Hispur cobalt column

cDNA corresponding to MOPV‐NP was cloned by recombination (Gateway; Invitrogen) into pETG20A expression vector, which adds a cleavable N‐terminal thioredoxin‐hexahistidine tag. Protein was overexpressed in Escherichia coli strain C41(DE3) (Merck) grown in 2YT medium (Sigma‐Aldrich) at 37 °C to an OD600 _nm of 0.5. Expression was induced with 0.5 mm IPTG, and bacteria were grown shaking at 210 r.p.m. overnight at 17 °C in presence of 100 µm of ZnCl₂. Bacteria were pelleted, frozen, and stored at −80 °C. The NP was purified by affinity chromatography using 2 mL of His pur^™ cobalt column (Thermo Scientific; 20 mm Tris pH7.5, 300 NaCl, 5 mm imidazole, 0.5 m TCEP and eluted with the same buffer with 250 mm imidazole). The tag was removed by cleavage with TEV protease followed by a purification on a second cobalt affinity chromatography. Proteins were further purified by gel filtration using Superdex 75 column (GE Healthcare) in 20 mm Tris pH 7.5, 300 mm NaCl.

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).

His-tagged RnpA was purified as previously described [33 (link)]. E. coli BL21 (DE3) cells harboring plasmid pEXP5-nt containing a hexahistidine tag fused to the N-terminus of S.aureus RnpA coding region were grown in LB supplemented with 50 µg mL⁻¹ ampicillin to OD₆₀₀ = 0.6 and induced with 1mM isopropyl β-D-1-thiogalactopyranoside (IPTG) for three hours to induce protein expression. E. coli cells were subjected to centrifugation at 1560× g for 20 min at 4 °C and the cell pellet was resuspended in 20 mL buffer A (300 mM NaCl, 50 mM Na₂HPO₄, pH 7.4) containing a mini EDTA-free protease inhibitor tablet (Roche; Bradford, CT, USA) and 20 mM imidazole. Cells were mechanically lysed 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 17,000× g for 10 min at 4 °C. Supernatants were collected, filtered through a 0.2 micron syringe filter, then loaded onto a 5 mL HisPur cobalt column (ThermoFisher Scientific, Waltham, MA, U.S.A.) using the BioRad Maximizer Duo-Flow Medium Pressure Chromatography System. Protein was eluted using an imidazole gradient (80 mM to 500 mM). Fractions were assessed for RnpA presence and purity via SDS-PAGE analysis, Coomassie staining and Western blotting using an anti-His antibody (Invitrogen; Grand Island, NY, USA).