(115a) Exceptional Total and Functional Yields of the Human Adenosine a2a Receptor Expressed in the Yeast Saccharomyces Cerevisiae | AIChE

(115a) Exceptional Total and Functional Yields of the Human Adenosine a2a Receptor Expressed in the Yeast Saccharomyces Cerevisiae


Robinson, A. S. - Presenter, University of Delaware
Niebauer, R. - Presenter, National Institutes of Health
O'Malley, M. - Presenter, University of Delaware

Membrane proteins are critical for cell-cell recognition, inter and intracellular signaling, and cell homeostasis, and are of great interest in understanding human disease and as drug targets. However, the majority of membrane protein structures have been obtained only from proteins available in natural abundance, due to the major difficulties with heterologous expression to date. Grisshammer and Tate recently (2003) underscored the magnitude of this daunting problem: ?Membrane protein structure determination remains one of the great challenges in structural biology ? Only once the overproduction of functional membrane proteins becomes easier, will we see exponential growth in the number of atomic resolution structures of integral membrane proteins.? Despite the clear and urgent need for better methods to express and study these proteins, there have been few if any systematic studies to identify the features that lead to the failures to recover high levels of active protein.

We have established a high-level expression system for human A2a, as well as a Green Fluorescent Protein (GFP) tagged form, A2a-GFP in Saccharomyces cerevisiae. A screen based on whole cell fluorescence was developed and a library of clones with various gene copy numbers were screened via flow cytometry to isolate clones with the highest protein expression levels. Approximately 120,000 functional A2a-GFP molecules per cell were present on the plasma membrane as determined by radioligand binding. Importantly, the amount of functionally expressed A2a-GFP per culture (~4 mg/L) is among the highest reported for any GPCR in any expression system. Recently, we have established a method to purify the A2aR-His10 protein to homogeneity while maintaining its activity based on radioligand binding, with yields of greater than 1 mg/L. Surprisingly, 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. mRNA levels remain high during expression, and protein degradation was low. Our expression efforts, as well as our studies aimed at understanding the cellular limitations to overexpression will be described in this talk.

Grisshammer, R. and C. G. Tate (2003). "Preface: Overexpression of integral membrane proteins." Biochem Biophys Acta 1610: 1.