A Bacterial Cell‐Free Expression System to Produce Membrane Proteins and Proteoliposomes: From cDNA to Functional Assay

Limitations in the production of folded membrane proteins represent the major bottleneck for functional and structural studies of this huge category of macromolecules. Cell-free expression systems provide an attractive alternative to the classical overexpression systems for producing membrane proteins. However, optimization of these systems remains a challenging task, considering the hydrophobic properties of these molecules. This unit describes the production of eukaryotic membrane proteins either in soluble form or integrated into liposomes using a bacterial cell-free expression system. Liposomes in the reaction mixture induce the direct insertion of freshly produced membrane proteins into the bilayer and allow the formation of functional proteoliposomes in which the membrane proteins are correctly folded.