Screening Methods for Cell-Free Synthesized GPCR/Nanoparticle Samples.

Abstract

Cell-free protein expression systems and lipid nanoparticle technologies are core platforms for membrane protein synthesis. The implementation of preassembled nanodiscs allows the co-translational insertion of membrane proteins into tailored lipid bilayers in the absence of any artificial hydrophobic compounds. This strategy is particularly interesting for detergent sensitive or otherwise critical membrane proteins such as G-protein-coupled receptors (GPCRs). Cell-free expression reactions are completed within a day and the formed GPCR/nanodisc particles can be purified directly out of the reaction mixture by affinity tags and without any further manipulation. The streamlined procedure reduces risk of GPCR denaturation and the sample quality can further be supported by supplying chaperones or other beneficial compounds directly into the expression reactions.GPCRs inserted into nanoparticle membranes are excellent tools for a variety of applications such as ligand screening, engineering or even structural characterization. In this chapter, we provide protocols for the reaction set-up and efficient cell-free production of functionally folded GPCRs reaching μM concentrations in the final expression reactions. We further exemplify the tuning of GPCR sample quality and discuss their application for throughput ligand screening and for the analysis of ligand-binding characteristics.