Expression, Characterization and purification of Recombinant Cannabinoid Receptor CB2 as a Fusion with Halo‐ and C‐terminal Rhodopsin Tags

Human peripheral cannabinoid receptor CB2 is a G protein‐coupled receptor (GPCR) involved in the immune response. Structural studies essential to exploit a potential of this receptor as a therapeutic target require large amounts of pure and functional protein. The purpose of the study is to develop tools for efficient purification and subsequent surface immobilization of the recombinant CB2 by testing different affinity tags. Usability of haloalkane dehalogenase protein (Halo tag), that enables purification via covalent capture and of rhodopsin C‐terminal nanopeptide (1D4 Rho tag), that allows purification via specific antibody‐binding were explored. Constructs expressing CB2 as a fusion with either of these tags were prepared. Expression level in E coli and accessibility of tags was determined by Western blotting. Different expression conditions were tested, and functional activity of CB2 produced by expression of best performing constructs was confirmed by the ligand binding experiments and in G‐protein activation assay. 1D4‐tagged CB2 was purified by anti‐1D4 antibody affinity chromatography, either by applying the crude extract directly to 1D4‐resin or after its pre‐purification on a Ni‐affinity column. In conclusion, we have identified several CB2‐fusion protein candidates for further optimization of expression, purification and subsequent structural analysis.