title: Epitope-tagging vectors for the expression and detection of recombinant proteins in mycobacteria.
abstract: New tools are required to study the growing number of uncharacterised genes derived from genome sequence projects that are specific to bacterial pathogens such as Mycobacterium tuberculosis. We have developed a series of vectors that permit the specific detection of recombinant proteins expressed in mycobacterial species. Gene expression in these vectors is driven by the strong hsp60 promoter of Mycobacterium bovis BCG and detection of expressed products is facilitated by C-terminal fusion of residues 409-419 of the human c-myc proto-oncogene. Using the M. tuberculosis Ag85B as a reporter of gene expression, we demonstrate that the vectors permit the specific detection of recombinant products expressed in the host species M. bovis BCG. BCG over-expressing Ag85B was a potent inducer of Ag85B-specific T cells in immunised mice, indicating that the C-terminal c-myc tag did not alter the characteristics of the recombinant protein. The versatility of the epitope-tagging vectors was demonstrated by the efficient secretion and detection of recombinant products in BCG. The vectors described in this study will facilitate the expression of foreign proteins in mycobacterial host systems.
authors.affiliation: Mycobacterial Research Group, Centenary Institute of Cancer Medicine and Cell Biology, Locked Bag No. 6, Newtown, NSW 2042, Australia.
authors.forename: Joanne M
authors.initials: JM
authors.lastname: Spratt
authors.forename: Anthony A
authors.initials: AA
authors.lastname: Ryan
authors.forename: Warwick J
authors.initials: WJ
authors.lastname: Britton
authors.forename: James A
authors.initials: JA
authors.lastname: Triccas
mesh_terms: D000818:Animals; D000942:Antigens, Bacterial / Q000235:genetics / Q000378:metabolism; D001500:BCG Vaccine / Q000032:analysis / Q000378:metabolism; D002874:Chromosome Mapping; D002876:Chromosomes, Bacterial; D000939:Epitopes; D015870:Gene Expression; D005798:Genes, Bacterial; D017930:Genes, Reporter; D005822:Genetic Vectors; D006801:Humans; D051379:Mice; D009161:Mycobacterium / Q000235:genetics / Q000378:metabolism*; D009163:Mycobacterium bovis / Q000235:genetics / Q000378:metabolism; D011401:Promoter Regions, Genetic; D000090063:Proto-Oncogene Mas; D016271:Proto-Oncogene Proteins c-myc / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism*; D013601:T-Lymphocytes / Q000276:immunology
chemical_list: D000942:Antigens, Bacterial; D001500:BCG Vaccine; D000939:Epitopes; C000715470:MAS1 protein, human; D000090063:Proto-Oncogene Mas; D016271:Proto-Oncogene Proteins c-myc; D011994:Recombinant Proteins