title: Association between diphtheria toxin A- and B-fragment and their fusion proteins.
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.
authors.affiliation: Institute for Cancer Research, Norwegian Radium Hospital, Oslo.
authors.forename: H
authors.initials: H
authors.lastname: Stenmark
authors.forename: B N
authors.initials: BN
authors.lastname: Afanasiev
authors.forename: S
authors.initials: S
authors.lastname: Ariansen
authors.forename: S
authors.initials: S
authors.lastname: Olsnes
mesh_terms: D000595:Amino Acid Sequence; D016632:Apolipoprotein A-I / Q000737:chemistry / Q000235:genetics; D001692:Biological Transport; D002462:Cell Membrane / Q000378:metabolism; D004167:Diphtheria Toxin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004220:Disulfides / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010455:Peptides / Q000737:chemistry / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013205:Staphylococcal Protein A / Q000737:chemistry / Q000235:genetics; D013762:Tetrahydrofolate Dehydrogenase / Q000737:chemistry / Q000235:genetics
chemical_list: D016632:Apolipoprotein A-I; D004167:Diphtheria Toxin; D004220:Disulfides; D010446:Peptide Fragments; D010455:Peptides; D011993:Recombinant Fusion Proteins; D013205:Staphylococcal Protein A; C029795:diphtheria toxin fragment A; C039171:diphtheria toxin fragment B; D013762:Tetrahydrofolate Dehydrogenase