Baculovirus method

Single chain construct of Fab16 (scFv16) was expressed and purified as previously described^{19 (link)}. In brief, a C-terminal 6× histidine tagged scFv16 was expressed in secreted form from Trichuplusia ni Hi5 insect cells using the baculovirus method (Expression Systems), and purified by Ni-NTA (Qiagen) chromatography. Supernatant from baculovirus-infected cells was pH balanced by addition of Tris pH 8.0. The C-terminal 6x His-tag of Ni-NTA eluent was cleaved by 3C protease, and the protein was dialyzed into a buffer consisting of 20 mM HEPES pH 7.5 and 100 mM NaCl. The sample was reloaded onto the Ni-NTA column to capture the cleaved His₆. The flow-through containing scFv16 was collected, concentrated and purified through gel-filtration chromatography in a final buffer (100 mM NaCl and 20 mM HEPES pH 7.5). Monomeric fractions were pooled, concentrated to ~100 mg/ml.

Human full-length CB1 with N-terminal FLAG and C-terminal hexahistadine tag was expressed in Spodoptera frugiperda Sf9 insect cells using the baculovirus method (Expression Systems). The same construct with an eGFP (CB1-eGFP) at the C-terminus was used for small-scale coupling and FSEC studies. The receptor was extracted from insect cell membranes with 1% lauryl maltose neopentyl glycol (L-MNG) and purified by nickel-chelating sepharose chromatography. The Ni-NTA pure eluate was applied to a Ml anti-FLAG immunoaffinity resin and washed with progressively decreasing concentration of inverse agonist, SR and increasing concentration of agonist FUB. 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), 2 μM FUB, FLAG peptide and 5 mM EDTA. The final step of purification was 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, and 2 μM FUB. Finally, agonist-bound CB1 was concentrated to ~500 μM and flash frozen and stored in −80 °C.