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- W2079632735 abstract "The human Cannabinoid Receptor 2 (CB2) is a Rhodopsin family G protein coupled receptor (GPCR) expressed highly in immune cells. Recent studies demonstrate the role of the CB2 receptor in attenuating bone cancer induced pain, reducing microglial activation in Alzheimer's disease and regulation of bone mass in osteoporosis. In this regard, detailed structure function knowledge of the mechanism of receptor activation will allow understanding of the ligand binding site, resolve the molecular basis of ligand functionality and also lead to the rational designing of more potent ligand. However majority of biophysical techniques which can be employed require large amounts of purified, native CB2. This poses the one of the biggest challenge for further studies. To this end we are using TrpLE, an N' terminal fusion partner with the CB2 receptor to direct its expression to inclusion bodies in the E. coli. High yielding GPCR inclusion bodies were solubilized and extracted to homogeneity in the presence of denaturing detergent concentrations. Inactive receptors were analyzed for its ligand binding ability and extent of secondary structure. The inactive receptor fusion protein was detergent exchanged for the removal of fusion partner by factor Xa cleavage. Purified CB2 receptor inclusion bodies are currently under refolding trials using detergents (DDM), lipids (POPC/POPS) and amphipols (A8-35) alone and in combination. These studies are being conducted in parallel and will allow determining the best refolding environment for the membrane protein receptor. Understanding of the refolding process would greatly enhance CB2 structure function studies which are currently limited by protein availability and facilitate understanding of protein lipid interactions during membrane protein refolding and/or stabilization. (The work is supported by NIH RO1 DA025612 to X.Q. Xie)" @default.
- W2079632735 created "2016-06-24" @default.
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- W2079632735 date "2013-01-01" @default.
- W2079632735 modified "2023-09-27" @default.
- W2079632735 title "Production, Characterization and Refolding of G-Protein Coupled CB2 Receptor from Inclusion Bodies" @default.
- W2079632735 doi "https://doi.org/10.1016/j.bpj.2012.11.1232" @default.
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