A generic selection system for improved expression and thermostability of G protein-coupled receptors by directed evolution

Christoph Klenk, Janosch Ehrenmann, Marco Schütz, Andreas Plückthun
2016 Scientific Reports  
Structural and biophysical studies as well as drug screening approaches on G protein-coupled receptors (GPCRs) have been largely hampered by the poor biophysical properties and low expression yields of this largest class of integral membrane proteins. Thermostabilisation of GPCRs by introduction of stabilising mutations has been a key factor to overcome these limitations. However, labelled ligands with sufficient affinity, which are required for selective binding to the correctly folded
more » ... , are often not available. Here we describe a novel procedure to improve receptor expression and stability in a generic way, independent of specific ligands, by means of directed evolution in E. coli. We have engineered a homogenous fluorescent reporter assay that only detects receptors which are correctly integrated into the inner cell membrane and, thus, discriminates functional from non-functional receptor species. When we combined this method with a directed evolution procedure we obtained highly expressing mutants of the neurotensin receptor 1 with greatly improved thermostability. By this procedure receptors with poor expression and/or low stability, for which no ligands or only ones with poor binding properties are available, can now be generated in quantities allowing detailed structural and biophysical analysis. By relaying signals from the exterior to the interior of the cell, integral membrane proteins (IMPs) play a central role in the physiological and pathophysiological processes of our body. With more than 800 members, G protein-coupled receptors (GPCRs) represent the largest superfamily of IMPs and are considered as one of the most important drug targets 1,2 . However, most GPCRs exhibit several unfavourable features which make them difficult to study. Major reasons for this are, on the one hand, the very low levels of endogenous expression and also the low achievable expression levels in most heterologous systems, resulting in low functional protein yields that can be isolated. On the other hand, owing to their inherent flexibility, most GPCRs are extremely unstable, and instability is further increased after solubilisation of the protein in detergents. Thus, the number of structural studies and studies requiring purified protein of GPCRs has remained limited, compared to the many investigations within the past decades that have employed cell-biological and biochemical methods in whole cells 3 . Likewise, the development of new drugs targeting GPCRs using structure-based drug design or fragment-based drug discovery has remained a challenge 4 , as these methods also require access to purified protein of sufficient stability. Among other methods, conformational stabilisation by introducing thermostabilising mutations has been a major advancement to improve the biophysical properties of GPCRs. Such modified receptors were the basis for several high resolution crystal structures 5-9 , and are well suited for extensive in vitro drug screening trials 10 . Stabilising mutations can be found, e.g., by performing successive alanine scans over the whole protein followed by screening for improved receptor stability of each individual mutant. Combinations of several beneficial mutations then lead to highly stabilized receptors [11] [12] [13] [14] [15] [16] [17] . Recently, we have developed a more comprehensive approach to
doi:10.1038/srep21294 pmid:26887595 pmcid:PMC4758055 fatcat:nnf5eh2kenen3nythbobm75aoy