PglH Protein Purification from E. coli

The gene encoding PglH was cloned into a modified pET-19b vector (Novagen) with a N-terminal His10 affinity tag fused to PglH. The protein was overexpressed in E. coli BL21-Gold (DE3) (stratagene) cells in Terrific Broth medium supplemented with 1% glucose (w/v). Cells were grown at 37 °C to A600 of 3.0 before the culture was induced by the addition of 0.5 mM IPTG and transfer to 18 °C for 16 h. All following steps were performed at 4 °C unless specified differently. Cells were harvested by centrifugation, re-suspended in 50 mM Tris-HCl, pH 8.0; 200 mM NaCl; 20 mM Imidazole; 0.5 mM PMSF and disrupted in a M-110L microfluidizer (Microfluidics) at 15,000 p.s.i. chamber pressure followed by addition of 1% TritonX-100 (w/v)42 (link). After centrifugation the supernatant was loaded onto a NiNTA superflow affinity column (Qiagen), washed once with the same buffer but containing 50 mM Imidazole. Elution was performed in buffer containing 50 mM Tris-HCl, pH 8.0; 500 mM Imidazole. The protein was desalted into 50 mM Tris-HCl, pH 8.0; 150 mM NaCl.