Directed evolution for high functional production and stability of a challenging G protein-coupled receptor

Membrane proteins such as G protein-coupled receptors (GPCRs) carry out many fundamental biological functions, are involved in a large number of physiological responses, and are thus important drug targets. To allow detailed biophysical and structural studies, most of these important receptors have to be engineered to overcome their poor intrinsic stability and low expression levels. However, those GPCRs with especially poor properties cannot be successfully optimised even with the current technologies. Here, we present an engineering strategy, based on the combination of three previously developed directed evolution methods, to improve the properties of particularly challenging GPCRs. Application of this novel combination approach enabled the successful selection for improved and crystallisable variants of the human oxytocin receptor, a GPCR with particularly low intrinsic production levels. To analyse the selection results and, in particular, compare the mutations enriched in different hosts, we developed a Next-Generation Sequencing (NGS) strategy that combines long reads, covering the whole receptor, with exceptionally low error rates. This study thus gave insight into the evolution pressure on the same membrane protein in prokaryotes and eukaryotes. Our long-read NGS strategy provides a general methodology for the highly accurate analysis of libraries of point mutants during directed evolution.