title: Pertussis holotoxoid formed in vitro with a genetically deactivated S1 subunit.
abstract: The cytotoxicity of pertussis toxin, a multisubunit exotoxin produced by Bordetella pertussis, is believed to be due to the ADP-ribosyltransferase activity of the S1 subunit. We have previously described the recombinant expression of each of the five individual pertussis toxin subunits in Escherichia coli and the production of an enzymatically deficient form of the S1 subunit by site-directed mutagenesis. We now report the in vitro assembly of holotoxin from native pertussis toxin B oligomer and recombinant S1 subunits, the latter purified and refolded from insoluble inclusion bodies. Holotoxin assembled with recombinant S1 of authentic amino acid sequence was indistinguishable from native pertussis toxin in its electrophoretic migration and ability to elicit a cytopathic response in cultured Chinese hamster ovary cells; in contrast, holotoxin assembled with the genetically deactivated analog of recombinant S1 displayed greatly diminished cytopathicity. These results verify that the in vitro cytopathic effects of pertussis toxin are the result of the enzymatic activity of the S1 subunit and illustrate the potential for constructing complex quaternary protein structures in vitro from insoluble, unfolded polypeptides derived from expression in recombinant systems.
authors.affiliation: Amgen Inc., Thousand Oaks, CA 91320.
authors.forename: T D
authors.initials: TD
authors.lastname: Bartley
authors.forename: D W
authors.initials: DW
authors.lastname: Whiteley
authors.forename: V L
authors.initials: VL
authors.lastname: Mar
authors.forename: D L
authors.initials: DL
authors.lastname: Burns
authors.forename: W N
authors.initials: WN
authors.lastname: Burnette
mesh_terms: D000818:Animals; D001120:Arginine; D001886:Bordetella pertussis / Q000235:genetics*; D002449:Cell Aggregation / Q000187:drug effects; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D005798:Genes, Bacterial; D008239:Lysine; D046911:Macromolecular Substances; D009154:Mutation; D037342:Pertussis Toxin; D013329:Structure-Activity Relationship; D010566:Virulence Factors, Bordetella / Q000235:genetics* / Q000494:pharmacology
chemical_list: D046911:Macromolecular Substances; D010566:Virulence Factors, Bordetella; D001120:Arginine; D037342:Pertussis Toxin; D008239:Lysine