Purification of the B-subunit oligomer of Escherichia coli heat-labile enterotoxin by heterologous expression and secretion in a marine vibrio.
Heat-labile enterotoxins (Etx) are plasmid-encoded, multimeric proteins produced by certain diarrheagenic strains of Escherichia coli. The nontoxic, receptor-binding B subunit (EtxB) of such toxins may be useful as a component of vaccines against enterotoxigenic E. coli, or as a carrier for the delivery of heterologous epitopes to the mucosal immune system. Here we describe a simple method for the purification of EtxB from a marine vibrio harboring a broad-host range controlled expression vector containing the etxB gene. Induction of EtxB resulted in its specific secretion to the medium, to a concentration of greater than 25 mg/liter of culture. The techniques of ultrafiltration and hydrophobic interaction chromatography were used to purify EtxB to homogeneity from the medium of this organism (with a yield of 60.7%). EtxB-epitope fusion proteins were also successfully expressed and secreted in this marine vibrio, suggesting that this system may be of general use in the preparation of EtxB-based vaccines.
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Biological Laboratory, The University, Canterbury, Kent, United Kingdom.
Hirst
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D001427:Bacterial Toxins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D002853:Chromatography, Liquid; D017077:Culture Media, Conditioned / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D004768:Enterotoxins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics*; D029968:Escherichia coli Proteins; D015964:Gene Expression Regulation, Bacterial / Q000187:drug effects; D005822:Genetic Vectors; D007544:Isopropyl Thiogalactoside / Q000494:pharmacology; D014462:Ultrafiltration; D014733:Vibrio / Q000378:metabolism*
D001427:Bacterial Toxins; D017077:Culture Media, Conditioned; D004768:Enterotoxins; D029968:Escherichia coli Proteins; D007544:Isopropyl Thiogalactoside; C027538:heat-labile enterotoxin, E coli