Heterologous expression of genes in mycobacteria.
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.
Triccas
James A
JA
Discipline of Infectious Diseases and Immunology, University of Sydney, Camperdown, NSW 2006, Australia. jamiet@infdis.usyd.edu.au
Ryan
Anthony A
AA
D015153:Blotting, Western / Q000379:methods; D002846:Chromatography, Affinity / Q000379:methods; D003001:Cloning, Molecular / Q000379:methods; D015870:Gene Expression; D005798:Genes, Bacterial; D005822:Genetic Vectors / Q000235:genetics*; D009163:Mycobacterium bovis / Q000235:genetics*; D020102:Mycobacterium smegmatis / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics* / Q000302:isolation & purification*
D011994:Recombinant Proteins