Purification of Recombinant Human CB1 Receptor

CB1 was expressed and purified as described previously^{13 (link)}. Briefly, human full-length CB1 containing an N-terminal FLAG tag and C-terminal histidine tag was expressed in Spodoptera frugiperda Sf9 insect cells with the baculovirus method (Expression Systems). Receptor was extracted using 1% lauryl maltose neopentyl glycol (L-MNG) and purified by nickel-chelating Sepharose chromatography. The eluant from the Ni column was applied to an M1 anti-FLAG immunoaffinity resin. After washing to progressively decreasing the concentration of L-MNG, the receptor was eluted in a buffer consisting of 20 mM HEPES pH 7.5, 150 mM NaCl, 0.05% L-MNG, 0.005% cholesterol hemisuccinate (CHS), FLAG peptide and 5 mM EDTA. Finally, CB1 was purified with size exclusion chromatography, on Superdex 200 10/300 gel filtration column (GE) in 20 mM HEPES pH 7.5, 150 mM NaCl, 0.02% L-MNG, 0.002% CHS. Ligand-free CB1 was concentrated to ~500 µM and stored at −80 °C.