For expression of PFO/D4-GFP, the plasmid pRT10
22 was freshly transformed to expression competent cells (BL21 Star [DE3]pLysS One Shot Chemically Competent
E. coli ; Invitrogen, Carlsbad, CA, USA) resulting in
E. coli BL21 PFO/D4-GFP. Transformed cells were grown on LB-Agar containing 100 μg/mL ampicillin for selection. A fresh overnight culture of
E. coli BL21 PFO/D4-GFP in LB with 100 μg/mL ampicillin (LB
amp) was diluted in 1 L of LB
amp with a final optical density (OD
600) of 0.05. Cells were cultured at 37°C overnight and PFO/D4-GFP expression was induced by IPTG and arabinose after 4 hours of incubation. Cells were purified and resuspended in PBS buffer containing 1 mM EDTA and protease inhibitors (Complete EDTA free, protease inhibitor cocktail tablets; Roche, Mannheim, Germany).
E. coli BL21 PFO/D4-GFP cells were lysed by ultrasonication for 1 minute. The lysate was mixed with glutathion agarose/beads (Sigma-Aldrich Corp., Taufkirchen, Germany) and incubated overnight at 4°C under mild shaking. Glutathion agarose/beads were harvested and washed three times with PBS buffer. Perfringolysin O/D4-GFP was eluated with 10 mM reduced glutathion and DTT was added. The PFO/D4-GFP eluate was enriched using a filter unit (Amicon Ultra 4, Centrifugal Filter Devices; Millipore Corp., Schwalbach, Germany). Protein concentration was determined using a Bradford assay (Quick Start Bradford, Dye; Bio-Rad, Hercules, CA, USA).