title: Application of HaloTag technology to expression and purification of cannabinoid receptor CB2.
abstract: Expression of milligram quantities of functional, stable G protein-coupled receptors (GPCR) for high-resolution structural studies remains a challenging task. The goal of this work was to evaluate the usefulness of the HaloTag system (Promega) for expression and purification of the human cannabinoid receptor CB(2), an important target for development of drugs for treatment of immune disorders, inflammation, and pain. Here we investigated expression in Escherichia coli cells of the integral membrane receptor CB(2) as a fusion with the 34 kDa HaloTag at N- or C-terminal location, either in the presence or in the absence of the N-terminal maltose-binding protein (MBP). The CB(2) was flanked at both ends by the tobacco etch virus (TEV) protease cleavage sites to allow for subsequent removal of expression partners. Expression by induction with either IPTG (in E. coli BL21(DE3) cell cultures) or by auto-induction (in E. coli KRX cells) were compared. While the N-terminal location of the HaloTag resulted in high levels of expression of the fusion CB(2), the recombinant receptor was not functional. However, when the HaloTag was placed in the C-terminal location, a fully active receptor was produced irrespective of induction method or bacterial strain used. For purification, the fusion protein was captured onto HaloLink resin in the presence of detergents. Treatment with specific TEV protease released the CB(2) upon washing. To our knowledge, this study represents the first example of expression, surface immobilization and purification of a functional GPCR using HaloTag technology.
authors.affiliation: Laboratory of Membrane Biochemistry and Biophysics, National Institute on Alcohol Abuse and Alcoholism, National Institutes of Health, Bethesda, MD 20892, USA.
authors.forename: Silvia
authors.initials: S
authors.lastname: Locatelli-Hoops
authors.forename: Fangmin C
authors.initials: FC
authors.lastname: Sheen
authors.forename: Lioudmila
authors.initials: L
authors.lastname: Zoubak
authors.forename: Klaus
authors.initials: K
authors.lastname: Gawrisch
authors.forename: Alexei A
authors.initials: AA
authors.lastname: Yeliseev
mesh_terms: D003902:Detergents / Q000378:metabolism; D004926:Escherichia coli; D006801:Humans; D007154:Immune System Diseases / Q000628:therapy; D007249:Inflammation / Q000628:therapy; D058619:Maltose-Binding Proteins / Q000737:chemistry / Q000235:genetics*; D043885:Receptor, Cannabinoid, CB2 / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*
chemical_list: D003902:Detergents; D058619:Maltose-Binding Proteins; D043885:Receptor, Cannabinoid, CB2; D011993:Recombinant Fusion Proteins