title: Stable expression of pertussis toxin in Bordetella bronchiseptica under the control of a tightly regulated promoter.
abstract: Pertussis toxin (PT) is an essential component of accellular vaccines against whooping cough. However, the industrial production of PT from Bordetella pertussis is impaired by slow growth and poor yields. To overcome these problems, we have constructed a minitransposon containing the tox operon under the control of a tightly regulated promoter responsive to an aromatic inducer. The expression cassettes have been integrated into the chromosome of Bordetella bronchiseptica 5376 and ATCC 10580 bvg. Five recombinant clones containing the tox operon under the control of the Psal promoter, which is activated by the product of nahR, were further characterized. The recombinant clones expressed PT after only 3 h of induction with sodium salicylate at levels similar to those of B. pertussis grown for 24 h. The stability of the engineered phenotype was 100% after 72 h of growth without selective pressure. The growth pattern was not modified either under noninducing conditions or in the presence of the inducer at low concentrations, suggesting that strain performance would not be affected in bioreactors when uncoupled from gene expression. Recombinant PT, which was localized mainly in the periplasm, was purified by affinity chromatography. The recombinant protein was immunologically indistinguishable from wild-type PT and retained its biological activity as determined by the CHO cell-clustering test. These recombinant clones appear to be useful tools for the cost-effective production of PT under conditions of improved biosafety, as demonstrated by the inducible expression of PT uncoupled from the bacterial biomass in a nonvirulent and fast-growing B. bronchiseptica background.
authors.affiliation: Division of Microbiology, GBF-National Research Centre for Biotechnology, Braunschweig, Germany.
authors.forename: A
authors.initials: A
authors.lastname: Suarez
authors.forename: L H
authors.initials: LH
authors.lastname: Staendner
authors.forename: M
authors.initials: M
authors.lastname: Rohde
authors.forename: G
authors.initials: G
authors.lastname: Piatti
authors.forename: K N
authors.initials: KN
authors.lastname: Timmis
authors.forename: C A
authors.initials: CA
authors.lastname: Guzmán
mesh_terms: D000818:Animals; D001483:Base Sequence; D016950:Bordetella bronchiseptica / Q000235:genetics* / Q000378:metabolism / Q000648:ultrastructure; D016466:CHO Cells; D006224:Cricetinae; D004269:DNA, Bacterial / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial; D006801:Humans; D016253:Microscopy, Immunoelectron; D009876:Operon; D037342:Pertussis Toxin; D010567:Pertussis Vaccine / Q000235:genetics / Q000302:isolation & purification; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D015183:Restriction Mapping; D014614:Vaccines, Synthetic / Q000235:genetics / Q000302:isolation & purification; D010566:Virulence Factors, Bordetella / Q000096:biosynthesis / Q000235:genetics*
chemical_list: D004269:DNA, Bacterial; D010567:Pertussis Vaccine; D011994:Recombinant Proteins; D014614:Vaccines, Synthetic; D010566:Virulence Factors, Bordetella; D037342:Pertussis Toxin