Purification of Nhp6 (pRJ1228. The glycerol stock for this purification was provided by Xx Xxxxxxxxx Xxxxx. Cells were grown in 2 x 1 L of LB with 100 μg/mL ampicillin at 37°C. Protein expression was induced once the cultures reached an OD600 of 0.6 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 20 mM Tris-HCl pH 7.5, 500 mM NaCl, 2 mM EDTA, 10% glycerol, 1 mM βME (buffer Z + 500 mM NaCl) + protease inhibitors (see Table 2-9). Cells were lysed via sonication (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). Trichloroacetic acid (TCA) was added slowly to the supernatant to a final concentration of 2% (the volume of 50% TCA to add was determined by multiplying the volume of the supernatant by 0.0417) and the solution was stirred for 30 minutes at 4°C. Insoluble material was cleared by centrifugation at 15,000 rpm for 30 minutes and the TCA concentration in the supernatant was increased to 10% (the volume of 50% TCA to add was calculated by multiplying the volume of the supernatant by 0.19) with stirring for 30 minutes at 4°C. Precipitated protein was collected via centrifugation at 15,000 rpm for 30 minutes at 4°C. The pellet was washed with acetone, vortexed, and centrifuged at 15,000 rpm for 5 minutes and then dried under vacuum (at 30°C). It was then resuspended in and dialysed against buffer Z + 300 mM NaCl (3 x 1 L for 2 hours at 4°C). The dialysed sample was filtered through a 0.2 μm Millex-GP Sterile Syringe Filter (Thermo Scientific) and applied to a 1 xX XxxxX column (GE Healthcare) pre-equilibrated in buffer Z + 300 mM NaCl. Nhp6 was eluted over a 35 CV gradient from 300 mM NaCl to 1400 mM NaCl in buffer Z. Peak fractions were pooled, dialysed against 600 mL of buffer containing 25 mM HEPES-KOH pH 7.6, 200 mM KOAc, 10% glycerol, 1 mM βME, concentrated with an Amicon Ultra 3,000 MWCO centrifugal filter (Millipore), and stored in aliquots at -80°C. Buffer Z: 20 mM Tris-HCl pH 7.5, 2 mM EDTA, 10% glycerol, 1 mM βME
