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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.

SUBMITTER: Locatelli-Hoops S

PROVIDER: S-EPMC3627748 | biostudies-literature | 2013 May

REPOSITORIES: biostudies-literature

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Project description:The endocannabinoid system (ECS) consists particularly of cannabinoid receptors 1 and 2 (CB1 and CB2), their endogenous ligands, and enzymes that synthesize and degrade their ligands. It acts in a variety of organs and disease states ranging from cancer progression over neuropathic pain to neurodegeneration. Protein components engaged in the signaling, trafficking, and homeostasis machinery of the G-protein coupled CB2, are however largely unknown. It is therefore important to identify further interaction partners to better understand CB2 receptor functions in physiology and pathophysiology. For this purpose, we used an affinity purification and mass spectrometry-based proteomics approach of Strep-HA-CB2 receptor in HEK293 cells. After subtraction of background interactions and protein frequency library assessment we could identify 83 proteins that were classified by the identification of minimally 2 unique peptides as highly probable interactors. A functional protein association network analysis obtained an interaction network with a significant enrichment of proteins functionally involved in protein metabolic process, in endoplasmic reticulum, response to stress but also in lipid metabolism and membrane organization. The network especially contains proteins involved in biosynthesis and trafficking like calnexin, Sec61A, tubulin chains TUBA1C and TUBB2B, TMED2, and TMED10. Six proteins that were only expressed in stable CB2 expressing cells were DHC24, DHRS7, GGT7, HECD3, KIAA2013, and PLS1. To exemplify the validity of our approach, we chose a candidate having a relatively low number of edges in the network to increase the likelihood of a direct protein interaction with CB2 and focused on the scaffold/phagosomal protein p62/SQSTM1. Indeed, we independently confirmed the interaction by co-immunoprecipitation and immunocytochemical colocalization studies. 3D reconstruction of confocal images furthermore showed CB2 localization in close proximity to p62 positive vesicles at the cell membrane. In summary, we provide a comprehensive repository of the CB2 interactome in HEK293 cells identified by a systematic unbiased approach, which can be used in future experiments to decipher the signaling and trafficking complex of this cannabinoid receptor. Future studies will have to analyze the exact mechanism of the p62-CB2 interaction as well as its putative role in disease pathophysiology.

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Application of HaloTag technology to expression and purification of cannabinoid receptor CB2.

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