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. 1992 Feb 1;281(Pt 3):619–625. doi: 10.1042/bj2810619

Association between diphtheria toxin A- and B-fragment and their fusion proteins.

H Stenmark 1, B N Afanasiev 1, S Ariansen 1, S Olsnes 1
PMCID: PMC1130734  PMID: 1536643

Abstract

Natural diphtheria toxin is synthesized as a single polypeptide chain that is activated by cleavage into an A- and a B-fragment, which are linked by a disulphide bond. In the present work the ability of independently translated A- and B-fragments to associate was investigated. Low amounts of A- and B-fragments synthesized in vitro were mixed under conditions that allowed formation of a disulphide bridge between the fragments. Under these conditions toxin was reconstituted in close to 100% yield and found to be as toxic to Vero cells as natural diphtheria toxin. Efficient association between the A- and B-fragment was dependent on the formation of a disulphide bridge. Reconstituted toxin obtained from one [35S]methionine-labelled fragment and one unlabelled fragment proved useful in translocation studies. Addition of a number of different polypeptides to the N- and C-termini of either fragment did not, in most cases, prevent reconstitution. The ready reconstitution allows easy manipulations with the toxin to form targeted molecules and to develop diphtheria toxin as a vector for translocation of peptides to the cytosol. The fact that the reconstituted toxin does not need to be nicked with proteinases to be active allows experimentation with proteinase-sensitive constructs.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

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