membrane-spanning proteins that mediate the information flow of the extracellular side with the interior of the cell are prime targets for drug development methods that would allow screening techniques or high throughput formats are of particular interest. Here we describe a systematic approach to the liposome-assisted cell-free synthesis of functional membrane proteins. We demonstrate the synthesis of bacteriorhodopsin (bR cf) in presence of small unilamellar liposomes. The yield of bR cf per volume cell culture is comparable to that of bacteriorhodopsin in its native host. The functional analysis of bR cf was performed directly using the cell-free reaction mixture. Photocycle measurements reveal kinetic data similar to that determined for bR in Halobacterium salinarum cell-envelope vesicles. The liposomes can be attached directly to black lipid membranes (BLM), which allows measuring light activated photocurrents in situ. The results reveal a functional proton pump with properties identical to those established for the native protein.