Abstract

G
protein-coupled receptors (GPCRs) are the largest superfamily of cell surface
receptors. Their central role in health and disease is underlined by the large
number of human diseases linked to dysfunctional GPCR signalling, and that approximately
35 % of existing drugs target GPCRs. To study protein-drug interactions and
facilitate structure-based drug design (SBDD), a target GPCR must be produced
in sufficient quantities in a functional state. Despite advances in recombinant
protein expression and structure solution by X-ray crystallography and Cryo-Electron
Microscopy, many GPCRs remain refractory to study, owing to their poor levels
of expression, and inherent instability once removed from the lipid bilayer in
detergent. Sosei Heptares proprietary research platform focusses on the engineering of
functional, stabilised GPCRs (StaR® protein) in their natural pharmacological
conformations (agonist or antagonist) by combinatorial mutagenesis to form a highly
expressed, stable and rigid protein that is amenable to biophysical and
structural analysis; whilst retaining key features of the wild-type.

Saccharomyces
based
receptor evolution (SaBRE) is an alternative method to engineering GPCRs for
enhanced expression and stability, forming an integral part of the Sosei
Heptares proprietary research platform. Combining the utility of Saccharomyces
cerevisiae as a microbial eukaryotic expression system with directed
evolution allows the efficient investigation of a vast sequence space, offering
solutions to enhance the expression and stability of even the most challenging
GPCR targets for SBDD. Here an example of SaBRE is detailed for a structurally
uncharacterised GPCR. This includes an overview of the molecular biology to
prepare yeast for directed evolution, the selection of the fittest receptors
using a fluorescent ligand and FACs, and the assessment of the enriched
receptors for downstream characterisation by biophysical and structural
analysis at Sosei Heptares.