Cell-Free Expression of GPCRs into Nanomembranes for Functional and Structural Studies

Despite their importance in many essential physiological processes of living cells, G protein-coupled receptors (GPCRs) are often difficult to express and purify in sufficient quality and quantity. We demonstrate cell-free protein synthesis as an interesting alternative to classical cell-based expression systems. We focus on a recently developed detergent-free expression mode by co-translational integration of nascent GPCRs into provided nanodisc membranes of defined composition. The protocol is in particular suitable for detergent sensitive targets and allows the synthesis of full-length as well as modified GPCRs. As a basic blueprint for the cell-free synthesis of GPCRs and potentially other membrane proteins as well, we describe the production of the human endothelin-B receptor. Subsequent purification strategies are streamlined by implementing complementary affinity chromatography steps. We further show the evaluation and optimization of the final GPCR samples for homogeneity and activity through a radioligand binding assay.