Protein purification

All pET28a 6x-HisSumo constructs—including SidJ 89–853, CaM, SdeA 211–1152, and point mutants—were transformed into Escherichia coli Rosetta (DE3) cells. Single colonies were then cultured in Luria-Bertani (LB) medium containing 50 µg/ml kanamycin to a density between 0.6 and 0.8 OD₆₀₀. Cultures were induced with 0.1 mM isopropyl-B-D-thiogalactopyranoside (IPTG) at 18°C for 12 hr. Cells were collected by centrifugation at 3,500 rpm for 15 min at 4°C and sonicated to lyse bacteria. To separate insoluble cellular debris, lysates were then centrifuged at 16,000 rpm for 45 min at 4°C. The supernatant was incubated with cobalt resin (Gold Biotechnology) for 2 hr at 4°C to bind proteins and washed extensively with purification buffer [20 mM Tris (pH 7.5), 150 mM NaCl]. Proteins of interest were then digested on the resin with SUMO-specific protease Ulp1 to release the protein from the His-SUMO tag and resin. The digested protein was concentrated and purified further by fast protein liquid chromatography (FPLC) size exclusion chromatography using a Superdex S200 column (GE Life Science) in purification buffer. Pure fractions were collected and concentrated in Amicon Pro Purification system concentrators.