Expression in Escherichia coli of two mutated genes encoding the cholera toxin B subunit.
To allow subsequent genetically mediated fusion of foreign antigens to cholera toxin B subunit (CTB), two mutated CTB encoding genes (ctxB) were constructed and overexpressed in Escherichia coli. The signal peptide coding sequence was deleted and restriction sites were created at both ends of the modified sequence. Both synthesized CTBs contain additional amino acid(s) at the N terminus (one and three). They were purified as insoluble products and refolded into the natural pentameric CTB structure by a denaturation-renaturation cycle. After renaturation, both recombinant proteins recovered CTB antigenicity and the ability to bind to GM1 gangliosides, as shown by in vitro analysis. Preliminary data indicated that both properties were unaltered by fusion of a foreign peptide to the mutated CTBs.
L'hoir
C
C
Laboratoire Central de Génie Génétique, Université de Liège, Belgium.
Renard
A
A
Martial
J A
JA
D001483:Base Sequence; D002772:Cholera Toxin / Q000235:genetics*; D002876:Chromosomes, Bacterial; D004269:DNA, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D005798:Genes, Bacterial; D005822:Genetic Vectors; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D009154:Mutation; D010957:Plasmids; D021382:Protein Sorting Signals / Q000235:genetics; D015183:Restriction Mapping; D014734:Vibrio cholerae / Q000235:genetics*
D004269:DNA, Bacterial; D046911:Macromolecular Substances; D021382:Protein Sorting Signals; D002772:Cholera Toxin