Protein Engineering of G-Protein Coupled Receptors Using Directed Evolution in Saccharomyces cerevisiae
By Daniel Jones | Nov 15, 2019
About the Conference
Drug Discovery 2019
The European Laboratory Research & Innovation Group (ELRIG)
5 – 6 November 2019
ACC, Liverpool, UK
Dr. Daniel Jones, Research Scientist, Protein Engineering at Sosei Heptares, recently attended The European Laboratory Research & Innovation Group (ELRIG) conference named “Drug Discovery 2019” at ACC in Liverpool, UK on November 5 - 6 and presented on “Protein Engineering of G-Protein Coupled Receptors Using Directed Evolution in Saccharomyces cerevisiae”.
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.