Decreases in yeast expression yields of the human adenosine A2a receptor are a result of translational or post-translational events.
The human adenosine receptor (A2a), a G-protein-coupled receptor (GPCR), was C-terminally tagged with the green fluorescent protein (GFP) and expressed in the yeast Saccharomyces cerevisiae to gain an understanding of the expression limitations of this medically relevant class of membrane proteins. The A2a-GFP protein was able to bind adenosine analogs indicating that the GFP tag did not alter the ligand binding activity of the receptor. A screen based on whole cell fluorescence was developed and a library of clones with various gene copy numbers was screened via flow cytometry to isolate clones with the highest protein expression levels. All clones studied exhibited a decrease in the net A2a-GFP protein production rate over time as determined by whole cell fluorescence, Western blotting, confocal microscopy, and ligand binding. Quantitative PCR showed that A2a-GFP mRNA levels remained relatively high even as the protein production rate decreased. A cycloheximide chase experiment showed that the mature protein was stable over time and was not significantly degraded. Taken together, these results suggest that heterologous expression of GPCRs is limited by a translational or post-translational bottleneck that is unique from expression limitations seen for soluble proteins.
Department of Chemical Engineering, University of Delaware, Newark, DE 19716, USA.
Ronald T
RT
Niebauer
Alison
A
Wedekind
Anne Skaja
AS
Robinson
D005434:Flow Cytometry; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008024:Ligands; D011485:Protein Binding; D014176:Protein Biosynthesis; D046188:Protein Modification, Translational; D011499:Protein Processing, Post-Translational; D021381:Protein Transport / Q000502:physiology; D012333:RNA, Messenger / Q000378:metabolism; D043705:Receptor, Adenosine A2A / Q000235:genetics* / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D015203:Reproducibility of Results; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*
D008024:Ligands; D012333:RNA, Messenger; D043705:Receptor, Adenosine A2A; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins