2.4 Protein purification
Each lysate generated as described above was cleared by centrifugation,
and the target protein was purified by nucleophilic chromatography on a
column of chelating Sepharose Fast Flow (GE Healthcare Bio-Sciences
Corp.) charged with Ni2+ ion. The column was
pre-equilibrated with the Lysis Buffer (50 mM Tris, 10 mM imidazole, 300
mM NaCl, pH 7.8), loaded with 30 mL of a lysate at a flow rate of 1.0
mL/min, and then washed with a Wash Buffer containing 30 mM imidazole,
0.3 M NaCl, and 50 mM Tris-HCl (pH 7.8), eluted with an Elution Buffer
containing 300 mM imidazole, 0.3 M NaCl, and 50 mM Tris-HCl (pH 7.8) to
obtain a purified fraction containing the target protein. After
dialyzing against Tris-HCl (50 mM, pH 7.5), the purified protein
fraction was collected and stored at -80°C. Protein concentration was
measured using the BCA Protein Quantitation Kit.