Skip to main content
NCBI home page
Infection and Immunity logoLink to Infection and Immunity
. 1995 Jan;63(1):301–308. doi: 10.1128/iai.63.1.301-308.1995

Expression and purification of Shiga-like toxin II B subunits.

D W Acheson 1, S A De Breucker 1, M Jacewicz 1, L L Lincicome 1, A Donohue-Rolfe 1, A V Kane 1, G T Keusch 1
PMCID: PMC172992  PMID: 7806370

Abstract

Shiga-like toxins (SLTs), which are produced by certain strains of Escherichia coli, are composed of enzymatically active A and B subunit multimers responsible for the toxin's binding. We have previously purified large amounts of the SLT-I B subunit by using a hyperexpression vector in Vibrio cholerae under the control of the trc promoter. In this study we examined various expression vectors to maximize yields of the SLT-II B subunit. The SLT-II B subunit has been expressed by using both the T7 promoter and the tac promoter in E. coli. When expressed from a plasmid containing the structural gene for SLT-II B deleted of the leader sequence, SLT-II B was able to form multimers when cross-linked, although SLT-II B production from this plasmid was unreproducible. SLT-II B expressed in all three systems appeared to form unstable multimers, which did not readily bind to a monoclonal antibody which preferentially recognizes B subunit multimers. SLT-II B expression was not increased by moving any of the plasmids into V. cholerae. Polyclonal antibodies raised to SLT-II B in rabbits recognized B subunit in SLT-II holotoxin yet were poorly neutralizing. SLT-II B was also expressed as a fusion protein with maltose-binding protein and could be cleaved from maltose-binding protein with factor Xa. Although the expression vectors were able to make large amounts of SLT-II B, as determined by Western blotting (immunoblotting), the levels of purified SLT-II B subunit were low compared with those obtained previously for SLT-I B subunit, probably because of instability of the multimeric SLT-II B subunit.

Full Text

The Full Text of this article is available as a PDF (270.9 KB).

Selected References

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

  1. Acheson D. W., Calderwood S. B., Boyko S. A., Lincicome L. L., Kane A. V., Donohue-Rolfe A., Keusch G. T. Comparison of Shiga-like toxin I B-subunit expression and localization in Escherichia coli and Vibrio cholerae by using trc or iron-regulated promoter systems. Infect Immun. 1993 Mar;61(3):1098–1104. doi: 10.1128/iai.61.3.1098-1104.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Acheson D. W., Jacewicz M., Kane A. V., Donohue-Rolfe A., Keusch G. T. One step high yield affinity purification of shiga-like toxin II variants and quantitation using enzyme linked immunosorbent assays. Microb Pathog. 1993 Jan;14(1):57–66. doi: 10.1006/mpat.1993.1006. [DOI] [PubMed] [Google Scholar]
  3. Calderwood S. B., Acheson D. W., Goldberg M. B., Boyko S. A., Donohue-Rolfe A. A system for production and rapid purification of large amounts of the Shiga toxin/Shiga-like toxin I B subunit. Infect Immun. 1990 Sep;58(9):2977–2982. doi: 10.1128/iai.58.9.2977-2982.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Donohue-Rolfe A., Acheson D. W., Kane A. V., Keusch G. T. Purification of Shiga toxin and Shiga-like toxins I and II by receptor analog affinity chromatography with immobilized P1 glycoprotein and production of cross-reactive monoclonal antibodies. Infect Immun. 1989 Dec;57(12):3888–3893. doi: 10.1128/iai.57.12.3888-3893.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Donohue-Rolfe A., Keusch G. T., Edson C., Thorley-Lawson D., Jacewicz M. Pathogenesis of Shigella diarrhea. IX. Simplified high yield purification of Shigella toxin and characterization of subunit composition and function by the use of subunit-specific monoclonal and polyclonal antibodies. J Exp Med. 1984 Dec 1;160(6):1767–1781. doi: 10.1084/jem.160.6.1767. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Endo Y., Tsurugi K., Yutsudo T., Takeda Y., Ogasawara T., Igarashi K. Site of action of a Vero toxin (VT2) from Escherichia coli O157:H7 and of Shiga toxin on eukaryotic ribosomes. RNA N-glycosidase activity of the toxins. Eur J Biochem. 1988 Jan 15;171(1-2):45–50. doi: 10.1111/j.1432-1033.1988.tb13756.x. [DOI] [PubMed] [Google Scholar]
  7. Gunzer F., Karch H. Expression of A and B subunits of Shiga-like toxin II as fusions with glutathione S-transferase and their potential for use in seroepidemiology. J Clin Microbiol. 1993 Oct;31(10):2604–2610. doi: 10.1128/jcm.31.10.2604-2610.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Jacewicz M., Clausen H., Nudelman E., Donohue-Rolfe A., Keusch G. T. Pathogenesis of shigella diarrhea. XI. Isolation of a shigella toxin-binding glycolipid from rabbit jejunum and HeLa cells and its identification as globotriaosylceramide. J Exp Med. 1986 Jun 1;163(6):1391–1404. doi: 10.1084/jem.163.6.1391. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Karmali M. A. Infection by verocytotoxin-producing Escherichia coli. Clin Microbiol Rev. 1989 Jan;2(1):15–38. doi: 10.1128/cmr.2.1.15. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Keusch G. T., Donohue-Rolfe A., Jacewicz M., Kane A. V. Shiga toxin: production and purification. Methods Enzymol. 1988;165:152-62, 399-401. doi: 10.1016/s0076-6879(88)65025-7. [DOI] [PubMed] [Google Scholar]
  11. Lopilato J., Bortner S., Beckwith J. Mutations in a new chromosomal gene of Escherichia coli K-12, pcnB, reduce plasmid copy number of pBR322 and its derivatives. Mol Gen Genet. 1986 Nov;205(2):285–290. doi: 10.1007/BF00430440. [DOI] [PubMed] [Google Scholar]
  12. O'Brien A. D., Newland J. W., Miller S. F., Holmes R. K., Smith H. W., Formal S. B. Shiga-like toxin-converting phages from Escherichia coli strains that cause hemorrhagic colitis or infantile diarrhea. Science. 1984 Nov 9;226(4675):694–696. doi: 10.1126/science.6387911. [DOI] [PubMed] [Google Scholar]
  13. Ostroff S. M., Tarr P. I., Neill M. A., Lewis J. H., Hargrett-Bean N., Kobayashi J. M. Toxin genotypes and plasmid profiles as determinants of systemic sequelae in Escherichia coli O157:H7 infections. J Infect Dis. 1989 Dec;160(6):994–998. doi: 10.1093/infdis/160.6.994. [DOI] [PubMed] [Google Scholar]
  14. Pugsley A. P. The complete general secretory pathway in gram-negative bacteria. Microbiol Rev. 1993 Mar;57(1):50–108. doi: 10.1128/mr.57.1.50-108.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Ramotar K., Boyd B., Tyrrell G., Gariepy J., Lingwood C., Brunton J. Characterization of Shiga-like toxin I B subunit purified from overproducing clones of the SLT-I B cistron. Biochem J. 1990 Dec 15;272(3):805–811. doi: 10.1042/bj2720805. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Samuel J. E., Perera L. P., Ward S., O'Brien A. D., Ginsburg V., Krivan H. C. Comparison of the glycolipid receptor specificities of Shiga-like toxin type II and Shiga-like toxin type II variants. Infect Immun. 1990 Mar;58(3):611–618. doi: 10.1128/iai.58.3.611-618.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Scotland S. M., Willshaw G. A., Smith H. R., Rowe B. Properties of strains of Escherichia coli belonging to serogroup O157 with special reference to production of Vero cytotoxins VT1 and VT2. Epidemiol Infect. 1987 Dec;99(3):613–624. doi: 10.1017/s0950268800066462. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Sjogren R., Neill R., Rachmilewitz D., Fritz D., Newland J., Sharpnack D., Colleton C., Fondacaro J., Gemski P., Boedeker E. Role of Shiga-like toxin I in bacterial enteritis: comparison between isogenic Escherichia coli strains induced in rabbits. Gastroenterology. 1994 Feb;106(2):306–317. doi: 10.1016/0016-5085(94)90587-8. [DOI] [PubMed] [Google Scholar]
  19. Stein P. E., Boodhoo A., Tyrrell G. J., Brunton J. L., Read R. J. Crystal structure of the cell-binding B oligomer of verotoxin-1 from E. coli. Nature. 1992 Feb 20;355(6362):748–750. doi: 10.1038/355748a0. [DOI] [PubMed] [Google Scholar]
  20. Waddell T., Head S., Petric M., Cohen A., Lingwood C. Globotriosyl ceramide is specifically recognized by the Escherichia coli verocytotoxin 2. Biochem Biophys Res Commun. 1988 Apr 29;152(2):674–679. doi: 10.1016/s0006-291x(88)80091-3. [DOI] [PubMed] [Google Scholar]

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

RESOURCES