Journal
PROTEIN EXPRESSION AND PURIFICATION
Volume 24, Issue 2, Pages 302-312Publisher
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1006/prep.2001.1556
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Funding
- NCI NIH HHS [N01-CO-56000] Funding Source: Medline
- OFFICE OF THE DIRECTOR, NCI [N01CO056000] Funding Source: NIH RePORTER
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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. (C) 2002 Elsevier Science (USA).
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