Amicon ultra 0.5 10 kda cutoffspin filters

Wild-type and mutant B. subtilis Spo0J (BsSpo0J) proteins were expressed with an N-terminal His6-SUMO tag in BL21 (DE3) pLysS cells and purified by a two-step tandem affinity method (26 (link)) with modified buffer conditions as described here. Briefly, cell pellets were lysed by sonication in lysis buffer (20 mM Tris at pH 8.0, 1 M NaCl, 50 mM imidazole and 5 mM 2-mercaptoethanol (BME)) supplemented with 1 mM PMSF, and supernatants were clarified by ultracentrifugation. The clarified supernatant was bound to Ni-NTA resin (Qiagen) and washed extensively with lysis buffer and sequentially with salt-reduction buffer (lysis buffer with only 350 mM NaCl). His6-SUMO fusion proteins were manually eluted with elution buffer (20 mM Tris at pH 8.0, 350 mM NaCl, 250 mM imidazole and 5 mM BME) in a series of 1 ml aliquots. Peak fractions were collected and dialyzed overnight at 4°C against dialysis/storage buffer (20 mM Tris at pH 8.0, 350 mM NaCl, 10% glycerol, 10 mM imidazole and 5 mM BME) in the presence of His6-Ulp1 protease (26 (link)). The cleaved His6-SUMO tag and His6-Ulp1 were then removed from the proteins by incubating with Ni-NTA resin again on the second day. The flow-through, containing untagged BsSpo0J proteins, was collected and concentrated by centrifugation in Amicon Ultra-0.5 10 kDa cutoff spin filters (EMD Millipore) before being stored at −80°C.