Protein Expression and Purification from Sf9 Cells

Proteins were expressed and purified as described in ref. ^{59 (link)}. Proteins were coexpressed by Baculovirus expression in Sf9 insect cells. Cells were infected at a density of 1 × 10⁶ cells per ml and grown for 72 h. Cells were collected in lysis buffer (50 mM TRIS pH 8, 150 mM NaCl, 2 mM TCEP) and complete EDTA-free protease inhibitor (Sigma) was added. Cells were lysed by sonication and sample was cleared by centrifugation at 21,000 × g for 30 min. Supernatant was loaded on His-affinity column (GE Healthcare) and column was washed with lysis buffer + 50 mM Imidazol. Sample was eluted with 500 mM Imidazol. The eluted sample was then loaded on a Strep-affinity column (IBA Life Science) and eluted using lysis buffer + 2.5 mM desthiobiotin. The sample was then purified on a Superdex 200 size exclusion column (GE Healthcare) in lysis buffer. Samples were concentrated and stored at − 80 °C.