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The harvested cells were resuspended in the lysis buffer containing 50 mM Tris–HCl (pH 8.0), 100 mM NaCl, 1 mM  CuCl2, 1 mg/L of lysozyme, and 10 µg/L of benzonase. After  30-min incubation at ambient temperature, the cells were  sonicated and the lysates were clarified by centrifugation at  8000 g for 20 min at 4 °C, and then loaded onto an immobilized metal affinity chromatography (IMAC) column (BioRad, Hercules, CA, USA) equipped with a protein purification system (JP-blupadStar, Shanghai, China).

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