200 10 300 gel filtration column

The mini-Gαq_iN chimeric construct was designed for the binding of scFv16 and sub-cloned into a designed vector that help form heterotrimeric G complex in situ (Figure S1). For the expression, Sf9 insect cells were infected with one virus, encoding three subunits including the mini-Gαq_iN subunit and Gβ1/γ2 subunits with histidine tag inserted at the amino terminus of the β subunit. Cells expressing the heterotrimeric G-protein were harvested 72 hours post infection. Cells were lysed in lysis buffer containing 20 mM HEPES (pH 7.5), 100 mM NaCl, 30 mM imidazole, 5 mM β-mercaptoethanol, 0.1 mM GDP, 1 mM MgCl₂, 0.2 % (v/v) Triton X-100 and protease inhibitors, and the soluble fraction was isolated by ultra-centrifugation at 40,000 rpm at 4°C for 50 min. The heterotrimer containing soluble fraction was purified using Ni-NTA chromatography. Human Rhinovirus 3C protease (HRV3C protease) was added and the histidine tag was cleaved at 4° C for overnight. The histidine tag removed heterotrimeric G protein was further purified by size exclusion chromatography on a Superdex 200 10/300 gel filtration column (GE) with SEC buffer containing 20 mM HEPES (pH 7.5), 100 mM NaCl, 0.001% (w/v) LMNG, 0.0001% (w/v) CHS, 0.00025% (w/v) GDN, 100 uM TCEP, 1 mM MgCl₂, and 10 uM GDP, and collected and concentrated to ~25 mg/ml, and stored at −80°C until use.