Purification of GINS (pFJD5 Sample Clauses

Purification of GINS (pFJD5. The pFJD12/GINS plasmid was transformed into BL21 (DE3) Rosetta cells (Millipore) and the resultant colonies were used to inoculate 2 x 1 L of LB with 50 μg/mL kanamycin and 34 μg/mL chloramphenicol at 37°C. Protein expression was induced once the cultures reached an OD600 of 0.5 with 1 mM IPTG for 3 hours at 37°C. Cells were then harvested by centrifugation for 10 minutes at 6,000 rpm in a SLA-3000 rotor (Thermo Scientific) and the pellet was resuspended in 50 mM HEPES-KOH pH 7.6, 200 mM KCl, βME (7 μL/50 mL), 10 mM MgOAc, 10% glycerol, 1 mM EDTA, 1 mM EGTA, 0.05% NP-40-S (buffer V + 200 mM KCl + βME) + protease inhibitors (see Table 2-9). Lysozyme (100 μg/mL) was added and the solution was incubated for 30 minutes at 4°C. Cells were then sonicated (1 minute, 5 seconds on/5 seconds off, 40%) and insoluble material was cleared by centrifugation at 15,000 rpm for 30 minutes (SS34 rotor; Thermo Scientific). The supernatant was recovered and rotated with 5 mL Ni-NTA agarose (Thermo Scientific) pre- equilibrated in buffer V + 200 mM KCl for 1 hour at 4°C. The resin was then collected and washed with 50 CV of buffer V + 200 mM KCl + 50 mM imidazole followed by elution of GINS with 10 x 1 mL of buffer V + 200 mM imidazole + 200 mM KCl. Following this, the eluate was pooled and dialysed against 2 x 1 L buffer V + 50 mM KCl for 2 hours at 4°C. The dialysed sample was then loaded onto a 1 mL MonoQ column (GE Healthcare) equilibrated in buffer V + 50 mM KCl and the protein was eluted over a 20 CV gradient from 50 mM KCl to 500 mM KCl in buffer V. Peak fractions were pooled, concentrated with an Amicon Ultra 30,000 MWCO centrifugal filter (Millipore), and applied to a Superdex 200 Increase 10/300 GL column (GE Healthcare) pre-equilibrated in 25 mM HEPES-KOH pH 7.6, 200 mM KOAc, 1 mM EDTA, 0.02% NP-40-S, 10% glycerol, 1 mM DTT. Fractions containing GINS were then pooled, concentrated, and stored in aliquots at -80°C. Buffer V: 50 mM HEPES-KOH pH 7.6, 10 mM MgOAc, 10% glycerol, 1 mM EDTA, 1 mM EGTA, 0.05% NP-40-S