Recombinant protein purification from E. coli

Rosetta2 E. coli were transformed with pET-16 plasmids encoding N-terminal 10xHis-tagged and C-terminal 1xFlag-tagged constructs. Liquid cultures were grown to an OD600 of 0.6 and then induced with 1 mM IPTG for 24 h at 20°C. Proteins were extracted under native conditions in N-250 buffer (50 mM Tris at pH 7.5, 250 mM NaCl, 15 mM imidazole, 0.2% igepal, 0.2% Tween-20, 0.2% Triton X-100, 1× Sigma protease inhibitor no. P8849, 1 mM PMSF) supplemented with 1 mg/mL lysozyme and 25 U/mL benzonase nuclease (Sigma, no. E1014-25KU). Lysates were clarified by centrifugation at 40,000g for 20 min at 4°C (Beckman Coulter 45-Ti Rotor), and protein extracts were incubated with Ni-NTA agarose beads (Qiagen) for 3 h. Beads were washed three times with 50 bead volumes of N-250 buffer, and proteins were eluted with His elution buffer (50 mM Tris at pH 7.5, 250 mM NaCl, 300 mM imidazole, 0.2% igepal, 0.2% Tween-20, 0.2% Triton X-100, 25% glycerol, 1 mM PMSF, 1× Sigma protease inhibitor no. P8849). After elution, EDTA was added to 2 mM, and proteins were concentrated using Amicon Ultra centrifugal filters (Millipore)