An attenuated, recombinant form of Staphylococcus enterotoxin B (rSEB) was overexpressed in Escherichia coli under transcriptional control of the T7 promoter. The 28-kDa rSEB was partially purified from soluble, intracellular protein by tangential flow filtration and differential ammonium sulfate precipitation. The intermediate product was then further purified using low-pressure liquid chromatography including hydrophobic interaction, cation exchange, and size-exclusion matrices. The final vialed product was >95% pure as determined by Coomassie blue-stained sodium dodecyl sulfate-polyacrylamide gel electrophoresis, high-pressure size-exclusion chromatography, and capillary zonal electrophoresis. The endotoxin level was <0.6 EU/mg. Final estimated yield of purified rSEB was 147 mg/L of starting culture. Purified rSEB was stable, elicited an immune response in mice, and protected mice against a lethal challenge with the native toxin.
Copyright 2002 Elsevier Science (USA).