Purification of Mutant DEK Proteins

The mutated DEK sequences (DEK SUMOmut 61-62, 144, 145 or 261) were inserted into a GST expression plasmid (pGEX 4T-1), transformed into Escherichia coli BL21(DE3) (Thermo Fisher Scientific) and purified as described previously (Ganz et al., 2019 (link)). Briefly, protein expression was induced with 0.5 mM IPTG (Sigma-Aldrich) for 1.5 h. Bacteria were harvested via centrifugation at 4600 g for 15 min at 4°C and resuspended in resuspension buffer (20 mM Tris-HCl pH 8.0, 1 M NaCl, 0.5 mM EDTA and 1 mM DTT) and frozen in liquid nitrogen. After thawing, bacteria were sonicated on ice and 0.5% NP-40 (Sigma-Aldrich) was added and centrifuged at 18,000 g for 30 min at 4°C. The supernatant was added to 200 µl Glutathione-Sepharose 4B-beads (GE Healthcare) and incubated for 2 h at 4°C. After centrifugation, beads were washed with wash buffer with decreasing NaCl concentrations (500 mM, 300 mM and 20 mM) and GST-tagged DEK was eluted with 200 µl elution buffer [200 mM Tris-HCl pH 8.0, 200 mM NaCl, 40 mM reduced glutathione (Sigma-Aldrich) and 10% glycerol] for 1 h at 4°C. The supernatant was frozen in liquid nitrogen and stored at −80°C.