Employing Rhodobacter sphaeroides to functionally express and purify human G protein-coupled receptors
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Abstract
G protein-coupled receptors (GPCRs) represent the largest class of cell surface receptors and play crucial roles in many cellular and physiological processes. Functional production of recombinant GPCRs is one of the main bottlenecks to obtaining structural information. Here, we report the use of a novel bacterial expression system based on the photosynthetic bacterium Rhodobacter sphaeroides for the production of human recombinant GPCRs. The advantage of employing R. sphaeroides as a host lies in the fact that it provides much more membrane surface per cell compared to other typical expression hosts. The system was tailored to overexpress recombinant receptors under the control of the moderately strong and highly regulated superoperonic photosynthetic promoter pufQ. We tested this system for the expression of some class A GPCRs, namely, the human adenosine A2a receptor (A2aR), the human angiotensin AT1a receptor (AT1aR) and the human bradykinin B2 receptor (B2R). Several different constructs were examined and functional production of the recombinant receptors was achieved. The best-expressed receptor, AT1aR, was solubilized and affinity-purified. To the best of our knowledge, this is the first report of successful use of a bacterial host – R. sphaeroides – to produce functional recombinant GPCRs under the control of a photosynthetic gene promoter.
©2008 by Walter de Gruyter Berlin New York
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Articles in the same Issue
- A counterintuitive approach to treat enzyme deficiencies: use of enzyme inhibitors for restoring mutant enzyme activity
- Reassessing the role of a 3′-UTR-binding translational inhibitor in regulation of circadian bioluminescence rhythm in the dinoflagellate Gonyaulax
- Cloning, functional analysis, and mitochondrial localization of Trypanosoma brucei monothiol glutaredoxin-1
- A new subfamily of bacterial glutamate/aspartate receptors
- Synthesis and characterization of a functional intact IgG in a prokaryotic cell-free expression system
- Collagen IV regulates Caco-2 cell spreading and p130Cas phosphorylation by FAK-dependent and FAK-independent pathways
- Differential effects of novel tumour-derived p53 mutations on the transformation of NIH-3T3 cells
- Employing Rhodobacter sphaeroides to functionally express and purify human G protein-coupled receptors
- Heparinase selectively sheds heparan sulphate from the endothelial glycocalyx
- Inhibition of human μ-calpain by conformationally constrained calpastatin peptides
- Cloning, expression and characterization of insulin-degrading enzyme from tomato (Solanum lycopersicum)
