Cell-Free Expression of GPCRs

G-protein-coupled receptors (GPCRs) are cell-surface membrane proteins that mediate various signal transduction processes through G-protein activation. The human endothelin receptor type A (ETA) and B (ETB) are prototypic GPCRs distributed among multiple endothelial cell types as well as in smooth muscle cells. The ubiquitous distribution of these receptors implicates their involvement in a wide variety of physiological and pathological processes.
In our laboratory we have established protocols for the high-level production of ETA, ETB and other GPCRs in an individual Continuous Exchange Cell-Free System (CECF). This expression system based on Escherichia coli cell extracts has been demonstrated to provide a highly promising tool for the preparative-scale production of membrane proteins. The quality of the GPCRs was modulated by their production in three different modes (P-CF, D-CF and L-CF)in presence of detergents or lipids.The effects of different detergents on protein homogeneity and stability was evaluated and ligand binding competence of the samples reconstituted into liposome was assessed. Taking advantage of the fusion construct ETB-GFP, the quality of the samples produced in L-CF mode was furthermore analyzed with fluorescent assays and confocal microscopy.
For structural analysis of ETB, mutants were created in order to improve sample homogeneity and stability. We demonstrate the systematic optimization of yield and quality of human GPCRs synthesized in individual cell-free expression systems based on E. coli extracts.