Systematic optimization of cell-free synthesized human endothelin B receptor folding

Publication date: Available online 2 February 2018 Source:Methods Author(s): Ralf-Bernhardt Rues, Fang Dong, Volker Dötsch, Frank Bernhard Cell-free production of G-protein coupled receptors is becoming attractive for biochemical characterization, ligand screening or even structural purposes. However, despite high production levels within the range of mg/mL, the fraction of functionally folded receptor is frequently below 1%. In synthetic cell-free reactions, numerous factors that affect the efficient folding and stability of translated membrane proteins can be addressed by the appropriate design of the synthetic expression environment. We demonstrate the systematic quality optimization of the cell-free synthesized human endothelin B receptor by a combined approach of lipid screening, redox optimization, and molecular engineering. Key parameters for receptor folding are the implementation of nanodiscs, the selection of suitable lipid environments for co-translational solubilization, as well as providing an optimized redox system for essential disulfide bridge formation. In addition, enrichment with chaperones as well as receptor engineering by thermostabilization further supported the folding into ligand binding conformation. In summary, we provide evidence that the initial co-translational folding process rather than long-term stability of the receptor is limiting. The folding efficiency could be improved by more than 103-fold and under optimized conditions, up to 1.6...
Source: Methods - Category: Molecular Biology Source Type: research