Purification of S. pombe E1, E2 Enzymes and Nse Proteins

The S. pombe E1 protein Uba1 and E2 enzymes (Ubc1, Ubc2, Ubc4, Ubc7, Ubc8, and Ubc13) were expressed in E. coli strain BL21 (DE3) RIL and purified similarly as described [21 (link)]. Cultures containing the Mms2-pGEX-6P-1 plasmid were grown at 37 °C to OD₆₀₀ ~ 1 and induced by 1 mM IPTG at 37 °C for 3 h. The cell pellet was resuspended in buffer A containing 20 mM Tris-HCl (pH 8.0), 150 mM NaCl, 10% glycerol, 1 mM DTT, and protease inhibitors. The cells were lysed by sonication, centrifuged, and supernatant mixed with Glutathione Sepharose 4 Fast Flow resin (GE Healthcare, Chicago, IL, USA) at 4 °C for 1 h. Proteins were eluted by gravity using buffer A containing 10 mM glutathione. The GST tag was cleaved off using PreScission protease overnight at 4 °C. The protein mixture was then subjected to gel filtration on Superdex 75 (GE Healthcare) in buffer A. Fractions containing Mms2 were concentrated and snap-frozen in liquid nitrogen. The Nse1/3/4 trimer and its vRING mutant were prepared as described [22 (link)]. The S. pombe Nse1 protein, its mutants, and Nse1/3 dimer were expressed and purified similarly as in the case of human Nse1/3 dimer [22 (link)].