Heterologous Expression of Genes in Mycobacteria

  • James A. TriccasEmail author
  • Anthony A. Ryan
Part of the Methods in Molecular Biology book series (MIMB, volume 465)


Elucidating the function of mycobacterial proteins, determining their contribution to virulence, and developing new vaccine candidates has been facilitated by systems permitting the heterologous expression of genes in mycobacteria. Mycobacterium bovis bacille Calmette Guérin (BCG) and Mycobacterium smegmatis have commonly been employed as host systems for the heterologous expression of mycobacterial genes as well as genes from other bacteria, viruses, and mammalian cells. Vectors that permit strong, constitutive expression of genes have been developed, and more recently systems that allow tightly regulated induction of gene expression have become available. In this chapter, we describe two complementary techniques relevant to the field of gene expression in mycobacteria. We first outline the methodology used for the expression and specific detection of recombinant products expressed in BCG. The expression vectors described use an epitope tag fused to the C-terminal end of the foreign protein, ablating the need for additional reagents to detect the recombinant product. Second, we describe the inducible expression of genes in recombinant M. smegmatis and the subsequent purification of gene products using affinity chromatography.


c-myc gene expression Mycobacteria Mycobacterium bovis BCG Mycobacterium smegmatis Ni2+-affinity chromatography protein purification recombinant protein Western blotting 



Work described in his chapter was supported by the National Health and Medical Research Council of Australia.


  1. 1.
    Dietrich, G., Viret, J.F., and Hess, J (2003) Novel vaccination strategies based on recombinant Mycobacterium bovis BCG. Int. J. Med. Microbiol. 292, 441–451.Google Scholar
  2. 2.
    Ohara, N. and Yamada, T. (2001) Recombinant BCG vaccines. Vaccine 19, 4089–4098.PubMedCrossRefGoogle Scholar
  3. 3.
    Stover, C.K., de la Cruz, V.F., Fuerst, T.R., Burlein, J.E., Benson, L.A., Bennett, L.T., Bansal, G.P., Young, J.F., Lee, M.H., and Hatfull, G.F. (1991) New use of BCG for recombinant vaccines. Nature 351, 456–460.PubMedCrossRefGoogle Scholar
  4. 4.
    Winter, N., Lagranderie, M., Rauzier, J., Timm, J., Leclerc, C., Guy, B., Kieny, M.P., Gheorghiu, M., and Gicquel, B. (1991) Expression of heterologous genes in Mycobacterium bovis BCG: induction of a cellular response against HIV-1 Nef protein. Gene 109, 47–54.PubMedCrossRefGoogle Scholar
  5. 5.
    Parish, T. and Stoker, N.G. (1997) Development and use of a conditional antisense mutagenesis system in mycobacteria. FEMS Microbiol. Lett. 154, 151–157.PubMedCrossRefGoogle Scholar
  6. 6.
    Manabe, Y.C., Chen, J.M., Ko, C.G., Chen, P., and Bishai, W.R. (1999) Conditional sigma factor expression, using the inducible acetamidase promoter, reveals that the Mycobacterium tuberculosis sigF gene modulates expression of the 16-kilodalton alpha-crystallin homologue. J. Bacteriol. 181, 7629–7633.PubMedGoogle Scholar
  7. 7.
    Triccas, J.A., Parish, T., Britton, W.J., and Gicquel, B. (1998) An inducible expression system permitting the efficient purification of a recombinant antigen from Mycobacterium smegmatis. FEMS Microbiol. Lett. 167, 151–156.PubMedCrossRefGoogle Scholar
  8. 8.
    Spratt, J.M., Ryan, A.A., Britton, W.J., and Triccas, J.A. (2005) Epitope-tagging vectors for the expression and detection of recombinant proteins in mycobacteria. Plasmid 53, 269–273.PubMedCrossRefGoogle Scholar
  9. 9.
    Al-Zarouni, M. and Dale, J.W. (2002) Expression of foreign genes in Mycobacterium bovis BCG strains using different promoters reveals instability of the hsp60 promoter for expression of foreign genes in Mycobacterium bovis BCG strains. Tuberculosis 82, 283–291.PubMedCrossRefGoogle Scholar
  10. 10.
    Roche, P.W., Winter, N., Triccas, J.A., Feng, C.G., and Britton, W.J. (1996) Expression of Mycobacterium tuberculosis MPT64 in recombinant Myco. smegmatis: purification, immunogenicity and application to skin tests for tuberculosis. Clin. Exp. Immunol. 103, 226–232.PubMedCrossRefGoogle Scholar
  11. 11.
    Triccas, J.A., Roche, P.W., Winter, N., Feng, C.G., Butlin, C.R., and Britton, W.J. (1996) A 35-kilodalton protein is a major target of the human immune response to Mycobacterium leprae. Infect. Immun. 64, 5171–5177.PubMedGoogle Scholar
  12. 12.
    Sambrook, J., Fritsch, E.F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press.Google Scholar
  13. 13.
    Dennehy, M. and Williamson, A.L. (2005) Factors influencing the immune response to foreign antigen expressed in recombinant BCG vaccines. Vaccine 23, 1209–1224.PubMedCrossRefGoogle Scholar
  14. 14.
    Mederle, I., Bourguin, I., Ensergueix, D., Badell, E., Moniz-Peireira, J., Gicquel, B., and Winter, N. (2002) Plasmidic versus insertional cloning of heterologous genes in Mycobacterium bovis BCG: impact on in vivo antigen persistence and immune responses. Infect. Immun. 70, 303–314.PubMedCrossRefGoogle Scholar
  15. 15.
    Timm, J.M., Perilli, G., Duez, C., Trias, J., Orefici, G., Fattorini, L., Amicosante, G., Oratore, A., Joris, B., Frere, J.M., Pugsley, A.P., and Gicquel, B. (1994) Transcription and expression analysis, using lacZ and phoA gene fusions, of Mycobacterium fortuitum β-lactamase genes cloned from a natural isolate and a high-level, β-lactamase producer. Mol. Microbiol. 12, 491–504.PubMedCrossRefGoogle Scholar
  16. 16.
    Triccas, J.A., Winter, N., Roche P.W., Gilpin, A., and Britton, W.J. (1998) Molecular and immunological analyses of the Mycobacterium avium homologue of the highly antigenic Mycobacterium leprae 35 kDa protein. Infect. Immun. 66, 2684–2690.PubMedGoogle Scholar
  17. 17.
    Daugelat, S., Kowall, J., Mattow, J., Bumann, D., Winter, R., Hurwitz, R., and Kaufmann, S.H. (2003) The RD1 proteins of Mycobacterium tuberculosis: expression in Mycobacterium smegmatis and biochemical characterization. Microbes Infect. 5, 1082–1095.PubMedCrossRefGoogle Scholar
  18. 18.
    Brown, A.C. and Parish, T. (2006) Instability of the acetamide-inducible expression vector pJAM2 in Mycobacterium tuberculosis. Plasmid 55, 81–86.PubMedCrossRefGoogle Scholar
  19. 19.
    Carroll, P., Muttucumaru, D.G., and Parish, T. (2005) Use of a tetracycline-inducible system for conditional expression in Mycobacterium tuberculosis and Mycobacterium smegmatis. Appl. Environ. Microbiol. 71, 3077–3084.PubMedCrossRefGoogle Scholar
  20. 20.
    Ehrt, S., Guo, X.V., Hickey, C.M., Ryou, M., Monteleone, M., Riley, L.W., and Schnappinger, D. (2005) Controlling gene expression in mycobacteria with anhydrotetracycline and Tet repressor. Nucleic Acids Res. 33, e21.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press, a part of Springer Science+Business Media, LLC 2009

Authors and Affiliations

  1. 1.Discipline of Infectious Diseases and ImmunologyUniversity of SydneyCamperdownAustralia
  2. 2.Centenary Institute of Cancer Medicine and Cell BiologyUniversity of SydneySydneyAustralia

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