Production and purification of a recombinant Staphylococcal enterotoxin B vaccine candidate expressed in Escherichia coli.
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.
Coffman
J Daniel
JD
Biopharmaceutical Development Program, SAIC Frederick, National Cancer Institute at Frederick, Maryland 21702-1201, USA.
Zhu
Jianwei
J
Roach
John M
JM
Bavari
Sina
S
Ulrich
Robert G
RG
Giardina
Steven L
SL
D000942:Antigens, Bacterial / Q000096:biosynthesis* / Q000276:immunology / Q000302:isolation & purification; D002845:Chromatography; D004768:Enterotoxins / Q000096:biosynthesis* / Q000276:immunology / Q000302:isolation & purification; D004926:Escherichia coli; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000276:immunology / Q000302:isolation & purification; D013209:Staphylococcal Vaccines
D000942:Antigens, Bacterial; D004768:Enterotoxins; D011994:Recombinant Proteins; D013209:Staphylococcal Vaccines; C031818:enterotoxin B, staphylococcal