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. 1989 May;27(5):1031–1035. doi: 10.1128/jcm.27.5.1031-1035.1989

Rapid purification of staphylococcal enterotoxin B by high-pressure liquid chromatography.

M P Strickler 1, R J Neill 1, M J Stone 1, R E Hunt 1, W Brinkley 1, P Gemski 1
PMCID: PMC267477  PMID: 2745678

Abstract

The Staphylococcus aureus enterotoxins represent a group of proteins that cause emesis and diarrhea in humans and other primates. We have developed a rapid two-step high-pressure liquid chromatography (HPLC) procedure for purification of staphylococcal enterotoxin B (SEB). Sterile filtrates (2.5 liters) of strain 10-275 were adsorbed directly onto a reversed-phase column (50 mm by 30 cm Delta Pak; 300 A [30 nm], 15 microns, C18). SEB was obtained by using a unique sequential gradient system. First, an aqueous ammonium acetate to acetonitrile gradient followed by an aqueous trifluoroacetic acid (TFA) wash was used to remove contaminants. A subsequent TFA to acetonitrile-TFA gradient eluted the bound SEB. Further purification was obtained by rechromatography on a cation-exchange column. From 35 to 45% of the SEB in starting filtrates was recovered. Analysis by immunoblotting of samples separated on sodium dodecyl sulfate-polyacrylamide gels indicated that HPLC-purified SEB exhibited immunological and biochemical properties similar to those of the SEB standard. Induction of an emetic response in rhesus monkeys showed that the HPLC-purified toxin also retained biological activity.

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Selected References

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  1. Betley M. J., Mekalanos J. J. Nucleotide sequence of the type A staphylococcal enterotoxin gene. J Bacteriol. 1988 Jan;170(1):34–41. doi: 10.1128/jb.170.1.34-41.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Brunson K. W., Watson D. W. Pyrogenic specificity of streptococcal exotoxins, staphylococcal enterotoxin, and gram-negative endotoxin. Infect Immun. 1974 Aug;10(2):347–351. doi: 10.1128/iai.10.2.347-351.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. CASMAN E. P., BERGDOLL M. S., ROBINSON J. DESIGNATION OF STAPHYLOCOCCAL EXTEROTOXINS. J Bacteriol. 1963 Mar;85:715–716. doi: 10.1128/jb.85.3.715-716.1963. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Chang P. C., Dickie N. Fractionation of staphylococcal enterotoxin B by isoelectric focusing. Biochim Biophys Acta. 1971 May 25;236(2):367–375. doi: 10.1016/0005-2795(71)90217-0. [DOI] [PubMed] [Google Scholar]
  5. Couch J. L., Soltis M. T., Betley M. J. Cloning and nucleotide sequence of the type E staphylococcal enterotoxin gene. J Bacteriol. 1988 Jul;170(7):2954–2960. doi: 10.1128/jb.170.7.2954-2960.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Ende I. A., Terplan G., Kickhöfen B., Hammer D. K. Chromatofocusing: a new method for purification of staphylococcal enterotoxins B and C1. Appl Environ Microbiol. 1983 Dec;46(6):1323–1330. doi: 10.1128/aem.46.6.1323-1330.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Huang I. Y., Bergdoll M. S. Primary structure of staphylococcal enterotoxin B. I. Isolation, composition, and sequence of tryptic peptides from oxidized enterotoxin B. J Biol Chem. 1970 Jul 25;245(14):3493–3510. [PubMed] [Google Scholar]
  8. Huang I. Y., Bergdoll M. S. The primary structure of staphylococcal enterotoxin B. 3. The cyanogen bromide peptides of reduced and aminoethylated enterotoxin B, and the complete amino acid sequence. J Biol Chem. 1970 Jul 25;245(14):3518–3525. [PubMed] [Google Scholar]
  9. Huang I. Y., Bergdoll M. S. The primary structure of staphylococcal enterotoxin B. II. Isolation, composition, and sequence of chymotryptic peptides. J Biol Chem. 1970 Jul 25;245(14):3511–3517. [PubMed] [Google Scholar]
  10. Huang I. Y., Hughes J. L., Bergdoll M. S., Schantz E. J. Complete amino acid sequence of staphylococcal enterotoxin A. J Biol Chem. 1987 May 25;262(15):7006–7013. [PubMed] [Google Scholar]
  11. Jones C. L., Khan S. A. Nucleotide sequence of the enterotoxin B gene from Staphylococcus aureus. J Bacteriol. 1986 Apr;166(1):29–33. doi: 10.1128/jb.166.1.29-33.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Kent T. H. Staphylococcal enterotoxin gastroenteritis in rhesus monkeys. Am J Pathol. 1966 Mar;48(3):387–407. [PMC free article] [PubMed] [Google Scholar]
  13. 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]
  14. Lei Z., Reiser R. F., Bergdoll M. S. Chromatofocusing in the purification of staphylococcal enterotoxin D. J Clin Microbiol. 1988 Jun;26(6):1236–1237. doi: 10.1128/jcm.26.6.1236-1237.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Metzger J. F., Johnson A. D., Collins W. S., 2nd Fractionation and purification of Staphylococcus aureus enterotoxin B by electrofocusing. Biochim Biophys Acta. 1972 Jan 26;257(1):183–186. doi: 10.1016/0005-2795(72)90269-3. [DOI] [PubMed] [Google Scholar]
  16. Peavy D. L., Adler W. H., Smith R. T. The mitogenic effects of endotoxin and staphylococcal enterotoxin B on mouse spleen cells and human peripheral lymphocytes. J Immunol. 1970 Dec;105(6):1453–1458. [PubMed] [Google Scholar]
  17. Ranelli D. M., Jones C. L., Johns M. B., Mussey G. J., Khan S. A. Molecular cloning of staphylococcal enterotoxin B gene in Escherichia coli and Staphylococcus aureus. Proc Natl Acad Sci U S A. 1985 Sep;82(17):5850–5854. doi: 10.1073/pnas.82.17.5850. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Schantz E. J., Roessler W. G., Wagman J., Spero L., Dunnery D. A., Bergdoll M. S. Purification of staphylococcal enterotoxin B. Biochemistry. 1965 Jun;4(6):1011–1016. doi: 10.1021/bi00882a005. [DOI] [PubMed] [Google Scholar]
  19. Schantz E. J., Roessler W. G., Woodburn M. J., Lynch J. M., Jacoby H. M., Silverman S. J., Gorman J. C., Spero L. Purification and some chemical and physical properties of staphylococcal enterotoxin A. Biochemistry. 1972 Feb 1;11(3):360–366. doi: 10.1021/bi00753a009. [DOI] [PubMed] [Google Scholar]
  20. Sidberry H., Kaufman B., Wright D. C., Sadoff J. Immunoenzymatic analysis by monoclonal antibodies of bacterial lipopolysaccharides after transfer to nitrocellulose. J Immunol Methods. 1985 Feb 11;76(2):299–305. doi: 10.1016/0022-1759(85)90307-2. [DOI] [PubMed] [Google Scholar]
  21. Smith B. G., Johnson H. M. The effect of staphylococcal enterotoxins on the primary in vitro immune response. J Immunol. 1975 Aug;115(2):575–578. [PubMed] [Google Scholar]
  22. Thompson N. E., Ketterhagen M. J., Bergdoll M. S. Monoclonal antibodies to staphylococcal enterotoxins B and C: cross-reactivity and localization of epitopes on tryptic fragments. Infect Immun. 1984 Jul;45(1):281–285. doi: 10.1128/iai.45.1.281-285.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. 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]
  24. Warren J. R., Spero L., Metzger J. F. Isothermal denaturation of aqueous staphylococcal enterotoxin B by guanidine hydrochloride, urea, and acid pH. Biochemistry. 1974 Apr 9;13(8):1678–1683. doi: 10.1021/bi00705a019. [DOI] [PubMed] [Google Scholar]
  25. Williams R. R., Wehr C. T., Rogers T. J., Bennett R. W. High-performance liquid chromatography of staphylococcal enterotoxin B. J Chromatogr. 1983 Aug 26;266:179–186. doi: 10.1016/s0021-9673(01)90891-x. [DOI] [PubMed] [Google Scholar]

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