Skip to main content
NCBI home page
Infection and Immunity logoLink to Infection and Immunity
. 1991 May;59(5):1627–1632. doi: 10.1128/iai.59.5.1627-1632.1991

Synthesis of tetanus toxin fragment C in insect cells by use of a baculovirus expression system.

I G Charles 1, B C Rodgers 1, A J Makoff 1, S N Chatfield 1, D E Slater 1, N F Fairweather 1
PMCID: PMC257894  PMID: 2019434

Abstract

The baculovirus expression vector p36C was used to express in cells of the insect Spodoptera frugiperda fragment C of tetanus toxin under the control of the strong polyhedrin promoter. Fragment C was expressed intracellularly at a high level and was soluble, allowing it to be purified by affinity chromatography with monoclonal antibody TT08. Purified fragment C from baculovirus was used to immunize mice and was shown to successfully prevent the symptoms of tetanus following a toxin challenge. The ganglioside-binding properties of baculovirus-derived fragment C were compared with those of intact tetanus toxin and native fragment C and were found to be dissimilar.

Full text

PDF
1627

Images in this article

1630Image
on p.1630
1630Image
on p.1630

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Ahnert-Hilger G., Weller U., Dauzenroth M. E., Habermann E., Gratzl M. The tetanus toxin light chain inhibits exocytosis. FEBS Lett. 1989 Jan 2;242(2):245–248. doi: 10.1016/0014-5793(89)80478-8. [DOI] [PubMed] [Google Scholar]
  2. Angus P. W., Mihaly G. W., Morgan D. J., Smallwood R. A. Oxygen dependence of omeprazole clearance and sulfone and sulfide metabolite formation in the isolated perfused rat liver. J Pharmacol Exp Ther. 1989 Sep;250(3):1043–1047. [PubMed] [Google Scholar]
  3. Birnboim H. C., Doly J. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 1979 Nov 24;7(6):1513–1523. doi: 10.1093/nar/7.6.1513. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bizzini B. Tetanus toxin. Microbiol Rev. 1979 Jun;43(2):224–240. doi: 10.1128/mr.43.2.224-240.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Boquet P., Duflot E. Tetanus toxin fragment forms channels in lipid vesicles at low pH. Proc Natl Acad Sci U S A. 1982 Dec;79(24):7614–7618. doi: 10.1073/pnas.79.24.7614. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Charles I. G., Dougan G., Pickard D., Chatfield S., Smith M., Novotny P., Morrissey P., Fairweather N. F. Molecular cloning and characterization of protective outer membrane protein P.69 from Bordetella pertussis. Proc Natl Acad Sci U S A. 1989 May;86(10):3554–3558. doi: 10.1073/pnas.86.10.3554. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Eisel U., Jarausch W., Goretzki K., Henschen A., Engels J., Weller U., Hudel M., Habermann E., Niemann H. Tetanus toxin: primary structure, expression in E. coli, and homology with botulinum toxins. EMBO J. 1986 Oct;5(10):2495–2502. doi: 10.1002/j.1460-2075.1986.tb04527.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Fairweather N. F., Lyness V. A., Maskell D. J. Immunization of mice against tetanus with fragments of tetanus toxin synthesized in Escherichia coli. Infect Immun. 1987 Nov;55(11):2541–2545. doi: 10.1128/iai.55.11.2541-2545.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Fairweather N. F., Lyness V. A. The complete nucleotide sequence of tetanus toxin. Nucleic Acids Res. 1986 Oct 10;14(19):7809–7812. doi: 10.1093/nar/14.19.7809. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Habermann E., Dreyer F. Clostridial neurotoxins: handling and action at the cellular and molecular level. Curr Top Microbiol Immunol. 1986;129:93–179. doi: 10.1007/978-3-642-71399-6_2. [DOI] [PubMed] [Google Scholar]
  11. Halpern J. L., Habig W. H., Neale E. A., Stibitz S. Cloning and expression of functional fragment C of tetanus toxin. Infect Immun. 1990 Apr;58(4):1004–1009. doi: 10.1128/iai.58.4.1004-1009.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Helting T. B., Nau H. H. Analysis of the immune response to papain digestion products of tetanus toxin. Acta Pathol Microbiol Immunol Scand C. 1984 Feb;92(1):59–63. doi: 10.1111/j.1699-0463.1984.tb00052.x. [DOI] [PubMed] [Google Scholar]
  13. Helting T. B., Parschat S., Engelhardt H. Structure of tetanus toxin. Demonstration and separation of a specific enzyme converting intracellular tetanus toxin to the extracellular form. J Biol Chem. 1979 Nov 10;254(21):10728–10733. [PubMed] [Google Scholar]
  14. Helting T. B., Zwisler O. Structure of tetanus toxin. I. Breakdown of the toxin molecule and discrimination between polypeptide fragments. J Biol Chem. 1977 Jan 10;252(1):187–193. [PubMed] [Google Scholar]
  15. Helting T. B., Zwisler O., Wiegandt H. Structure of tetanus toxin. II. Toxin binding to ganglioside. J Biol Chem. 1977 Jan 10;252(1):194–198. [PubMed] [Google Scholar]
  16. Iacono-Connors L. C., Schmaljohn C. S., Dalrymple J. M. Expression of the Bacillus anthracis protective antigen gene by baculovirus and vaccinia virus recombinants. Infect Immun. 1990 Feb;58(2):366–372. doi: 10.1128/iai.58.2.366-372.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Kagan B. L., Finkelstein A., Colombini M. Diphtheria toxin fragment forms large pores in phospholipid bilayer membranes. Proc Natl Acad Sci U S A. 1981 Aug;78(8):4950–4954. doi: 10.1073/pnas.78.8.4950. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Kang C. Y. Baculovirus vectors for expression of foreign genes. Adv Virus Res. 1988;35:177–192. doi: 10.1016/s0065-3527(08)60711-3. [DOI] [PubMed] [Google Scholar]
  19. Kozak M. At least six nucleotides preceding the AUG initiator codon enhance translation in mammalian cells. J Mol Biol. 1987 Aug 20;196(4):947–950. doi: 10.1016/0022-2836(87)90418-9. [DOI] [PubMed] [Google Scholar]
  20. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  21. Makoff A. J., Oxer M. D., Romanos M. A., Fairweather N. F., Ballantine S. Expression of tetanus toxin fragment C in E. coli: high level expression by removing rare codons. Nucleic Acids Res. 1989 Dec 25;17(24):10191–10202. doi: 10.1093/nar/17.24.10191. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Matsuda M., Yoneda M. Isolation and purification of two antigenically active, "complimentary" polypeptide fragments of tetanus neurotoxin. Infect Immun. 1975 Nov;12(5):1147–1153. doi: 10.1128/iai.12.5.1147-1153.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Matsuura Y., Possee R. D., Overton H. A., Bishop D. H. Baculovirus expression vectors: the requirements for high level expression of proteins, including glycoproteins. J Gen Virol. 1987 May;68(Pt 5):1233–1250. doi: 10.1099/0022-1317-68-5-1233. [DOI] [PubMed] [Google Scholar]
  24. Miller L. K. Baculoviruses as gene expression vectors. Annu Rev Microbiol. 1988;42:177–199. doi: 10.1146/annurev.mi.42.100188.001141. [DOI] [PubMed] [Google Scholar]
  25. Page M. J. p36C: an improved baculovirus expression vector for producing high levels of mature recombinant proteins. Nucleic Acids Res. 1989 Jan 11;17(1):454–454. doi: 10.1093/nar/17.1.454. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Penner R., Neher E., Dreyer F. Intracellularly injected tetanus toxin inhibits exocytosis in bovine adrenal chromaffin cells. Nature. 1986 Nov 6;324(6092):76–78. doi: 10.1038/324076a0. [DOI] [PubMed] [Google Scholar]
  27. Pennock G. D., Shoemaker C., Miller L. K. Strong and regulated expression of Escherichia coli beta-galactosidase in insect cells with a baculovirus vector. Mol Cell Biol. 1984 Mar;4(3):399–406. doi: 10.1128/mcb.4.3.399. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Sheppard A. J., Cussell D., Hughes M. Production and characterization of monoclonal antibodies to tetanus toxin. Infect Immun. 1984 Feb;43(2):710–714. doi: 10.1128/iai.43.2.710-714.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. van Heyningen S. Binding of ganglioside by the chains of tetanus toxin. FEBS Lett. 1976 Sep 15;68(1):5–7. doi: 10.1016/0014-5793(76)80391-2. [DOI] [PubMed] [Google Scholar]

Articles from Infection and Immunity are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES