Expression of a synthetic pertussis toxin operon in Escherichia coli.
Bordetella pertussis is the causative agent of whooping cough, a severe disease of infants characterised by repeated 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 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 five PT subunits individually 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 beta-lactamase leader sequence. We have developed a stepwise cloning method 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, S1 was partially processed, whilst the majority of S2 and S3 remained unprocessed. Periplasmic extracts contained soluble S1 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. This work should allow an investigation of the potential of E. coli to produce detoxified PT in a background free of other pertussis virulence factors that may contribute to the side-effects of some vaccine preparations currently in use.
Pozza
T D
TD
Department of Biological Sciences, University of Wollongong, New South Wales, Australia.
Yan
H
H
Walker
M J
MJ
D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D003001:Cloning, Molecular; D004274:DNA, Recombinant / Q000096:biosynthesis*; D004926:Escherichia coli / Q000235:genetics*; D005813:Genes, Synthetic; D005822:Genetic Vectors / Q000138:chemical synthesis*; D008969:Molecular Sequence Data; D009876:Operon; D010452:Peptide Biosynthesis; D037342:Pertussis Toxin; D011817:Rabbits; D011994:Recombinant Proteins / Q000096:biosynthesis; D010566:Virulence Factors, Bordetella / Q000096:biosynthesis* / Q000235:genetics*
D004274:DNA, Recombinant; D011994:Recombinant Proteins; D010566:Virulence Factors, Bordetella; D037342:Pertussis Toxin