Preparation of Culture Filtrate Proteins from Mycobacterium tuberculosis

  • Ida Rosenkrands
  • Peter Andersen
Part of the Methods in Molecular Medicine book series (MIMM, volume 54)


Culture filtrates obtained by in vitro cultivation of Mycobacterium tuberculosis have been studied for more than 20 years to identify and characterize proteins of immunological relevance. Culture filtrate preparations have been shown to induce a protective immune response in mice and guinea pigs by several groups (1, 2, 3, 4). Stimulated by these findings, the isolation and evaluation of culture filtrate antigens have been the subjects of intensified effort in several laboratories in recent years. This work has resulted in an increasing number of novel antigens such as MPT59 (5, 6, 7), ESAT-6 (8), MPT64 (9), MTB12 (10), MTB8.4 (11), CFP29 (12), TB10.4 (13), among several others.


Culture Filtrate Starter Culture Ferric Ammonium Citrate Culture Filtrate Protein Filter Clotting 
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  1. 1.
    Andersen P. (1994) Effective vaccination of mice against Mycobacterium tuberculosis infection with a soluble mixture of secreted mycobacterial proteins. Infect. Immun. 62, 2536–2544.PubMedGoogle Scholar
  2. 2.
    Hubbard R. D., Flory C. M., and Collins F. M. (1992) Immunization of mice with mycobacterial culture filtrate proteins. Clin. Exp. Immunol. 87, 94–98.CrossRefPubMedGoogle Scholar
  3. 3.
    Pal P. G. and Horwitz M. A. (1992) Immunization with extracellular proteins of Mycobacterium tuberculosis induces cell-mediated immune responses and substantial protective immunity in an guinea pig model of pulmonary tuberculosis. Infect. Immun. 60, 4781–4792.PubMedGoogle Scholar
  4. 4.
    Roberts A. D., Sonnenberg M. G., Ordway D. J., Furney S. K., Brennan P. J., Belisle J. T., and Orme I. M. (1995) Characteristics of protective immunity engendered by vaccination of mice with purified culture filtrate protein antigens of Mycobacterium tuberculosis. Immunology 85, 502–508.PubMedGoogle Scholar
  5. 5.
    Nagai S., Wiker H. G., Harboe M., and Kinomoto M. (1991) Isolation and partial characterization of major protein antigens in the culture fluid of Mycobacterium tuberculosis. Infect. Immun. 59, 372–382.PubMedGoogle Scholar
  6. 6.
    Andersen P., Andersen Å. B., Sørensen A. L., and Nagai S. (1995) Recall of long-lived immunity to Mycobacterium tuberculosis infection in mice. J. Immunol. 154, 3359–3372.PubMedGoogle Scholar
  7. 7.
    Horwitz M. A., Lee B.-W. E., Dillon B. J., and Harth G. (1995) Protective immunity against tuberculosis induced by vaccination with major extracellular proteins of Mycobacterium tuberculosis. Proc. Natl. Acad. Sci. USA 92, 1530–1534.CrossRefPubMedGoogle Scholar
  8. 8.
    Sørensen A. L., Nagai S., Houen G., Andersen P., and Andersen Å. B. (1995) Purification and characterization of a low-molecular-mass T-cell antigen secreted by Mycobacterium tuberculosis. Infect. Immun. 63, 1710–1717.PubMedGoogle Scholar
  9. 9.
    Oettinger T., Holm A., Mtoni I. M., Andersen Å. B., and Hasløv K. (1995) Mapping of the delayed-type hypersensitivity-inducing epitope of secreted MPT64 from Mycobacterium tuberculosis. Infect. Immun. 63, 4613–4618.PubMedGoogle Scholar
  10. 10.
    Webb J. R., Vedvick T. S., Alderson M. R., Guderian J. A., Jen S. S., Ovendale P. J., Johnson S. M., Reed S. G., and Skeiky Y. A. W. (1998) Molecular cloning, expression, and immunogenicity of MTB12, a novel low-molecular-weight antigen secreted by Mycobacterium tuberculosis. Infect. Immun. 66, 4208–4214.PubMedGoogle Scholar
  11. 11.
    Coler R. N., Skeiky Y. A., Vedvick T., Bement T., Campos-Neto A., Alderson M. R., and Reed S. G. (1998) Molecular cloning and immunologic reactivity of a novel low molecular mass antigen of Mycobacterium tuberculosis. J. Immunol. 161, 2356–2364.PubMedGoogle Scholar
  12. 12.
    Rosenkrands I., Rasmussen P. B., Carnio M., Jacobsen S., Theisen M., and Andersen P. (1998) Identification and characterization of a 29-kilodalton protein from Mycobacterium tuberculosis culture filtrate recognized by mouse memory effector cells. Infect. Immun. 66, 2728–2735.PubMedGoogle Scholar
  13. 13.
    Skjet R. L. V., Oettinger T., Rosenkrands I., Ravn P., Brock I., Jacobsen S., and Andersen P. (2000) Comparative evaluation of low mass T-cell antigens from Mycobacterium tuberculosis identify ESAT-6 family members as immunodominant. Infect. Immun. 68, 214–220.CrossRefGoogle Scholar
  14. 14.
    Harth G., and Horwitz M. A. (1999) Export of recombinant Mycobacterium tuberculosis superoxide dismutase is dependent upon both information in the protein and mycobacterial export machinery. A model for studying export of leaderless proteins by pathogenic mycobacteria. J. Biol. Chem. 274, 4281–4292.CrossRefPubMedGoogle Scholar
  15. 15.
    Harth G., and Horwitz M. A. (1997) Expression and efficient export of enzymatically active Mycobacterium tuberculosis glutamine synthetase in Mycobacterium smegmatis and evidence that the information for export is contained within the protein. J. Biol. Chem. 272, 22,728–22,735.CrossRefPubMedGoogle Scholar
  16. 16.
    Andersen P., Askgaard D., Ljungqvist L., Bennedsen J., and Heron I. (1991) Proteins released from Mycobacterium tuberculosis during growth. Infect. Immun. 59, 1905–1910.PubMedGoogle Scholar
  17. 17.
    Rosenkrands I., Weldingh K., Jacobsen S., Hansen C. V., Florio W., Gianetri I., and Andersen P. (2000) Mapping and identification of Mycobacterium tuberculosis proteins by two-dimensional electrophoresis, microsequencing and immunodetection. Electrophoresis 21, 935–948.CrossRefPubMedGoogle Scholar
  18. 18.
    Wiker H. G., Harboe M., and Nagai S. (1991) A localization index for distinction between extracellular and intracellular antigens of Mycobacterium tuberculosis. J. Gen. Microbiol. 137, 875–884.PubMedGoogle Scholar
  19. 19.
    Wiker H. G., Michell S. L., Hewinson R. G., Spierings E., Nagai S., and Harboe M. (1999) Cloning, expression and significance of MPT53 for identification of secreted proteins of Mycobacterium tuberculosis. Microb. Pathog. 26, 207–219.CrossRefPubMedGoogle Scholar
  20. 20.
    Samanich K. M., Belisle J. T., Sonnenberg M. G., Keen M. A., Zolla-Pazner S., and Laal S. (1998) Delineation of human antibody responses to culture filtrate antigens of Mycobacterium tuberculosis. J. Infect. Dis. 178, 1534–1538.CrossRefPubMedGoogle Scholar
  21. 21.
    Sonnenberg M. G. and Belisle J. T. (1997) Definition of Mycobacterium tuberculosis culture filtrate proteins by two-dimensional polyacrylamide gel electrophoresis, N-terminal amino acid sequencing, and electrospray mass spectrometry. Infect. Immun. 65, 4515–4524.PubMedGoogle Scholar
  22. 22.
    Weldingh K., Rosenkrands I., Jacobsen S., Rasmussen P. B., Elhay M. J., and Andersen P. (1998) Two-dimensional electrophoresis for analysis of Mycobacterium tuberculosis culture filtrate and purification and characterization of six novel proteins. Infect. Immun. 66, 3492–3500.PubMedGoogle Scholar
  23. 23.
    Cole S. T., Brosch R., Parkhill J., Garnier T., Churcher C., Harris D., Gordon S. V., Eiglmeier K., Gas S., Barry C. E., 3rd, Tekaia F., Badcock K., Basham D., Brown D., Chillingworth T., Connor R., Davies R., Devlin K., Feltwell T., Gentles S., Hamlin N., Holroyd S., Hornsby T., Jagels K., and Barrell B. G. (1998) Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence. Nature 393, 537–544.CrossRefPubMedGoogle Scholar
  24. 24.
    Jungblut P. R., Schaible U. E., Mollenkopf H.-J., Zimny-Arndt U., Raupach B., Mattow J., Halada P., Lamer S., Hagens K., and Kaufmann S. H. E. (1999) Comparative proteome analysis of Mycobacterium tuberculosis and Mycobacterium bovis BCG strains: towards functional genomics of microbial pathogens. Mol. Microbiol. 33, 1103–1117.CrossRefPubMedGoogle Scholar
  25. 25.
    Sinha R. K., Verma I., and Khuller G. K. (1997) Immunobiological properties of a 30 kDa secretory protein of Mycobacterium tuberculosis H37Ra. Vaccine 15, 689–699.CrossRefPubMedGoogle Scholar
  26. 26.
    Hunter S. W., Rivoire B., Mehra V., Bloom B. R., and Brennan P. J. (1990) The major native proteins of the leprosy bacillus. J. Biol. Chem. 265, 14,065–14,068.PubMedGoogle Scholar
  27. 27.
    Laemmli U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685.CrossRefPubMedGoogle Scholar
  28. 28.
    Blum H., Beier H., and Gross H. J. (1987) Improved silver staining of plant proteins, RNA and DNA in polyacrylamide gels. Electrophoresis 8, 93–99.CrossRefGoogle Scholar

Copyright information

© Humana Press Inc., Totowa, NJ 2001

Authors and Affiliations

  • Ida Rosenkrands
    • 1
  • Peter Andersen
    • 1
  1. 1.Department of TB ImmunologyStatens Serum InstitutCopenhagenDenmark

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