pET-28b-hBCO1

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	A pET-28b plasmid vector containing the cDNA of human BCO1 with a C-terminal hexahistidine tag was a gift from Dr. William Blaner of Columbia University. The plasmid was transformed into E. coli BL21-Gold(DE3) (Stratagene) according to the manufacturer's instructions. The transformed bacterial cells were grown in LB broth (Sigma-Aldrich) to an A600 of 0.5–0.7 at 30 °C, and expression of the recombinant protein was induced by adding isopropyl β-d-1-thiogalactopyranoside (Gold Biotechnology) to a final concentration of 0.1 mm.

Protocol tips

A pET-28b plasmid vector containing the cDNA of human BCO1 with a C-terminal hexahistidine tag was a gift from Dr. William Blaner of Columbia University. The plasmid was transformed into E. coli BL21-Gold(DE3) (Stratagene) according to the manufacturer's instructions. The transformed bacterial cells were grown in LB broth (Sigma-Aldrich) to an A600 of 0.5–0.7 at 30 °C, and expression of the recombinant protein was induced by adding isopropyl β-d-1-thiogalactopyranoside (Gold Biotechnology) to a final concentration of 0.1 mm.

Publication protocol

A pET-28b plasmid vector containing the cDNA of human BCO1 with a C-terminal hexahistidine tag was a gift from Dr. William Blaner of Columbia University. The plasmid was transformed into E. coli BL21-Gold(DE3) (Stratagene) according to the manufacturer's instructions. The transformed bacterial cells were grown in LB broth (Sigma-Aldrich) to an A600 of 0.5–0.7 at 30 °C, and expression of the recombinant protein was induced by adding isopropyl β-d-1-thiogalactopyranoside (Gold Biotechnology) to a final concentration of 0.1 mm. Five liters of culture were grown for 24 h, and the cells were harvested by centrifugation. The cell pastes were frozen at −80 °C and thawed prior to lysis. The cells were lysed by gentle stirring with lysis buffer (50 mm phosphate, 50 mm NaCl, 10% (v/v) glycerol, 1% (v/v) Triton X-100, 1 mm β-mercaptoethanol, 2.5 mm imidazole, 10 mg/ml chicken egg white lysozyme (Sigma), 0.1 μl/ml benzonase nuclease HC (Novagen), 1 mm phenylmethanesulfonylfluoride, and 1 tablet of EDTA-free protease inhibitor mixture (Roche Diagnostics)/50 ml), using 5 ml of lysis buffer per gram of wet cell paste. The lysis was conducted in ice for 1 h. The lysate was centrifuged at 15,600 × g for 25 min, and the supernatant was filtered through a 0.22-μm syringe-driven filter. The NaCl concentration of the filtered supernatant was brought up to 300 mm using salt adjustment buffer (50 mm phosphate, 2,500 mm NaCl, 10% (v/v) glycerol, 1% (v/v) Triton X-100, 1 mm β-mercaptoethanol, 2.5 mm imidazole) and stirred with 0.5 ml of HisPur resin (Pierce) that has been previously washed with equilibration buffer (50 mm phosphate, 300 mm NaCl, 10% (v/v) glycerol, 1% (v/v) Triton X-100, 1 mm β-mercaptoethanol, 2.5 mm imidazole) for 1 h in an ice bath. The resin was harvested by centrifugation at 700 × g for 2 min and transferred to a gravity column. The resin was then eluted with the following solutions, which were prepared by combining the calculated amounts of equilibration buffer (2.5 mm imidazole) and elution buffer (same composition as equilibration buffer except for 150 mm imidazole): 4 ml of 2.5 mm imidazole (equilibration buffer), 1.5 ml of 10 mm imidazole, 1 ml each of 30, 60, 90, 120, and 150 mm imidazole (elution buffer). The fractions with pure protein were transferred into the final storage buffer (50 mm phosphate, 50 mm NaCl, 10% (v/v) glycerol, 1% (v/v) Triton X-100, 1 mm β-mercaptoethanol) using a PD-10 desalting column (GE Healthcare). SDS-PAGE was used to assess the purity of the protein throughout the purification process. The concentration of the protein solution was measured using the reducing agent-compatible bicinchoninic acid protein assay kit (Pierce). The protein was stored at −80 °C until used.

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