Employing Rhodobacter sphaeroides to functionally express and purify human G protein-coupled receptors.
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.
Roy
Ankita
A
Department of Molecular Membrane Biology, Max Planck Institute of Biophysics, Max-von-Laue Strasse 3, D-60438 Frankfurt/Main, Germany.
Shukla
Arun Kumar
AK
Haase
Winfried
W
Michel
Hartmut
H
D002462:Cell Membrane / Q000378:metabolism / Q000648:ultrastructure; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D015964:Gene Expression Regulation, Bacterial; D005822:Genetic Vectors; D006801:Humans; D007202:Indicators and Reagents; D008854:Microscopy, Electron; D010788:Photosynthesis; D011869:Radioligand Assay; D043705:Receptor, Adenosine A2A / Q000096:biosynthesis / Q000235:genetics; D044140:Receptor, Angiotensin, Type 1 / Q000096:biosynthesis / Q000235:genetics; D043782:Receptor, Bradykinin B2 / Q000096:biosynthesis / Q000235:genetics; D043562:Receptors, G-Protein-Coupled / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D012242:Rhodobacter sphaeroides / Q000378:metabolism* / Q000648:ultrastructure
D007202:Indicators and Reagents; D043705:Receptor, Adenosine A2A; D044140:Receptor, Angiotensin, Type 1; D043782:Receptor, Bradykinin B2; D043562:Receptors, G-Protein-Coupled; D011994:Recombinant Proteins