Purification of Mph Enzyme from E. coli

Mphs were purified as described previously for MphI^{36 (link)}. E. coli BL21(DE3) was cultured with shaking at 37 °C in 1 L of LB-Lennox until an OD₆₀₀ of 0.5–0.6, chilled in an ice bath for 20 min, and protein overexpression induced by the addition of 1 mM IPTG and incubation at 17 °C for 16 h. Cell mass was harvested by centrifuging at 10,000 × g for 20 min, washed with cold saline, and resuspended in 20 mL buffer A (50 mM HEPES pH 7.5, 150 mM NaCl, 5% glycerol, 10 mM imidazole). Cell lysis was performed using a One-shot Cell Disruptor (Constant Systems Limited) at 20,000 psi, and cell debris removed after adding 15 mL buffer A, 5 mg bovine bovine pancreas DNase, and 2.5 mg of bovine pancreas RNase by centrifugation at 50,000 × g for 45 min. Proteins were purified using 1 mL Ni²⁺-nitrilotriacetic acid column (Qiagen) and a linear gradient of 95% buffer A to 100% buffer B (buffer A with 250 mM imidazole) over 20 column volumes. Fractions with pure Mph were pooled and desalted into 50 mM HEPES pH 7.5 using a PD-10 desalting column (GE Scientific). Pure enzyme stocks were stored at 4 °C.