Purification of Histidine-Tagged S. aureus RnpA

His-tagged S. aureus RnpA was purified as previously described [14 (link)]. Briefly, E. coli BL21 (DE3) cells harboring plasmid pEXP5-nt [36 (link)], containing a hexahistadine tag fused to the N-terminus of the S. aureus RnpA coding region under the control of the plasmid’s T7 promoter, were cultured to an OD₆₀₀ of approximately 0.6 and then induced with 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) for three hours to induce protein production. E. coli cells were collected by centrifugation at 9000 rpm for 10 min at 4 °C and then suspended in 20 mL of buffer A (300 mM NaCl, 50 mM Na₂HPO₄, pH 7.4) containing a mini EDTA-free protease inhibitor tablet (Roche; Branford, CT, USA) and 20 mM imidazole. Cells were mechanically ruptured by three passes at 18,000 psi through a French Pressure Cell Press (SLM-Aminco; Pittsford, NY, USA), and cell debris was removed by centrifugation at 4 °C at 17,000 × g for 10 min at 4 °C. Supernatants were collected, filtered through a 0.2 μm syringe filter then loaded onto the BioRad Maximizer Duo-Flow Medium Pressure Chromatography System containing a 5 mL HisPur Cobalt Column (Thermo Scientific). Protein was eluted using an imidazole gradient (80 mM to 500 mM); fractions were assessed for RnpA presence and purity on SDS-PAGE gels via Coomassie staining and Western blotting using anti-His antibody (Invitrogen).