Purification of Recombinant Proteins from E. coli

rCI, rNTD and His-σ^A (primary sigma factor of S. aureus with a histidine tag) were purified from E. coli strains SAU1304, SAU1315, and SAU1283, respectively, using standard methods [7] (link), [37] (link). Briefly, IPTG-induced cells were ruptured in buffer B [20 mM Tris-HCl (pH 8.0), 300 mM NaCl, 10 mM imidazole, 5% glycerol and 10 µg/ml PMSF], followed by purification of the polyhistidine-tagged recombinant protein (rCI or rNTD or His-σ^A) from the crude extract by Ni-NTA column chromatography (Qiagen). The eluted proteins were dialyzed against buffer C [10 mM Tris-HCl (pH 8.0), 200 mM NaCl, 1 mM EDTA, and 5% glycerol] for 12–16 h at 4°C. All proteins in buffer C were stored on ice until use. The molar concentrations of the recombinant proteins were determined using the molecular masses of their respective monomeric forms.