1887

Abstract

Details of a relatively inexpensive method for the purification of K99 pili in their native conformation are reported. The method involved sequential precipitation of K99 pili with (NH)SO, followed by precipitation in 12% (w/v) mannose or sorbitol solution. The crude pili preparation was adsorbed with Bio-Gel A-5m and subjected to sequential gel filtration on Bio-Gel A-5m equilibrated with Tris/EDTA/NaN/NaCl buffer and KSCN/KCl solution, respectively. The K99 containing peak was subjected to sequential ion-exchange chromatography on DEAE-Bio-Gel A equilibrated with 0·02 -Tris/HCl, pH 8·6 containing 0·05 -NaCl and DEAE-Sephadex A-50 equilibrated with 0·05 -phosphate buffer, pH 7·2. The purified pili yielded a single band on SDS-PAGE with an estimated molecular weight of 13000. Attempts to purify pili by other methods evaluated, viz. MgCl precipitation and chromatofocusing were unsuccessful. While the amino acid composition of purified K99 pili was similar to that reported previously the -terminal amino acid was apparently blocked.

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/content/journal/micro/10.1099/00221287-129-6-1975
1983-06-01
2021-05-18
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References

  1. Altmann K., Pyliotis N. A., Mukkur T. K. S. 1982; A new method for the extraction and purification of K99 pili from enterotoxigenicEscherichia coli and their characterization. Biochemical Journal 201:505–513
    [Google Scholar]
  2. Brinton C. C. 1965; The structure, function, synthesis and genetic control of bacterial pili and a molecular model for DNA and RNA transport in gram-negative bacteria. Transactions of the New York Academy of Sciences 227:1003–1054
    [Google Scholar]
  3. Ceriotti G. 1952; A microchemical determination of deoxyribonucleic acid. Journal of Biological Chemistry 198:297–303
    [Google Scholar]
  4. Clements J. D., Finkelstein R. A. 1979; Isolation and characterization of homogeneous heat-labile enterotoxins with high specific activity from Escherichia coli cultures. Infection and Immunity 24:760–769
    [Google Scholar]
  5. Donta S. T., Moon H. W., Whipp S. C. 1974; Detection of heat-labile Escherichia coli enterotoxin with the use of adrenal cells in tissue culture. Science 183:334–336
    [Google Scholar]
  6. De Graaf F. K., Klemm P., Gaastra W. 1981; Purification, characterization and partial covalent structure of Escherichia coli adhesive antigen K99. Infection and Immunity 33:877–883
    [Google Scholar]
  7. GuinÉe P. A. M., Jansen W. H., Agterberg G. M. 1976; Detection of the K99 antigen by means of agglutination and immunoelectrophoresis in Escherichia coli isolates from calves and its correlation with enterotoxigenicity. Infection and Immunity 13:1369–1377
    [Google Scholar]
  8. Isaacson R. E. 1977; K99 surface antigen of Escherichia coli: purification and partial characterization. Infection and Immunity 15:272–279
    [Google Scholar]
  9. Mokrasch L. C. 1954; Analysis of hexose phosphates and sugar mixtures with the anthrone reagent. Journal of Biological Chemistry 208:55–59
    [Google Scholar]
  10. Moon H. W., Whipp S. C., Skartvedt S. M. 1976; Etiologic diagnosis of diarrheal diseases in calves: frequency and methods for detecting enterotoxin and K99 antigen production by Escherichia coli. American Journal of Veterinary Research 37:1025–1029
    [Google Scholar]
  11. Morris J. A., Thorns C. J., Sojka W. J. 1980; Evidence for two adhesive antigens on the K99 reference strain Escherichia coli B41. Journal of General Microbiology 118:107–113
    [Google Scholar]
  12. Mukkur T. K. S., Pyliotis N. A. 1981; The potassium thiocyanate extract of Pasteurella multocida: electron microscopy, and susceptibility of its immunogenic activity to some physical, chemical and enzymatic treatments. Journal of Comparative Pathology 91:427–437
    [Google Scholar]
  13. Myers L. L., GuinÉe P. A. M. 1976; Occurrence and characteristics of enterotoxigenicEscherichia coli isolated from calves with diarrhea. Infection and Immunity 13:1117–1119
    [Google Scholar]
  14. Rosmus J., Deyl Z. 1971; Chromatographic methods in the analysis of protein structure.The methods for identification of N-terminal amino acids in peptides and proteins. Part A. Chromatographic Reviews 13:163–302
    [Google Scholar]
  15. Sivaswamy G., Gyles C. L. 1976; Characterization of enterotoxigenic bovine Escherichia coli. Canadian Journal of Comparative Medicine 40:247–251
    [Google Scholar]
  16. Wevers P., Picken R., Schmidt G., Jann B., Jann K., Golecki J. R., Kist M. 1980; Characterization of pili associated with Escherichia coli 018ac. Infection and Immunity 29:685–691
    [Google Scholar]
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