High-level expression in Saccharomyces cerevisiae enables isolation and spectroscopic characterization of functional human adenosine A2a receptor.
The G-protein coupled receptors (GPCRs) are a class of membrane proteins that trigger cellular responses to external stimuli, and are believed to be targets for nearly half of all pharmaceutical drugs on the market. However, little is known regarding their folding and cellular interactions, as well as what factors are crucial for their activity. Further structural characterization of GPCRs has largely been complicated by problems with expression, purification, and preservation of activity in vitro. Previously, we have demonstrated high-level expression (approximately 4mg/L of culture) of functional human adenosine A(2)a receptor fused to a green fluorescent protein (A(2)aR-GFP) from Saccharomyces cerevisiae. In this work, we re-engineered A(2)aR with a purification tag, developed an adequate purification scheme, and performed biophysical characterization on purified receptors. Milligram amounts per liter of culture of A(2)aR and A(2)aR-GFP were functionally expressed in S. cerevisiae, with a C-terminal deca-histidine tag. Lysis procedures were developed for optimal membrane protein solubilization and recovery through monitoring fluorescence of A(2)aR-GFP-His(10). One-step purification of the protein was achieved through immobilized metal affinity chromatography. After initial solubilization in n-dodecyl-beta-d-maltoside (DDM), a combination of added cholesterol hemisuccinate (CHS) in 3-(3-cholamidopropyl)-dimethylammoniopropane sulfonate (CHAPS) was required to stabilize the functional state of the protein. Isolated A(2)aR under these conditions was found to be largely alpha-helical, and properly incorporated into a mixed-micelle environment. The A(2)a-His(10) receptor was purified in quantities of 6+/-2mg/L of culture, with ligand-binding yields of 1mg/L, although all protein bound to xanthine affinity resin. This represents the highest purified total and functional yields for A(2)aR yet achieved from any heterologous expression system.
O'Malley
Michelle A
MA
Department of Chemical Engineering, University of Delaware, 150 Academy Street, Newark, DE 19716, USA.
Lazarova
Tzvetana
T
Britton
Zachary T
ZT
Robinson
Anne S
AS
D002784:Cholesterol / Q000378:metabolism; D003001:Cloning, Molecular; D003902:Detergents / Q000737:chemistry; D006801:Humans; D008823:Micelles; D011487:Protein Conformation; D043705:Receptor, Adenosine A2A / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*
D003902:Detergents; D008823:Micelles; D043705:Receptor, Adenosine A2A; D011993:Recombinant Fusion Proteins; D002784:Cholesterol