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Expression of a biologically active diphtheria toxin fragment B in Escherichia coli

par Cabiaux, Véronique ;Phalipon, A;Wattiez, R;Falmagne, Paul ;Ruysschaert, Jean Marie ;Kaczorek, M
Référence Molecular microbiology, 2, 3, page (339-346)
Publication Publié, 1988-05

Article révisé par les pairs

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Résumé :

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.