Purification and Analysis of Rho‐1D4‐tagged Peripheral Cannabinoid Receptor CB2

Abstract

Human peripheral cannabinoid receptor CB2 is involved in regulation of the immune response. For studies requiring surface immobilization, pure and functional receptor with a reliable tag is required. The goal of this project is to test the usefulness of the C‐terminal epitope of rhodopsin (rho‐1D4‐tag) for purification and surface immobilization of recombinant CB2 using the monoclonal antibody 1D4.Expression of rho‐1D4‐tagged CB2 as a fusion with maltose binding protein (MBP) in E coli was evaluated in terms of protein levels, accessibility of the rho‐1D4 tag and activity. Highly pure tagged CB2 was obtained by rho‐1D4 and Ni‐NTA affinity chromatography. The reconstituted protein was functionally active by G protein activation assay. The interaction of rho‐1D4‐tagged CB2 with 1D4 antibody was characterized by surface plasmon resonance (SPR). Either purified or fusion CB2 from crude extracts was captured on the 1D4 antibody‐coated surface in a specific and quantitative fashion, and its presence confirmed by biding of an anti‐CB2 monoclonal antibody. Ligand binding to purified receptor in detergents was studied upon immobilization of fusion CB2 on 1D4 antibody resin. This methodology was also used to study thermal stability of the receptor in micelles.In conclusion, the rho‐1D4 tag was successfully utilized for purification, immobilization, biochemical and biophysical studies of functional CB2.