Purification and Characterization of scFv16

The codon-optimized nucleotide sequence of scFv16 was synthesized by GenScript and subcloned into an expression vector pFastBac1 with an 8x His tag at the C-terminus. The scFv16 used in this paper was the same as that used in the structures of the CB1-Gi-scFv16^{35 (link)}. In brief, scFv16 was expressed in secreted form from Trichuplusia ni Hi5 insect cells and purified by Ni-NTA chromatography. The supernatant was incubated with Ni-NTA resin (GenScript) at 4 °C for 2 h. The resin was then loaded to a gravity column and washed with 15 column volumes (CV) of wash I buffer containing 20 mM HEPES (pH7.5), 100 mM NaCl and 10 mM imidazole; followed by 15 CV of wash II buffer containing 20 mM HEPES (pH7.5), 100 mM NaCl and 30 mM imidazole. The protein was eluted with elute buffer containing 20 mM HEPES (pH7.5), 100 mM NaCl and 250 mM imidazole. The elute was collected and further purified using a Superdex 200 10/300 column (GE Healthcare). Monomeric fractions were pooled, concentrated 10 mg/mL with a 10-kDa cut-off concentrator (Millipore), and flash frozen in liquid nitrogen, then stored at −80 °C for further use.