The cell pellets were lysed in a buffer containing 20 mM HEPES, pH 7.5, 100 mM NaCl, and 2 mM MgCl2 supplemented with EDTA-free protease inhibitor cocktail (Bimake) by dounce homogenization. The complex formation was initiated by addition of 10 μg/mL scFv16, 50 mU/mL apyrase (NEB), and 100 mM Ghrelin. After incubation at room temperature for 1.5 h, the membranes were solubilized by addition of 0.5% (w/v) lauryl maltose neopentyl glycol (LMNG, Anatrace) and 0.1% (w/v) cholesteryl hemisuccinate TRIS salt (CHS, Anatrace) for 2 h at 4 °C. The supernatant was isolated by centrifugation at 30,000 × g for 30 min and then incubated 1 h at 4 °C with pre-equilibrated MBP resin. After binding, the resin was washed with 15 column volumes of 20 mM HEPES pH 7.5, 100 mM NaCl, 2 mM MgCl2, 0.01% (w/v) LMNG, 0.002% (w/v) CHS, and 10 mM Ghrelin. The complex was eluted with 5 column volumes of 20 mM HEPES pH 7.5, 100 mM NaCl, 2 mM MgCl2, 0.01% (w/v) LMNG, 0.002% (w/v) CHS, 10 mM maltose, and 10 mM ghrelin.
The protein was then concentrated and loaded onto a Superose™ 6 Increase column (GE Healthcare) pre-equilibrated with buffer containing 20 mM HEPES pH 7.5, 100 mM NaCl, 0.00075% (w/v) LMNG, 0.00025% (w/v) glyco-diosgenin (GDN, Anatrace), 0.0002% (w/v) CHS and 10 mM ghrelin. The fractions for the monomeric complex were collected and concentrated for electron microscopy experiments.
The protein was then concentrated and loaded onto a Superose™ 6 Increase column (GE Healthcare) pre-equilibrated with buffer containing 20 mM HEPES pH 7.5, 100 mM NaCl, 0.00075% (w/v) LMNG, 0.00025% (w/v) glyco-diosgenin (GDN, Anatrace), 0.0002% (w/v) CHS and 10 mM ghrelin. The fractions for the monomeric complex were collected and concentrated for electron microscopy experiments.