Recombinant Protein Purification from E. coli

WH indicator was expressed in Escherichia coli strain JM109 (DE3), and cultured at 23 °C for 69 h in 200 mL LB bacterial growth medium supplemented with 100 µg/mL carbenicillin. Cultured cells were suspended in PBS buffer, and lysed using a French press (ThermoFisher Scientific). The lysate was centrifuged (8000 rpm at 4 °C for 20 min), and recombinant proteins were purified from the supernatant using Ni-NTA agarose affinity columns (QIAGEN), followed by buffer exchange with 20 mM HEPES (pH 7.4) with the desalting column, PD-10 (GE Healthcare, Buckinghamshire, UK). After lysis, all protein purification processes were conducted on ice to avoid protein degradation. Protein concentration was determined by both the alkaline-denaturation method [36 (link),37 (link)] and the Bradford assay.