title: Mistic and TarCF as fusion protein partners for functional expression of the cannabinoid receptor 2 in Escherichia coli.
abstract: G protein coupled receptors (GPCRs) are key players in signal recognition and cellular communication making them important therapeutic targets. Large-scale production of these membrane proteins in their native form is crucial for understanding their mechanism of action and target-based drug design. Here we report the overexpression system for a GPCR, the cannabinoid receptor subtype 2 (CB2), in Escherichia coli C43(DE3) facilitated by two fusion partners: Mistic, an integral membrane protein expression enhancer at the N-terminal, and TarCF, a C-terminal fragment of the bacterial chemosensory transducer Tar at the C-terminal of the CB2 open reading frame region. Multiple histidine tags were added on both ends of the fusion protein to facilitate purification. Using individual and combined fusion partners, we found that CB2 fusion protein expression was maximized only when both partners were used. Variable growth and induction conditions were conducted to determine and optimize protein expression. More importantly, this fusion protein Mistic-CB2-TarCF can localize into the E. coli membrane and exhibit functional binding activities with known CB2 ligands including CP55,940, WIN55,212-2 and SR144,528. These results indicate that this novel expression and purification system provides us with a promising strategy for the preparation of biologically active GPCRs, as well as general application for the preparation of membrane-bound proteins using the two new fusion partners described.
authors.affiliation: Department of Pharmaceutical Sciences, School of Pharmacy, and Computational Chemical Genomics Screening Center, University of Pittsburgh, Pittsburgh, PA 15260, USA.
authors.forename: Ananda
authors.initials: A
authors.lastname: Chowdhury
authors.forename: Rentian
authors.initials: R
authors.lastname: Feng
authors.forename: Qin
authors.initials: Q
authors.lastname: Tong
authors.forename: Yuxun
authors.initials: Y
authors.lastname: Zhang
authors.forename: Xiang-Qun
authors.initials: XQ
authors.lastname: Xie
mesh_terms: D002462:Cell Membrane / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006639:Histidine / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008958:Models, Molecular; D011485:Protein Binding; D043885:Receptor, Cannabinoid, CB2 / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011956:Receptors, Cell Surface / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D029968:Escherichia coli Proteins; D043885:Receptor, Cannabinoid, CB2; D011956:Receptors, Cell Surface; D011993:Recombinant Fusion Proteins; C057331:Tar protein, E coli; D006639:Histidine