Roy Ankita, Shukla Arun Kumar, Haase Winfried, Michel Hartmut
Department of Molecular Membrane Biology, Max Planck Institute of Biophysics, Max-von-Laue Strasse 3, D-60438 Frankfurt/Main, Germany.
Biol Chem. 2008 Jan;389(1):69-78. doi: 10.1515/BC.2008.001.
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.
G蛋白偶联受体（GPCRs）是最大的一类细胞表面受体，在许多细胞和生理过程中发挥着关键作用。重组GPCRs的功能性表达是获取结构信息的主要瓶颈之一。在此，我们报道了一种基于光合细菌球形红杆菌的新型细菌表达系统用于生产人重组GPCRs。将球形红杆菌用作宿主的优势在于，与其他典型表达宿主相比，它每个细胞可提供更多的膜表面。该系统经过优化，可在中等强度且高度调控的超操纵子光合启动子pufQ的控制下过表达重组受体。我们测试了该系统对一些A类GPCRs的表达情况，即人腺苷A2a受体（A2aR）、人血管紧张素AT1a受体（AT1aR）和人缓激肽B2受体（B2R）。我们检测了几种不同的构建体，并实现了重组受体的功能性表达。表达效果最佳的受体AT1aR经过溶解和亲和纯化。据我们所知，这是首次报道成功利用细菌宿主——球形红杆菌——在光合基因启动子的控制下生产功能性重组GPCRs。
