Skip to main content
PMC home page
Infection and Immunity logoLink to Infection and Immunity
. 1988 Jul;56(7):1708–1714. doi: 10.1128/iai.56.7.1708-1714.1988

Purification and characterization of toxin B from Clostridium difficile.

J Meador 3rd 1, R K Tweten 1
PMCID: PMC259466  PMID: 3384474

Abstract

Toxin B from Clostridium difficile was purified to homogeneity and characterized. Purification of toxin B was achieved by gel filtration, chromatography on two consecutive anion-exchange columns, and chromatography on a high-resolution anion-exchange column in the presence of 50 mM CaCl2. The molecular weight of toxin B was estimated to be 250,000 by denaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and 500,000 by gel filtration. No subunits were apparent when the toxin was reduced and analyzed by SDS-PAGE. The estimated molecular weight of native toxin B indicated that dimers may form in solution. Toxin B was homogeneous by SDS-PAGE, native PAGE, and high-resolution anion-exchange chromatography. No secondary sequences were detected when the amino terminus of the toxin was sequenced, which also indicated that contaminating peptides were absent from the preparation. The amino terminus of toxin B was determined to be NH3-Trp-Leu-Val-Asn-Arg-Lys-Gln-Leu-Glu-Lys-Met-Ala-Asn-Val-ARg-Phe-Arg. One cytotoxic unit of toxin B was estimated to be 0.2 to 0.8 pg.

Full text

PDF
1712

Images in this article

1710Image
on p.1710
1711Image
on p.1711
1712Image
on p.1712
1712Image
on p.1712
1713Image
on p.1713

Selected References

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

  1. Banno Y., Kobayashi T., Kono H., Watanabe K., Ueno K., Nozawa Y. Biochemical characterization and biologic actions of two toxins (D-1 and D-2) from Clostridium difficile. Rev Infect Dis. 1984 Mar-Apr;6 (Suppl 1):S11–S20. doi: 10.1093/clinids/6.supplement_1.s11. [DOI] [PubMed] [Google Scholar]
  2. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1006/abio.1976.9999. [DOI] [PubMed] [Google Scholar]
  3. KRAUSE R. M., McCARTY M. Studies on the chemical structure of the streptococcal cell wall. II. The composition of group C cell walls and chemical basis for serologic specificity of the carbohydrate moiety. J Exp Med. 1962 Jan 1;115:49–62. doi: 10.1084/jem.115.1.49. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. 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]
  5. Lyerly D. M., Saum K. E., MacDonald D. K., Wilkins T. D. Effects of Clostridium difficile toxins given intragastrically to animals. Infect Immun. 1985 Feb;47(2):349–352. doi: 10.1128/iai.47.2.349-352.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Mitchell M. J., Laughon B. E., Lin S. Biochemical studies on the effect of Clostridium difficile toxin B on actin in vivo and in vitro. Infect Immun. 1987 Jul;55(7):1610–1615. doi: 10.1128/iai.55.7.1610-1615.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Pothoulakis C., Barone L. M., Ely R., Faris B., Clark M. E., Franzblau C., LaMont J. T. Purification and properties of Clostridium difficile cytotoxin B. J Biol Chem. 1986 Jan 25;261(3):1316–1321. [PubMed] [Google Scholar]
  8. Rolfe R. D., Finegold S. M. Purification and characterization of Clostridium difficile toxin. Infect Immun. 1979 Jul;25(1):191–201. doi: 10.1128/iai.25.1.191-201.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Sullivan N. M., Pellett S., Wilkins T. D. Purification and characterization of toxins A and B of Clostridium difficile. Infect Immun. 1982 Mar;35(3):1032–1040. doi: 10.1128/iai.35.3.1032-1040.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Taylor N. S., Bartlett J. G. Partial purification and characterization of a cytotoxin from Clostridium difficile. Rev Infect Dis. 1979 Mar-Apr;1(2):379–385. doi: 10.1093/clinids/1.2.379. [DOI] [PubMed] [Google Scholar]
  11. Taylor N. S., Thorne G. M., Bartlett J. G. Comparison of two toxins produced by Clostridium difficile. Infect Immun. 1981 Dec;34(3):1036–1043. doi: 10.1128/iai.34.3.1036-1043.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Tweten R. K., Iandolo J. J. Purification and partial characterization of a putative precursor to staphylococcal enterotoxin B. Infect Immun. 1981 Dec;34(3):900–907. doi: 10.1128/iai.34.3.900-907.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES