Expression of a biologically active diphtheria toxin fragment B in Escherichia coli |
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Authors: | V. Cabiaux A. Phalipon R. Wattiez P. Falmagne J. M. Ruysschaert M. Kaczorek |
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Affiliation: | Laboratoire de Chimie Physique des Macromolécules aux interfaces, CP 206/2 UniversitéLibre de Bruxelles, Boulevard du Triomphe, 1050 Bruxelles, Belgium.;Unitédes Applications de Genie Genetique, Institut Pasteur, 28 rue du Dr Roux, 75724 Parts Cedex 15, France.;Laboratoire de Chimie Biologique, Universitéde I'Etat a Mons, Avenue Maistriau 21, 7000 Mons, Belgium. |
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Abstract: | The toxB gene of Corynebacterium diphtheriae bacteriophage β 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. diphthsriae 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. |
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