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. 1987 Apr;55(4):963–967. doi: 10.1128/iai.55.4.963-967.1987

Expression and immunological properties of the five subunits of pertussis toxin.

A Nicosia, A Bartoloni, M Perugini, R Rappuoli
PMCID: PMC260445  PMID: 3549567

Abstract

Pertussis toxin, a protein composed of five different subunits, is responsible for the pathogenicity of Bordetella pertussis and is the main component of a new vaccine against whooping cough. The genes coding for the five subunits, recently cloned and sequenced, are organized as an operon. We approached the problem of expression of the five genes in Escherichia coli and, although we obtained high levels of transcription of the native pertussis toxin genes, the amount of proteins produced was very low or undetectable. To obtain suitable expression of each of the five subunits, we fused their genes to the gene coding for the DNA polymerase of MS2 in the expression vector pEx31. A total of 5 to 30 mg of purified fusion proteins could be obtained from 1 liter of culture. The purified fusion proteins were used to immunize rabbits to obtain sera against each of the five subunits. These sera, although able to recognize the toxin in an enzyme-linked immunosorbent assay and the corresponding subunits in Western blots, were not able to protect CHO cells from the action of pertussis toxin. Mice immunized with the five subunits were not protected from an intracerebral challenge with B. pertussis. Subunits S2 and S3, which are 67% homologous, were shown to cross-react immunologically. The fused subunit S1 was able to ADP-ribosylate transducin as efficiently as the native pertussis toxin.

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Selected References

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  1. Burnette W. N. "Western blotting": electrophoretic transfer of proteins from sodium dodecyl sulfate--polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A. Anal Biochem. 1981 Apr;112(2):195–203. doi: 10.1016/0003-2697(81)90281-5. [DOI] [PubMed] [Google Scholar]
  2. Cryz S. J., Welkos S. L., Holmes R. K. Immunochemical studies of diphtherial toxin and related nontoxic mutant proteins. Infect Immun. 1980 Dec;30(3):835–846. doi: 10.1128/iai.30.3.835-846.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Hewlett E. L., Sauer K. T., Myers G. A., Cowell J. L., Guerrant R. L. Induction of a novel morphological response in Chinese hamster ovary cells by pertussis toxin. Infect Immun. 1983 Jun;40(3):1198–1203. doi: 10.1128/iai.40.3.1198-1203.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Hsia J. A., Moss J., Hewlett E. L., Vaughan M. ADP-ribosylation of adenylate cyclase by pertussis toxin. Effects on inhibitory agonist binding. J Biol Chem. 1984 Jan 25;259(2):1086–1090. [PubMed] [Google Scholar]
  5. Katada T., Ui M. Direct modification of the membrane adenylate cyclase system by islet-activating protein due to ADP-ribosylation of a membrane protein. Proc Natl Acad Sci U S A. 1982 May;79(10):3129–3133. doi: 10.1073/pnas.79.10.3129. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Klinkert M. Q., Herrmann R., Schaller H. Surface proteins of Mycoplasma hyopneumoniae identified from an Escherichia coli expression plasmid library. Infect Immun. 1985 Aug;49(2):329–335. doi: 10.1128/iai.49.2.329-335.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Locht C., Keith J. M. Pertussis toxin gene: nucleotide sequence and genetic organization. Science. 1986 Jun 6;232(4755):1258–1264. doi: 10.1126/science.3704651. [DOI] [PubMed] [Google Scholar]
  8. Manning D. R., Fraser B. A., Kahn R. A., Gilman A. G. ADP-ribosylation of transducin by islet-activation protein. Identification of asparagine as the site of ADP-ribosylation. J Biol Chem. 1984 Jan 25;259(2):749–756. [PubMed] [Google Scholar]
  9. Munoz J. J., Arai H., Cole R. L. Mouse-protecting and histamine-sensitizing activities of pertussigen and fimbrial hemagglutinin from Bordetella pertussis. Infect Immun. 1981 Apr;32(1):243–250. doi: 10.1128/iai.32.1.243-250.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Nicosia A., Perugini M., Franzini C., Casagli M. C., Borri M. G., Antoni G., Almoni M., Neri P., Ratti G., Rappuoli R. Cloning and sequencing of the pertussis toxin genes: operon structure and gene duplication. Proc Natl Acad Sci U S A. 1986 Jul;83(13):4631–4635. doi: 10.1073/pnas.83.13.4631. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Pappenheimer A. M., Jr, Uchida T., Harper A. A. An immunological study of the diphtheria toxin molecule. Immunochemistry. 1972 Sep;9(9):891–906. doi: 10.1016/0019-2791(72)90163-2. [DOI] [PubMed] [Google Scholar]
  12. Remaut E., Stanssens P., Fiers W. Plasmid vectors for high-efficiency expression controlled by the PL promoter of coliphage lambda. Gene. 1981 Oct;15(1):81–93. doi: 10.1016/0378-1119(81)90106-2. [DOI] [PubMed] [Google Scholar]
  13. Sato H., Ito A., Chiba J., Sato Y. Monoclonal antibody against pertussis toxin: effect on toxin activity and pertussis infections. Infect Immun. 1984 Nov;46(2):422–428. doi: 10.1128/iai.46.2.422-428.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Sato Y., Kimura M., Fukumi H. Development of a pertussis component vaccine in Japan. Lancet. 1984 Jan 21;1(8369):122–126. doi: 10.1016/s0140-6736(84)90061-8. [DOI] [PubMed] [Google Scholar]
  15. Strebel K., Beck E., Strohmaier K., Schaller H. Characterization of foot-and-mouth disease virus gene products with antisera against bacterially synthesized fusion proteins. J Virol. 1986 Mar;57(3):983–991. doi: 10.1128/jvi.57.3.983-991.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Tamura M., Nogimori K., Murai S., Yajima M., Ito K., Katada T., Ui M., Ishii S. Subunit structure of islet-activating protein, pertussis toxin, in conformity with the A-B model. Biochemistry. 1982 Oct 26;21(22):5516–5522. doi: 10.1021/bi00265a021. [DOI] [PubMed] [Google Scholar]
  17. Tuomanen E., Weiss A. Characterization of two adhesins of Bordetella pertussis for human ciliated respiratory-epithelial cells. J Infect Dis. 1985 Jul;152(1):118–125. doi: 10.1093/infdis/152.1.118. [DOI] [PubMed] [Google Scholar]
  18. Van Dop C., Yamanaka G., Steinberg F., Sekura R. D., Manclark C. R., Stryer L., Bourne H. R. ADP-ribosylation of transducin by pertussis toxin blocks the light-stimulated hydrolysis of GTP and cGMP in retinal photoreceptors. J Biol Chem. 1984 Jan 10;259(1):23–26. [PubMed] [Google Scholar]
  19. Weiss A. A., Hewlett E. L., Myers G. A., Falkow S. Pertussis toxin and extracytoplasmic adenylate cyclase as virulence factors of Bordetella pertussis. J Infect Dis. 1984 Aug;150(2):219–222. doi: 10.1093/infdis/150.2.219. [DOI] [PubMed] [Google Scholar]
  20. Zucker D. R., Murphy J. R. Monoclonal antibody analysis of diphtheria toxin--I. Localization of epitopes and neutralization of cytotoxicity. Mol Immunol. 1984 Sep;21(9):785–793. doi: 10.1016/0161-5890(84)90165-2. [DOI] [PubMed] [Google Scholar]

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