Production and purification of a recombinant Staphylococcal enterotoxin B vaccine candidate expressed in Escherichia coli

Protein Expr Purif. 2002 Mar;24(2):302-12. doi: 10.1006/prep.2001.1556.

Abstract

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.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Antigens, Bacterial / biosynthesis*
  • Antigens, Bacterial / immunology
  • Antigens, Bacterial / isolation & purification
  • Chromatography
  • Enterotoxins / biosynthesis*
  • Enterotoxins / immunology
  • Enterotoxins / isolation & purification
  • Escherichia coli
  • Mutagenesis, Site-Directed
  • Recombinant Proteins / biosynthesis*
  • Recombinant Proteins / immunology
  • Recombinant Proteins / isolation & purification
  • Staphylococcal Vaccines*

Substances

  • Antigens, Bacterial
  • Enterotoxins
  • Recombinant Proteins
  • Staphylococcal Vaccines
  • enterotoxin B, staphylococcal