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.