Accelerate Functional GPCR Production

Application Note Active, Monomeric β1AR in <72h: Cell-Free GPCR Synthesis via Multiplexed Nanodisc Screening Thomas Guilliam, Elena Rahmani, and Ruben Tomás Nuclera Ltd, Cambridge UK Introduction G protein-coupled receptors (GPCRs) are targets for ~35% of approved drugs, but structurally characterized GPCRs remain disproportionately underrepresented relative to the size of the receptor family.1,2 A major contributor is the difficulty of producing stable, functional receptor in detergents: solubilization can strip stabilizing lipids, increase aggregation, and reduce ligand-binding competency. As a result, teams often spend weeks iterating detergent and construct conditions before obtaining assayor structure-grade material. Cell-free protein synthesis (CFPS) offers a powerful alternative by bypassing cellular toxicity and enabling the co-translational insertion of membrane proteins. By including pre-assembled membrane scaffold protein (MSP)-lipid nanodiscs directly in the translation mixture, nascent GPCRs insert spontaneously into a native-like lipid bilayer. This detergent-free, “stabilize as you synthesize” approach preserves key lipid–protein interactions and supports rapid exploration of a tunable design space (scaffold size and lipid chemistry) to maximize yield and functional folding. Mg2+ CA G G L V K Polymerase M ATP Cell-free Uracil core reagent Pre-assembled nanodiscs Setup cell-free expression Co-translational insertion into nanodisc Stable and active GPCR DNA Ribosome T7-RNA Polymerase DNA Template mRNA Figure 1. CFPS enables the co-translational insertion of GPCRs into defined lipid environments during translation.