Novel approaches to the expression and purification of G protein-coupled receptors

G protein-coupled receptors mediate cellular responses to extracellular cues and compose the largest family of eukaryotic membrane proteins. Due to their role in a number of physiological and pathophysiological processes, GPCRs are the targets of nearly 50% of all small molecule and biological drugs, which corresponds to approximately ;To address obstacles to the production of GPCRs for biophysical and structural characterization, novel approaches to GPCR expression and purification in two heterologous expression systems have been developed. In Part I of this dissertation, a robust system for the expression and purification of GPCRs transmembrane peptides in E. coli was developed for the characterization of specific subdomains of GPCRs. Using a model peptide of the adenosine A2a receptor, we have verified that GPCR peptides can be produced in this system in sufficient yields and purities for biophysical characterization. In Part II of this dissertation, expression and purification obstacles in S. cerevisiae were addressed by developing a novel plasmid system for the efficient generation and quantitative analysis of protein variants, where the selection of desired protein properties (expression level, trafficking, and stability) may be rationally designed. In so doing, we have gained an improved understanding of this host organism so that it may be better utilized for the high-yield production of GPCRs intended for structural characterization.