STABLE EXPRESSION OF PERTUSSIS TOXIN IN BORDETELLA-BRONCHISEPTICA UNDER THE CONTROL OF A TIGHTLY REGULATED PROMOTER

Pertussis toxin (PT) is an essential component of acellular vaccines a gainst whooping cough. However, the industrial production of PT from B ordetella pertussis is impaired by slow growth and poor yields. To ove rcome these problems, we have constructed a minitransposon containing the tox operon under the control of a tightly regulated promoter respo nsive to an aromatic inducer. The expression cassettes have been integ rated into the chromosome of Bordetella bronchiseptica 5376 and ATCC 1 0580 bvg. Five recombinant clones containing the tox operon under the control of the Psal promoter, which is activated by the product of nah R, were further characterized. The recombinant clones expressed PT aft er only 3 h of induction with sodium salicylate at levels similar to t hose of B. pertussis grown for 24 h. The stability of the engineered p henotype was 100% after 72 h of growth without selective pressure. The growth pattern was not modified either under noninducing conditions o r in the presence of the inducer at low concentrations, suggesting tha t strain performance would not be affected in bioreactors when uncoupl ed from gene expression. Recombinant PT, which was localized mainly in the periplasm, was purified by affinity chromatography. The recombina nt protein was immunologically indistinguishable from wild-type PT and retained its biological activity as determined by the CHO cell-cluste ring 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.