J 26 xp centrifuge

Protein expression and purification were performed as described by Van Loi et al. 1 with minor modifications. Briefly, the harvested cells were lysed using a Sonic VibraCell sonicator for 10 min, with 30 s sound/30 s pause with 61% amplitude. Cell debris was removed by centrifugation (45 min at 18,000 rpm, at 4°C; Avanti ® J-26xp centrifuge (BECKMAN COULTER ® )), and the supernatant was in-batch incubated with Ni 2+ -Sepharose 6 Fast Flow beads (Cytiva) equilibrated with the binding buffer (20 mM HEPES/NaOH pH 7.5, 0.5 M NaCl and 10 mM imidazole) for 1 h at 4°C. The beads were then packed in a column coupled to an AKTA™ Pure system (GE Healthcare, Life Sciences). HypR was eluted using a linear gradient with elution buffer: 20 mM HEPES pH 7.5, 0.5 M NaCl and 0 to 500 mM (0-100%) imidazole. Protein purity was assessed on a nonreducing SDS-PAGE gel, and the pure fractions were collected, dialyzed (~20 mL sample/2 L dialysis buffer) overnight at 4°C against 20 mM HEPES pH 7.5 and 250 mM NaCl, and stored at -80°C in 20% glycerol.