Ultrasonic disruptor

Recombinant Hpn was purified from harvested cells of 200 ml culture. Harvested cells were suspended in lysis buffer (50 mM Tris-HCl, pH 7.5, and 500 mM NaCl) and purification was done as described previously for His-tagged proteins with some modifications [38 (link)]. After sonication (TOMY Ultrasonic Disruptor, duty: 50, output: 4, time: 4 min x 6), supernatant fractions were filtered using Millex^®GV filter units of 0.22-μm-pore-size and applied onto a 1-ml HiTrap chelating column (GE Healthcare) that had been equilibrated with start buffer containing 20 mM imidazole, 50 mM Tris-HCl, and 500 mM NaCl. The column was washed with buffer (50 mM Tris, pH 7.5, 50 mM NaCl, and 40 mM imidazole), and then Hpn was eluted using start buffer with 400 mM instead of 20 mM imidazole. Eluted fractions were analyzed for purity using SDS-PAGE (15%), and the purest fractions were applied onto a 5-ml HiTrap desalting column (GE Healthcare) that had been equilibrated with desalting buffer (20 mM Hepes-KOH, pH 7.4, 100 mM NaCl, and 20% glycerol), and purified Hpn was stored at −80°C until use. The concentration and quality of purified Hpn was measured using bovine serum albumin (BSA) as the standard in a BCA assay (Bio-Rad) in accordance with the manufacturer’s instructions and SDS-PAGE (polyacrylamide 15%), respectively.