Halt proteinase inhibitor cocktail

The general expression and purification procedures were described previously (42 (link)). In brief, a single colony of BL21(DE3) containing the pET-SUMO-DprE1 plasmid was inoculated into 100 ml of lysogeny broth (LB) with kanamycin (50 µg/ml), followed by incubation at 37°C, 250 rpm overnight. The overnight culture was added into 500 ml of fresh LB broth with kanamycin (50 µg/ml) and 1 mM isopropyl β-D-1-thiogalactopyranoside. After incubation for up to 6 hours at 37°C and 250 rpm, bacteria were harvested and pellet was frozen at −80°C before resuspension in 22.5 ml of lysis buffer [25 mM Hepes (pH 7.4), 300 mM KCl, and 10% glycerol]. Lysozyme (2.5 ml of 7.5 mg of lysozyme per milliliter in lysis buffer) was used to lyse the bacteria. Halt proteinase inhibitor cocktail (Thermo Fisher Scientific) was added to the bacterial lysate before two rounds of metal-affinity purification using TALON metal affinity resin. The affinity purified DprE1_sm fraction was eluted using lysis buffer containing up to 500 mM imidazole. DprE1_sm was then dialyzed into Hepes buffer [25 mM Hepes and 10% glycerol (pH 7.4)] and stored at −80°C. Tag cleavage was achieved by overnight incubation with SUMO protease (Thermo Fisher Scientific), followed by a third-round affinity purification to remove both His₆-SUMO tag and SUMO protease.