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.