Exceptional total and functional yields of the human adenosine (A2a) receptor expressed in the yeast Saccharomyces cerevisiae.
The yeast Saccharomyces cerevisiae was used to express a medically relevant G-protein coupled receptor (GPCR), the human adenosine (A2a) receptor, with a C-terminal green fluorescent protein (GFP) fusion tag. In prior studies, we established an expression system for A2a-GFP. Here, we quantified the total A2a-GFP expression levels by correlating GFP levels as detected by fluorescence and densitometry to A2a-GFP molecules overexpressed in the system. We also quantified A2a-GFP functional levels by classical radioligand binding assays. Approximately, 120,000 functional A2a-GFP molecules per cell were present on the plasma membrane as determined by radioligand binding. Using whole cell GFP fluorescence, 340,000 A2a-GFP molecules per cell were detected; approximately 70% of those molecules were plasma membrane localized, as determined by using confocal microscopy analysis. These results show that a significant portion of the total expressed protein is functional. In addition, the quick and inexpensive whole cell fluorescence appears to provide a good approximation of functional receptor numbers for this case. Importantly, the amount of functionally expressed A2a-GFP per culture ( approximately 4 mg/L) is among the highest reported for any GPCR in any expression system.
Niebauer
Ronald T
RT
Department of Chemical Engineering, University of Delaware, Newark, DE 19716, USA.
Robinson
Anne Skaja
AS
D049452:Green Fluorescent Proteins / Q000096:biosynthesis / Q000235:genetics; D006801:Humans; D043705:Receptor, Adenosine A2A / Q000096:biosynthesis* / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000254:growth & development*
D043705:Receptor, Adenosine A2A; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins