Year

1997

Degree Name

Doctor of Philosophy

Department

Department of Biological Sciences

Abstract

Bordetella pertussis is the causative agent of whooping cough, a severe disease of infants characterised by repeated bouts of paroxysmal coughing. Pertussis toxin (PT) is a major virulence factor of B. pertussis 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 organised into an operon which is not expressed in Escherichia coli, thus hampering the use of this organism for vaccine production. In this research the five PT subunits were individually expressed in E. coli by replacing the wild-type transcriptional and translational signals, and in the case of the S4 subunit the leader peptide has been exchanged with a modified E. coli 6-lactamase leader sequence. In E. coli CAG629 all five PT subunits were at least partially processed, with S5 being completely processed. A stepwise cloning method has then been developed to construct a synthetic PT operon which simultaneously expresses the five PT subunits in E. coli. Western blot analysis indicated that in E. coli KS476 containing the synthetic PT operon, S4 and S5 were completely processed, SI was partially processed, whilst the majority of S2 and S3 remained unprocessed. Periplasmic extracts contained soluble SI and S3; however the processed form of S2, S4 and S5 were not detected, suggesting that these subunits may be membrane associated or in an insoluble form.

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