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• W2034715148 abstract "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 (∼4 mg/L of culture) of functional human adenosine A2a receptor fused to a green fluorescent protein (A2aR–GFP) from Saccharomyces cerevisiae. In this work, we re-engineered A2aR with a purification tag, developed an adequate purification scheme, and performed biophysical characterization on purified receptors. Milligram amounts per liter of culture of A2aR and A2aR–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 A2aR–GFP–His10. One-step purification of the protein was achieved through immobilized metal affinity chromatography. After initial solubilization in n-dodecyl-β-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 A2aR under these conditions was found to be largely α-helical, and properly incorporated into a mixed-micelle environment. The A2a–His10 receptor was purified in quantities of 6 ± 2 mg/L of culture, with ligand-binding yields of 1 mg/L, although all protein bound to xanthine affinity resin. This represents the highest purified total and functional yields for A2aR yet achieved from any heterologous expression system." @default.
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• W2034715148 date "2007-08-01" @default.
• W2034715148 modified "2023-09-26" @default.
• W2034715148 title "High-level expression in Saccharomyces cerevisiae enables isolation and spectroscopic characterization of functional human adenosine A2a receptor" @default.
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• W2034715148 doi "https://doi.org/10.1016/j.jsb.2007.05.001" @default.
• W2034715148 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/1994815" @default.
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