Expression of Foreign Genes in Mycobacteria

Jeanne E. Burlein; Shawn Offutt; Mark S. Hanson; C. Kendall Stover

doi:10.1128/9781555818357.ch17

Chapter 17 : Expression of Foreign Genes in Mycobacteria

Authors: Jeanne E. Burlein¹, Shawn Offutt¹, Mark S. Hanson¹, C. Kendall Stover²

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Affiliations: 1: Medlmmune, Inc., 35 West Watkins Mill Road, Gaithersburg, Maryland 20878; 2: PathoGenesis Corp., Department of Tuberculosis and Infectious Diseases, 201 Elliott Avenue, West, Seattle, Washington 98119

Content Type: Monograph

Publication Year: 1994

Category: Fungi and Fungal Pathogenesis; Clinical Microbiology

Book DOI: 10.1128/9781555818357

Chapter DOI: 10.1128/9781555818357.ch17

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Abstract:

This chapter reviews the development of genetic systems for the expression of foreign genes in mycobacteria. Mycobacteriophage have been used for many years to type mycobacterial isolates and have more recently been modified as vectors for efficient delivery of foreign DNA into mycobacteria. Plasmid-based expression systems extend the capabilities of phage-based systems by providing increased cloning capacity, increased copy number in the mycobacterial host, and ease of manipulation. Various derivatives of the pAL5000 plasmid replicon have been combined with Escherichia coli plasmid replication origins, such as ColEl and pl5A, by several groups to fashion E. coli-mycobacterium shuttle vectors. Expression of foreign proteins in recombinant organisms is influenced by many factors. Much of the increased interest in mycobacteria is a result of the emergence of multidrug-resistant Mycobacterium tuberculosis. Complex surface macromolecules, such as oligosaccharides and glycolipids, require the products of many genes and the correct cell envelope structure for proper assembly. BCG, the current vaccine against tuberculosis, has provided variable protective efficacy against tuberculosis in different vaccine trials. Immunization of chickens with recombinant Mycobacterium smegmatis expressing an Eimeria acervulina surface antigen elicits partial protection against coccidiosis.

Citation: Burlein J, Offutt S, Hanson M, Stover C. 1994. Expression of Foreign Genes in Mycobacteria, p 239-252. In Bloom B (ed), Tuberculosis. ASM Press, Washington, DC. doi: 10.1128/9781555818357.ch17

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Expression of Foreign Genes in Mycobacteria

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Figure 1

Mycobacterial extrachromosomal (pMV261) and integrative (pMV361) expression vectors (modified from Stover et al., 1991 ). Common elements in these two vectors include an expression cassette, the Tn905-derived aph gene (kanamycin resistance), and an origin of replication functional in E. coli (oriE) derived from pUC19. The expression cassette contains 0.4 kb of the 5ʹ end of the BCG hsp-60 gene, including the promoter region, ribosome-binding site, and first six codons (MAKTIA) as well as a multiple cloning site and the E. coli rmABt1 transcriptional terminator. The vectors differ by inclusion of either a mycobacterial plasmid replication origin (oriM) or the attP and int genes of mycobacteriophage L5.

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Figure 1

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Figure 2

Expression of foreign antigens in different BCG substrains. BCG culture lysates equivalent to approximately 5 x 106 bacteria were analyzed by SDS-PAGE and Coomassie blue staining (A) or by immunoblotting with antibodies specific for foreign antigens B. burgdorferi OspA (B, C) or HIV-1 gpl20 (D). Panel A shows the total proteins expressed by nonrecombinant BCG. Panels B through D show relevant portions of immunoblots of different BCG recombinants expressing the indicated foreign antigens.

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Figure 2

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