New Recombinant Mycobacterium bovis BCG Expression Vectors: Improving Genetic Control over Mycobacterial Promoters

Author(s)

Kanno, Alex I. ^{[1, 2]} ; Goulart, Cibelly ^{[1, 2]} ; Rofatto, Henrique K. ^{[1, 2]} ; Oliveira, Sergio C. ^[3] ; Leite, Luciana C. C. ^[1] ; McFadden, Johnjoe ^[4]

Affiliation

^[1] Inst Butantan, Ctr Biotecnol, Sao Paulo, SP - Brazil

^[2] USP IPT IB, Programa Posgrad Interunidades Biotecnol, Sao Paulo - Brazil

^[3] Univ Fed Minas Gerais, Dept Bioquim & Imunol, Belo Horizonte, MG - Brazil

^[4] Univ Surrey, Fac Hlth & Med Sci, Dept Microbial & Cellular Sci, Guildford GU2 5XH, Surrey - England

• Document type: Journal article
• Source: Applied and Environmental Microbiology; v. 82, n. 8, p. 2240-2246, APR 2016.
• Web of Science Citations: 4

Abstract

The expression of many antigens, stimulatory molecules, or even metabolic pathways in mycobacteria such as Mycobacterium bovis BCG or M. smegmatis was made possible through the development of shuttle vectors, and several recombinant vaccines have been constructed. However, gene expression in any of these systems relied mostly on the selection of natural promoters expected to provide the required level of expression by trial and error. To establish a systematic selection of promoters with a range of strengths, we generated a library of mutagenized promoters through error-prone PCR of the strong P-L5 promoter, originally from mycobacteriophage L5. These promoters were cloned upstream of the enhanced green fluorescent protein reporter gene, and recombinant M. smegmatis bacteria exhibiting a wide range of fluorescence levels were identified. A set of promoters was selected and identified as having high (pJK-F8), intermediate (pJK-B7, pJK-E6, pJK-D6), or low (pJK-C1) promoter strengths in both M. smegmatis and M. bovis BCG. The sequencing of the promoter region demonstrated that it was extensively modified (6 to 11%) in all of the plasmids selected. To test the functionality of the system, two different expression vectors were demonstrated to allow corresponding expression levels of the Schistosoma mansoni antigen Sm29 in BCG. The approach used here can be used to adjust expression levels for synthetic and/or systems biology studies or for vaccine development to maximize the immune response. (AU)

• FAPESP's process: 08/04631-7 - Recombinant BCG expressing Schistosoma mansoni surface antigens: immune response and protection assessment
• Grantee: Alex Issamu Kanno
• Support Opportunities: Scholarships in Brazil - Doctorate (Direct)