Protein expression and purification of OCT4 and SOX2

Human full-length OCT4 (residues 1 – 360), OCT4 DNA binding domain (residues 134-290) or human SOX2 DNA binding domain (residues 37 – 118) were subcloned into pAC-derived vectors  containing an N-terminal StrepII tag. An additional N-terminal EGFP tag and C-terminal sortase-6XHIS tag (LPETGGHHHHHH) were fused in frame with OCT4 full-length to improve purification and was also used 3 for cryo-EM sample preparation. Recombinant proteins were expressed in 2- 4 L cultures of Trichoplusia ni High Five cells using the Bac-to-Bac system (Thermo Fisher). Cells were cultured at 27°C, harvested 2 days after infection, resuspended in lysis buffer (50 mM Tris-HCl pH 8.0, 1M NaCl, 100 uM phenylmethylsulfonyl fluoride, 1 × protease inhibitor cocktail (Sigma), 250 uM TCEP), and lysed by sonication. The supernatant was harvested and the proteins were purified by Streptactin affinity chromatography (IBA) using a gravity flow column and a streptactin elution buffer (lysis buffer + 2.5mM desthiobiotin) and then purified by Heparin ion exchange chromatography on an FPLC (GE Healthcare). Heparin buffer A: 20mM sodium phosphate pH 7.4, 50mM NaCl, 5% glycerol, 0.5mM TCEP, Heparin buffer B: 20mM sodium phosphate pH 7.4, 2M NaCl, 5% glycerol, 0.5mM TCEP.  All proteins were further purified by size exclusion chromatography (Superdex 200; GE Healthcare) in GF buffer (20 mM HEPES pH 7.4, 150 mM NaCl, 5% glycerol, 500 uM TCEP) as a last purification step. The purified proteins were concentrated and stored at -80°C.