Protein Purification from Recombinant E. coli

Bacterial suspensions of recombinant E. coli used to overexpress proteins were lysed by ultrasonic vibration with a Branson sonifier 250 (Branson Ultrasonics, Brookfield, CT, USA), using 6 cycles of sonication of 30 s each with a 40% duty cycle, adding 2% (v/v) Triton X-100 before the last two sonication cycles. Centrifugation at 12,000× g for 30 min at 4 °C was then performed to remove non-soluble cell debris. The cleared supernatants were collected and kept at 4 °C. The BCAL2645 and BCAL2022 6× His-tagged proteins were then purified by affinity chromatography using HisTrap FF columns (GE Healthcare, Chicago, IL, USA). The columns were initially equilibrated by flowing 10 mL of Buffer A (20 mM sodium phosphate, 750 mM NaCl, 20 mM imidazole, 10% glycerol, pH 7.4) for BCAL2645 or Buffer B (20 mM sodium phosphate, 500 mM NaCl, 20 mM imidazole, pH 7.4) for BCAL2022. The proteins were then eluted with 5 mL of Buffer A or B, respectively, containing imidazole concentrations of 60, 100, 150, 200, 250, 300, 400 and 500 mM. Aliquots of 1 mL were collected for each protein, followed by their analysis by SDS-PAGE. Immunoblotting experiments were carried out as previously described using a commercial monoclonal anti-polyhistidine peroxidase conjugate clone HIS-1 antibody (Sigma; St. Louis, MO, USA) [26 (link)]. BCAL2958 purification was performed as previously described [26 (link)].