title: An inducible expression system for high-level expression of recombinant proteins in slow growing mycobacteria.
abstract: A novel protein expression vector utilising the inducible hspX promoter of Mycobacterium tuberculosis was constructed and evaluated in this study. High-level induction of three mycobacterial antigens, comprising up to 9% of bacterial sonicate, was demonstrated in recombinant Mycobacterium bovis BCG when grown under low-oxygen tension, which serves to enhance hspX promoter activity. Recombinant proteins were efficiently purified from bacterial lysates in a soluble form by virtue of a C-terminal 6-histidine tag. Purification of the immunodominant M. tuberculosis Ag85B antigen using this system resulted in a recombinant protein that stimulated significant IFN-γ release from Ag85B-reactive T cells generated after vaccination of mice with an Ag85B-expressing vaccine. Further, the M. tuberculosis L-alanine dehydrogenase (Ald) protein purified from recombinant BCG displayed strong enzymatic activity in recombinant form. This study demonstrated that high levels of native-like recombinant mycobacterial proteins can be produced in mycobacterial hosts, and this may aid the analysis of mycobacterial protein function and the development of new treatments.
authors.affiliation: Microbial Pathogenesis and Immunity Group, Department of Infectious Diseases and Immunology, Sydney Medical School, The University of Sydney, NSW 2006, Australia.
authors.forename: Lisa
authors.initials: L
authors.lastname: Leotta
authors.affiliation: Microbial Pathogenesis and Immunity Group, Department of Infectious Diseases and Immunology, Sydney Medical School, The University of Sydney, NSW 2006, Australia.
authors.forename: Joanne M
authors.initials: JM
authors.lastname: Spratt
authors.affiliation: Microbial Pathogenesis and Immunity Group, Department of Infectious Diseases and Immunology, Sydney Medical School, The University of Sydney, NSW 2006, Australia.
authors.forename: Carlyn U
authors.initials: CU
authors.lastname: Kong
authors.affiliation: Microbial Pathogenesis and Immunity Group, Department of Infectious Diseases and Immunology, Sydney Medical School, The University of Sydney, NSW 2006, Australia. Electronic address: jamie.triccas@sydney.edu.au.
authors.forename: James A
authors.initials: JA
authors.lastname: Triccas
mesh_terms: D000942:Antigens, Bacterial / Q000235:genetics / Q000276:immunology; D003001:Cloning, Molecular; D015964:Gene Expression Regulation, Bacterial; D023061:Gene Order; D005822:Genetic Vectors / Q000235:genetics; D009161:Mycobacterium / Q000235:genetics* / Q000254:growth & development*; D009163:Mycobacterium bovis / Q000235:genetics / Q000276:immunology; D010957:Plasmids / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology; D011994:Recombinant Proteins / Q000235:genetics*
chemical_list: D000942:Antigens, Bacterial; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins
keywords: Inducible expression; Mycobacterium; Protein purification; hspX promoter