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Expression of a biologically active diphtheria toxin fragment B in Escherichia coli
Author(s) -
Cabiaux V.,
Phalipon A.,
Wattiez R.,
Falmagne P.,
Ruysschaert J. M.,
Kaczorek M.
Publication year - 1988
Publication title -
molecular microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.857
H-Index - 247
eISSN - 1365-2958
pISSN - 0950-382X
DOI - 10.1111/j.1365-2958.1988.tb00037.x
Subject(s) - biology , diphtheria toxin , recombinant dna , escherichia coli , corynebacterium diphtheriae , microbiology and biotechnology , adp ribosylation , toxin , clostridium difficile toxin b , elongation factor , bacteriophage , gene , biochemistry , clostridium difficile toxin a , diphtheria , virology , enzyme , ribosome , rna , nad+ kinase , vaccination , clostridium difficile , antibiotics
Summary 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.