Engineering yeast cells for optimal expression of the human adenosine (A2a) receptor

G-protein coupled receptors (GPCRs) mediate cellular responses to diverse stimuli, represent about 60% of drug targets, and have been linked to numerous diseases including heart disease, cancer, AIDS, and diabetes. Despite their medical importance, structural information is limited due to the inability to express large quantities of functional protein. Although membrane proteins represent about 30% of all proteins, they represent only about 0.3% of the proteins for which crystal structures have been obtained. Our goals were to engineer a system for high-level GPCR expression, quantify the total and functional expression yields and gain an understanding of expression limitations for this class of proteins.; To achieve the first goal, Saccharomyces cerevisiae was used to express a model GPCR, the human adenosine (A2a) receptor, with a C-terminal green fluorescent protein (GFP) fusion tag to aid in quantification and localization studies. A highly expressing clone was isolated using flow cytometry. Using this clone the total expression levels were quantified using a series of calibration curves. Next, ligand binding was performed to quantify the functional expression level. The experiments revealed that a large portion of the A2aGFP protein that is expressed is functional. Importantly, the functional expression yields (∼4 mg/L) were found to be among the highest reported for any GPCR in any expression system.; To gain insight into expression limitations over time the A2aGFP expression and cell growth was monitored. The expression was characterized in several yeast strains by Western blotting, whole cell GFP fluorescence, confocal microscopy, and ligand binding. A decrease in the protein levels on a per cell basis was observed and possible causes of this decrease were investigated. A cycloheximide chase experiment suggested that the A2a protein was not significantly degraded over time. To ensure that the transcription levels (mRNA) were not effected, quantitative PCR was performed. Experiments suggest that there is a translational or post-translational limitation that is unique from other limitations seen for soluble protein expression. Taken together, exceptional total and functional yields of the human adenosine (A2a) receptor have been achieved in spite of expression limitations over time.