Ultrasonic processors sonicator 3000

Recombinant proteins were purified on a Ni-NTA resin (Cyrusbioscience, Taiwan), which was charged with nickel (Ni²⁺) ions. E. coli and P. pastoris cells were centrifuged at 6000× g for 20 min and resuspend in 20 mL 1× lysis buffer (50 mM NaH₂PO₄, pH 7.5, 10 mM imidazole, 100 mM NaCl and proper amount of protease inhibitors). E. coli and P. pastoris cells were mechanically disrupted by sonication using Ultrasonic Processors Sonicator 3000 (Misonix, Farmingdale, NY, USA) and by glass beads (0.5 mm diameter) using Mini-Bead Beater-16 (BioSpec Products, Bartlesville, OK, USA) [44 (link)], respectively. The lysate was clarified by centrifugation at 10,000× g for 10 min. Then, the supernatant was loaded into a Ni-NTA column and eluted stepwise with buffers containing 50, 125, 250 or 500 mM imidazole. The recombinant proteins extracted from E. coli and P. pastoris were designated as eBoPAL1/2 and pBoPAL1/2, respectively.

Recombinant BoPAL3 protein was affinity purified on a Ni-NTA resin (Cyrus Bioscience Inc., New Taipei City, Taiwan) and chelated with nickel (Ni²⁺) ions. E. coli cells stored at −20 °C were resuspended in 20 mL lysis buffer containing 50 mM Tris–HCl (pH 7.5), 10 mM imidazole, 100 mM NaCl, and the indicated amount of protease inhibitors [3 (link)]. An Ultrasonic Processors Sonicator 3000 (Misonix, Farmingdale, NY, USA) was used to sonicate cells, which mechanically disrupts the cells. The cell lysate was centrifuged at 10,000× g for 10 min. A Ni-NTA column was used to apply the supernatant, and buffers containing either 50, 125, 250, or 500 mM of imidazole were used to competitively elute the recombinant BoPAL3 protein. The purity of eluted protein was estimated using polyacrylamide gel electrophoresis.