An inducible expression system for high-level expression of recombinant proteins in slow growing mycobacteria

• The novel plasmid pJEX93 was constructed for inducible expression of recombinant proteins in slow growing mycobacteria.
• The Mycobacterium tuberculosis hspX promoter facilitates recombinant protein expression under low oxygen tension.
• Recombinant products can be purified from mycobacterial lysates by virtue of a C-terminal histidine tag.
• Recombinant proteins produced in this system display immunological and biochemical properties of their native counterparts.

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.