N-glycosylation and homodimeric folding significantly enhance the immunoreactivity of Mycobacterium tuberculosis virulence factor CFP32 when produced in the yeast Pichia pastoris

We previously reported that immunoreactivity of recombinant CFP32 (Rv0577), a virulence factor of Mycobacterium tuberculosis, was higher when produced in transformed Pichia pastoris as compared to transformed E. coli. In this study, we show that this difference is partly due to the N-glycosylation of the recombinant CFP32 (rCFP32) by the yeast Pichia pastoris. In addition, SDS-PAGE and western blotting analysis of Mycobacterium bovis BCG and yeast-produced rCFP32 showed the presence of a band corresponding to a homodimeric state of the protein, unlike that of rCFP32 produced in E. coli. Computational modeling indicates that a single cysteine residue at position 193 of each monomer might bond to stabilize the homodimeric state of CFP32. Computational study showed that this residue is buried inside the protein core of E. coli-produced rCFP32 suggesting that rCFP32 may adopt a different folding in P. pastoris and BCG, in which C193 is solvent exposed. Surprisingly, an enzyme-linked immunosorbent assay using a generated monoclonal antibody (14D4) reveals the presence of a differential epitope that appears to be the consequence of the protein dimerization of the yeast- and BCG-, but not E.coli– produced, CFP32 recombinant form. We conclude that, in addition to N-glycosylation, homodimeric folding significantly enhances the immunoreactivity of rCFP32 and may these post-translational modifications may factor into the structure and function of native M. tuberculosis CFP32. [Display omitted] •N-glycosylation is in part responsible for higher immunoreactivity of Mycobacterium tuberculosis virulence factor CFP32 when produced in transformed Pichia pastoris as compared to transformed E. coli.•Recombinant forms of CFP32 produced in Pichia pastoris and in BCG but not in E. coli showed the formation of homodimer.•In silico analysis of rCFP32 form produced in E. coli showed a folding unable to form homodimer. Thus, rCFP32 forms produced in P. pastoris and BCG may adopt a different folding allowing the formation of homodimer.•Monoclonal antibody 14D4 reveals the presence of a differential epitope that appears to be the consequence of the protein dimerization.