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The column was equilibrated with lysis buffer, the supernatant was loaded, the column was washed three times with washing buffer (50 mM NaH 2 PO 4 /Na 2 HPO 4 , 0.3 M NaCl, 40 mM imidazole, pH 8.0), and the recombinant proteins were eluted with elution buffer (50 mM Na 2 HPO 4 /Na 2 HPO 4 , 0.3 M NaCl, 250 mM imidazole, pH 8.0)