Protein purification

ESCO1 N-terminal tail cDNAs were cloned into a pET-based N-terminally 6His- and GFP-tagged expression vector with a PreScission Protease cut-site between the 6His and GFP tags. These plasmids were transformed into C41(DE3) E. Coli (Sigma Aldrich) and protein expression was induced with 100uM IPTG overnight at 18 °C. Induced cells were harvested and lysed via sonication in 20mM Tris pH 8.0, 500mM KCl, 0.01% Zwittergent 3–10 detergent (Sigma), 5mM Imidazole, 5mM β-mercaptoethanol, 1mM PMSF, and 250ug/ml lysozyme (VWR). Lysates were incubated with ProteinDex Ni-NTA Agarose beads (Marvelgent) for 2 hours rotating at 4 °C to bind the 6His tag, resuspended in 20mM Tris pH 8.0, 500mM NaCl, and 5mM Imidazole, transferred into a column, and washed 5 times with the same buffer. Columns were washed twice with 50mM Tris pH 8.0 and 150mM NaCl and then the GFP and ESCO1 proteins were cleaved off the 6His and nickel beads by 630nM PreScission Protease in 50mM Tris pH 8.0, 150mM NaCl, and 5mM β-mercaptoethanol overnight, rotating at 4 °C. The flow-through was collected, protease was removed by incubation with Glutathione Sepharose Agarose beads (Thomas Scientific) for 1 hour rotating at 4 °C, beads were spun down, and cleaved protein was collected from the bead supernatant. Proteins were dialyzed to dilution buffer (50mM HEPES pH 8.0, 200mM NaCl, 1mM MgCl₂, and 10% glycerol), concentrated, aliquoted, flash frozen in liquid nitrogen, and then stored at −80 °C.