Directed evolution of a G protein-coupled receptor for expression, stability, and binding selectivity

Abstract

We outline a powerful method for the directed evolution of integral membrane proteins in the inner membrane of Escherichia coli. For a mammalian G protein-coupled receptor, we arrived at a sequence with an order-of-magnitude increase in functional expression that still retains the biochemical properties of wild type. This mutant also shows enhanced heterologous expression in eukaryotes (12-fold in Pichia pastoris and 3-fold in HEK293T cells) and greater stability when solubilized and purified, indicating that the biophysical properties of the protein had been under the pressure of selection. These improvements arise from multiple small contributions, which would be difficult to assemble by rational design. In a second screen, we rapidly pinpointed a single amino acid substitution in wild type that abolishes antagonist binding while retaining agonist-binding affinity. These approaches may alleviate existing bottlenecks in structural studies of these targets by providing sufficient quantities of stable variants in defined conformational states.integral membrane proteins ͉ protein engineering ͉ protein folding

Genetic selection system for improving recombinant membrane protein expression in E. coli

“…8 Also, the membrane protein itself can be mutated to generate more stable variants that express at higher levels, but the disadvantage is that the product may not reflect the native structure and function. [9][10][11] Another approach is cell-free expression which can bypass deleterious changes in cell physiology. 12 The produced protein, however, is not necessarily in a folded, functional form, and this is particularly troublesome for membrane proteins, which tend to be difficult to refold.…”

Ice breaking in GPCR structural biology

“…www.nature.com/aps Zhao Q et al Acta Pharmacologica Sinica npg insect cells, and by this, higher expression and stability were then achieved [38,39] . These three groups tested stabilization mutant using 5 GPCRs independently and found some very interesting consistencies [37-39, 45, 46] .…”

“…The work of PLÜCKTHUN et al pushes these approaches to a new level. They developed a fluorescence-activated cell sorting (FACS) method that could enhance both the expression level and stability of GPCRs while retaining function and tailoring ligand selectivity [38] . Using this approach, the expression levels of multiple GPCRs were increased several folds, and a receptor analog that more prone to bind agonists vs antagonists was obtained, similar to results from the Tate group when they solved the active state A 2A adenosine receptor structure [39] .…”

“…Based on the understanding achieved from previous X-ray crystallographic work, several new amphiphiles and detergents have been developed by modifications of known surfac- Figure 2. A snake β 2 AR plot to brief summary of the mutagenesis studies aiming higher GPCR stability [37,38,[44][45][46]91] . The mutations on different receptors are applied to β 2 AR receptor based on the Ballesteros and Weinstein Number System [43] and labeled in red on the Figure.…”

“…Tate and Schertler have mapped almost every residue in the receptor and measured the thermal stability of the corresponding mutants. The most promising mutations were then combined and further screened for the highest stability to tolerate the harsh environment during extraction, purification and crystalliza tion [38,45] . Guided by this method, they solved the crystal structures of the β1 adrenergic receptor with different ligands and the A 2A adenosine receptor structure in an active state [4,16] .…”