EXPRESSION OF A SYNTHETIC PERTUSSIS TOXIN OPERON IN ESCHERICHIA-COLI

Bordetella pertussis is the causative agent of whooping cough, a sever e disease of infants characterised by repeated bouts of paroxysmal cou ghing. Pertussis toxin (PT) is a major virulence factor of B. pertussi s and is a typical A/B bacterial toxin consisting of five subunits S1- S5 in a ratio of 1:1:1:2:1. The PT subunit genes are organized into an operon which is not expressed in Escherichia coli, thus hampering the use of this organism for vaccine production. We have expressed the Ji ve PT subunits individually, in E. coli by replacing the wild-type tra nscriptional and translational signals, and in the case of the S4 subu nit the leader peptide has been exchanged with a modified E. coli beta -lactamase lender sequence. We have developed a stepwise cloning metho d to construct a synthetic PT operon which simultaneously expresses th e Jive PT subunits in E. coli. Western blot analysis indicated that in E. coli KS476 containing the synthetic PT operon, S4 and S5 were comp letely processed, SI was partially processed whilst the majority of S2 and S3 remained unprocessed. Periplasmic extracts contained soluble S I and S3; however, the processed form of S2, S4 and S5 were not detect ed suggesting that these subunits may be membrane associated or in an insoluble form. This work should allow an investigation of the potenti al of E. coli to produce detoxified PT in a background free of other p ertussis virulence factors that may contribute to the side-effects of some vaccine preparations currently in use. (C) 1997 Elsevier Science Ltd.