Expression of a biologically active diphtheria toxin fragment B in Escherichia coli.
The toxB gene of Corynebacterium diphtheriae bacteriophage beta encoding the B fragment of diphtheria toxin was cloned into an inducible expression vector. When expressed in Escherichia coli, fragment B was not proteolysed and was indistinguishable, by immunological criteria, from wild-type C. diphtheriae-derived fragment B. Soluble fragment B was partially purified from the cytoplasm by saline precipitation steps and was shown to compete with the wild-type diphtheria toxin for binding to receptors of sensitive eukaryotic cells. A complete diphtheria toxin was reconstituted by formation of the disulphide bridge between purified fragment A and recombinant fragment B, which migrates at the expected Mr on Western blots and which was able to block protein synthesis by ADP-ribosylation of elongation factor-2, thereby indicating that the recombinant fragment B had retained its biological activity.
D000818:Animals; D001667:Binding, Competitive; D003001:Cloning, Molecular; D003353:Corynebacterium diphtheriae / Q000235:genetics; D004167:Diphtheria Toxin / Q000138:chemical synthesis / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism / Q000633:toxicity; D004926:Escherichia coli / Q000235:genetics*; D005798:Genes, Bacterial; D005822:Genetic Vectors; D066257:Heparin-binding EGF-like Growth Factor; D036341:Intercellular Signaling Peptides and Proteins; D010446:Peptide Fragments / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D010957:Plasmids; D011956:Receptors, Cell Surface; D011950:Receptors, Cholinergic / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D014709:Vero Cells
D004167:Diphtheria Toxin; D066257:Heparin-binding EGF-like Growth Factor; D036341:Intercellular Signaling Peptides and Proteins; D010446:Peptide Fragments; D011956:Receptors, Cell Surface; D011950:Receptors, Cholinergic; D011994:Recombinant Proteins; C029795:diphtheria toxin fragment A; C039171:diphtheria toxin fragment B