Ultra edta free protease inhibitor

Purification of the Smc5/6^5mer was as described previously (5 (link)). S. cerevisiae cells containing the overexpression constructs for five subunits of the Smc5/6 complex, wherein the Smc5 subunit is tagged with CBP tag, were grown at 30 °C in YP-GL (yeast extract, peptone, 2% glycerol, and 2% lactic acid) media until the log phase. Protein expression was induced by the addition of 2% galactose for 4 h. Harvested cells were resuspended in buffer E (45 mM Hepes-KOH pH 7.6, 10% glycerol, and 0.02% NP40) supplemented with 100 mM NaCl, 1 mM DTT, protease inhibitor cocktail (Sigma), and Ultra EDTA-free protease inhibitor (Roche) and then broken by using a freezer mill (SPEX CertiPrep 6850 Freezer/Mill). Cell powder was resuspended with buffer E supplemented with 300 mM NaCl and 1 mM DTT before being centrifuged for 30 min at 40,000 rpm to uncover the supernatant. Complexes containing Smc5-CBP fusion in the supernatant were pulled down by adding 2 mM CaCl₂ and incubated with calmodulin resin for 2 h at 4 °C. After washing the resins with 10 bed volume of buffer E supplemented with 300 mM NaCl, 2 mM CaCl₂, and 1 mM DTT, proteins were eluted using the same buffer without CaCl₂ and supplemented with 1 mM EDTA and 2 mM EGTA. Peak fractions were pooled and subjected to gel-filtration on a Superose 6 Increase column. Peak fractions were collected and snap-frozen for storage.