Purification of Cra-His6 Protein from E. coli

Escherichia coli harboring pCra-His₆ was cultivated in LB broth and treated with 0.2% arabinose for 3 h for Cra-His₆ induction (81 (link)). Bacterial cells were harvested at 10,000 × g for 10 min and treated with 1 mg/mL lysozyme in lysis buffer (50 mM NaH₂PO₄, 300 mM NaCl, and 20 mM imidazole, pH 8.0) on ice for 30 min. The cell suspension was sonicated and centrifuged at 10,000 × g at 4°C for 20 min. The soluble lysate fraction was then mixed with Ni²⁺-nitrilotriacetic acid (Ni²⁺-NTA) agarose beads (Qiagen) and incubated at 4°C for 1 h with gentle agitation. The reactant was loaded onto a Ni²⁺-NTA agarose affinity column (Qiagen). The column was washed with washing buffer (50 mM NaH₂PO₄, 300 mM NaCl, and 40 mM imidazole, pH 8.0) three times, and Cra-His₆ bound to the column was eluted using elution buffer (50 mM NaH₂PO₄, 300 mM NaCl, and 300 mM imidazole, pH 8.0). The eluted fraction was placed in a dialysis membrane tubing with 10 K MWCO (SnakeSkin Dialysis tubing, Thermo Scientific Inc.) and incubated in dialysis buffer (20 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.1 mM EDTA, 5 mM DTT, and 20% glycerol) at 4°C. Protein concentration was measured using the Bradford assay (Bio-Rad) (82 (link)).