Skip to main content
PMC home page
Biochemical Journal logoLink to Biochemical Journal
. 1990 Dec 15;272(3):805–811. doi: 10.1042/bj2720805

Characterization of Shiga-like toxin I B subunit purified from overproducing clones of the SLT-I B cistron.

K Ramotar 1, B Boyd 1, G Tyrrell 1, J Gariepy 1, C Lingwood 1, J Brunton 1
PMCID: PMC1149779  PMID: 2268304

Abstract

The cistron encoding the B subunit of Escherichia coli Shiga-like toxin I (SLT-I) was cloned under control of the tac promoter in the expression vector pKK223-3 and the SLT-I B subunit was expressed constitutively in a wild-type background and inducibly in a lacIq background. The B subunit was located in the periplasmic space, and less than 10% was found in the culture medium after 24 h incubation. Polymyxin B extracts contained as much as 160 micrograms of B subunit/ml of culture. B subunit was purified to homogeneity by ion-exchange chromatography followed by chromatofocusing. Cross-linking analysis of purified native B subunit showed that it exists as a pentamer. In gels containing 0.1% SDS the native protein dissociated into monomers. B subunit was found to have the same glycolipid-receptor-specificity as SLT-I holotoxin. Competitive binding studies showed that B subunit and holotoxin had the same affinity for the globotriosylceramide receptor. We conclude that this recombinant plasmid is a convenient source of large amounts of purified SLT-I B subunit, which could be used for biophysical and structural studies or as a natural toxoid.

Full text

PDF
806

Images in this article

808Fig. 2.
on p.808
809Fig. 5.
on p.809
810Fig. 6.
on p.810

Selected References

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

  1. Achtman M., Mercer A., Kusecek B., Pohl A., Heuzenroeder M., Aaronson W., Sutton A., Silver R. P. Six widespread bacterial clones among Escherichia coli K1 isolates. Infect Immun. 1983 Jan;39(1):315–335. doi: 10.1128/iai.39.1.315-335.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Amann E., Brosius J., Ptashne M. Vectors bearing a hybrid trp-lac promoter useful for regulated expression of cloned genes in Escherichia coli. Gene. 1983 Nov;25(2-3):167–178. doi: 10.1016/0378-1119(83)90222-6. [DOI] [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. Cohen A., Hannigan G. E., Williams B. R., Lingwood C. A. Roles of globotriosyl- and galabiosylceramide in verotoxin binding and high affinity interferon receptor. J Biol Chem. 1987 Dec 15;262(35):17088–17091. [PubMed] [Google Scholar]
  5. Davies G. E., Stark G. R. Use of dimethyl suberimidate, a cross-linking reagent, in studying the subunit structure of oligomeric proteins. Proc Natl Acad Sci U S A. 1970 Jul;66(3):651–656. doi: 10.1073/pnas.66.3.651. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. De Grandis S., Ginsberg J., Toone M., Climie S., Friesen J., Brunton J. Nucleotide sequence and promoter mapping of the Escherichia coli Shiga-like toxin operon of bacteriophage H-19B. J Bacteriol. 1987 Sep;169(9):4313–4319. doi: 10.1128/jb.169.9.4313-4319.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. DeGrandis S., Law H., Brunton J., Gyles C., Lingwood C. A. Globotetraosylceramide is recognized by the pig edema disease toxin. J Biol Chem. 1989 Jul 25;264(21):12520–12525. [PubMed] [Google Scholar]
  8. 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]
  9. Donohue-Rolfe A., Jacewicz M., Keusch G. T. Isolation and characterization of functional Shiga toxin subunits and renatured holotoxin. Mol Microbiol. 1989 Sep;3(9):1231–1236. doi: 10.1111/j.1365-2958.1989.tb00273.x. [DOI] [PubMed] [Google Scholar]
  10. 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]
  11. 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]
  12. Gannon V. P., Gyles C. L., Wilcock B. P. Effects of Escherichia coli Shiga-like toxins (verotoxins) in pigs. Can J Vet Res. 1989 Jul;53(3):306–312. [PMC free article] [PubMed] [Google Scholar]
  13. Gyles C. L., De Grandis S. A., MacKenzie C., Brunton J. L. Cloning and nucleotide sequence analysis of the genes determining verocytotoxin production in a porcine edema disease isolate of Escherichia coli. Microb Pathog. 1988 Dec;5(6):419–426. doi: 10.1016/0882-4010(88)90003-4. [DOI] [PubMed] [Google Scholar]
  14. Harari I., Donohue-Rolfe A., Keusch G., Arnon R. Synthetic peptides of Shiga toxin B subunit induce antibodies which neutralize its biological activity. Infect Immun. 1988 Jun;56(6):1618–1624. doi: 10.1128/iai.56.6.1618-1624.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Hardy S. J., Holmgren J., Johansson S., Sanchez J., Hirst T. R. Coordinated assembly of multisubunit proteins: oligomerization of bacterial enterotoxins in vivo and in vitro. Proc Natl Acad Sci U S A. 1988 Oct;85(19):7109–7113. doi: 10.1073/pnas.85.19.7109. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Hawkes R., Niday E., Gordon J. A dot-immunobinding assay for monoclonal and other antibodies. Anal Biochem. 1982 Jan 1;119(1):142–147. doi: 10.1016/0003-2697(82)90677-7. [DOI] [PubMed] [Google Scholar]
  17. Head S. C., Karmali M. A., Roscoe M. E., Petric M., Strockbine N. A., Wachsmuth I. K. Serological differences between verocytotoxin 2 and shiga-like toxin II. Lancet. 1988 Sep 24;2(8613):751–751. doi: 10.1016/s0140-6736(88)90228-0. [DOI] [PubMed] [Google Scholar]
  18. Huang A., de Grandis S., Friesen J., Karmali M., Petric M., Congi R., Brunton J. L. Cloning and expression of the genes specifying Shiga-like toxin production in Escherichia coli H19. J Bacteriol. 1986 May;166(2):375–379. doi: 10.1128/jb.166.2.375-379.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Ito H., Yutsudo T., Hirayama T., Takeda Y. Isolation and some properties of A and B subunits of Vero toxin 2 and in vitro formation of hybrid toxins between subunits of Vero toxin 1 and Vero toxin 2 from Escherichia coli O157:H7. Microb Pathog. 1988 Sep;5(3):189–195. doi: 10.1016/0882-4010(88)90021-6. [DOI] [PubMed] [Google Scholar]
  20. 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]
  21. Jacewicz M., Feldman H. A., Donohue-Rolfe A., Balasubramanian K. A., Keusch G. T. Pathogenesis of Shigella diarrhea. XIV. Analysis of Shiga toxin receptors on cloned HeLa cells. J Infect Dis. 1989 May;159(5):881–889. doi: 10.1093/infdis/159.5.881. [DOI] [PubMed] [Google Scholar]
  22. Karmali M. A., Petric M., Lim C., Fleming P. C., Arbus G. S., Lior H. The association between idiopathic hemolytic uremic syndrome and infection by verotoxin-producing Escherichia coli. J Infect Dis. 1985 May;151(5):775–782. doi: 10.1093/infdis/151.5.775. [DOI] [PubMed] [Google Scholar]
  23. Konowalchuk J., Dickie N., Stavric S., Speirs J. I. Comparative studies of five heat-labile toxic products of Escherichia coli. Infect Immun. 1978 Dec;22(3):644–648. doi: 10.1128/iai.22.3.644-648.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. 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]
  25. Lindberg A. A., Brown J. E., Strömberg N., Westling-Ryd M., Schultz J. E., Karlsson K. A. Identification of the carbohydrate receptor for Shiga toxin produced by Shigella dysenteriae type 1. J Biol Chem. 1987 Feb 5;262(4):1779–1785. [PubMed] [Google Scholar]
  26. Lingwood C. A., Law H., Richardson S., Petric M., Brunton J. L., De Grandis S., Karmali M. Glycolipid binding of purified and recombinant Escherichia coli produced verotoxin in vitro. J Biol Chem. 1987 Jun 25;262(18):8834–8839. [PubMed] [Google Scholar]
  27. Lopez E. L., Diaz M., Grinstein S., Devoto S., Mendilaharzu F., Murray B. E., Ashkenazi S., Rubeglio E., Woloj M., Vasquez M. Hemolytic uremic syndrome and diarrhea in Argentine children: the role of Shiga-like toxins. J Infect Dis. 1989 Sep;160(3):469–475. doi: 10.1093/infdis/160.3.469. [DOI] [PubMed] [Google Scholar]
  28. Merril C. R., Goldman D., Sedman S. A., Ebert M. H. Ultrasensitive stain for proteins in polyacrylamide gels shows regional variation in cerebrospinal fluid proteins. Science. 1981 Mar 27;211(4489):1437–1438. doi: 10.1126/science.6162199. [DOI] [PubMed] [Google Scholar]
  29. Migasena S., Desakorn V., Suntharasamai P., Pitisuttitham P., Prayurahong B., Supanaranond W., Black R. E. Immunogenicity of two formulations of oral cholera vaccine comprised of killed whole vibrios and the B subunit of cholera toxin. Infect Immun. 1989 Jan;57(1):117–120. doi: 10.1128/iai.57.1.117-120.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Mobassaleh M., Donohue-Rolfe A., Jacewicz M., Grand R. J., Keusch G. T. Pathogenesis of shigella diarrhea: evidence for a developmentally regulated glycolipid receptor for shigella toxin involved in the fluid secretory response of rabbit small intestine. J Infect Dis. 1988 May;157(5):1023–1031. doi: 10.1093/infdis/157.5.1023. [DOI] [PubMed] [Google Scholar]
  31. Rokeach L. A., Haselby J. A., Hoch S. O. Molecular cloning of a cDNA encoding the human Sm-D autoantigen. Proc Natl Acad Sci U S A. 1988 Jul;85(13):4832–4836. doi: 10.1073/pnas.85.13.4832. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Sanger F., Coulson A. R., Hong G. F., Hill D. F., Petersen G. B. Nucleotide sequence of bacteriophage lambda DNA. J Mol Biol. 1982 Dec 25;162(4):729–773. doi: 10.1016/0022-2836(82)90546-0. [DOI] [PubMed] [Google Scholar]
  33. Schägger H., von Jagow G. Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal Biochem. 1987 Nov 1;166(2):368–379. doi: 10.1016/0003-2697(87)90587-2. [DOI] [PubMed] [Google Scholar]
  34. Smith P. K., Krohn R. I., Hermanson G. T., Mallia A. K., Gartner F. H., Provenzano M. D., Fujimoto E. K., Goeke N. M., Olson B. J., Klenk D. C. Measurement of protein using bicinchoninic acid. Anal Biochem. 1985 Oct;150(1):76–85. doi: 10.1016/0003-2697(85)90442-7. [DOI] [PubMed] [Google Scholar]
  35. Strockbine N. A., Marques L. R., Holmes R. K., O'Brien A. D. Characterization of monoclonal antibodies against Shiga-like toxin from Escherichia coli. Infect Immun. 1985 Dec;50(3):695–700. doi: 10.1128/iai.50.3.695-700.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Strockbine N. A., Marques L. R., Newland J. W., Smith H. W., Holmes R. K., O'Brien A. D. Two toxin-converting phages from Escherichia coli O157:H7 strain 933 encode antigenically distinct toxins with similar biologic activities. Infect Immun. 1986 Jul;53(1):135–140. doi: 10.1128/iai.53.1.135-140.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. 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]
  39. Weinstein D. L., Jackson M. P., Samuel J. E., Holmes R. K., O'Brien A. D. Cloning and sequencing of a Shiga-like toxin type II variant from Escherichia coli strain responsible for edema disease of swine. J Bacteriol. 1988 Sep;170(9):4223–4230. doi: 10.1128/jb.170.9.4223-4230.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES