Nickel activated chelating sepharose column

DNA sequences of the five antigens were amplified by PCR using genomic DNA of S. aureus strain USA300. The PCR products were then cloned into pET21a or pET28a vector. Next, the recombinant plasmids were transformed into E. coli BL21(DE3). Bacterial pellets harvested from 1 liter of bacterial culture were harvested by centrifugation and resuspended in binding buffer (Tris-HCl [pH 8.0], 20 mM imidazole) followed by sonication. After centrifugation at 15,000 × g for 30 min at 4°C, the soluble cell extracts were collected and filtered using a 0.22-μm-pore-size membrane. The samples were then loaded on a nickel-activated chelating Sepharose column (GE Healthcare). After washing, the bound proteins were eluted with elution buffer (Tris-HCl [pH 8.0], 250 mM imidazole). The purified proteins were identified using SDS-PAGE analysis and subjected to endotoxin removal afterward (Pierce).

DNA sequences of KOMPs were amplified by PCR using genomic DNA of K. pneumoniae 260 strain as template. The PCR products were then cloned into pET28a vector. Next, the recombinant plasmids were transformed into E. coli BL21 strain (DE). A single transformed BL21 clone was subjected to induction with IPTG followed by testing for protein expression by SDS-PAGE analysis. Bacteria were harvested by centrifugation and resuspend in binding buffer (Tris–HCl, pH 8.0, 20 mM imidazole) followed by sonication. After centrifugation at 15,000 × g for 30 min at 4°C, the soluble cell extracts were collected and filtered using a 0.22-μm membrane. The samples were then loaded on a nickel-activated chelating Sepharose column (GE Healthcare). After washing, the bound proteins were eluted with elution buffer (Tris–HCl, pH 8.0, 250 mM imidazole). The expression of purified proteins was identified using SDS-PAGE analysis with anti-histidine antibodies. The proteins were subject to endotoxin removal afterwards (Pierce).