Expression and purification of the Mycobacterium tuberculosis complex-restricted antigen CFP21 to study its immunoprophylactic potential in mouse model

Protein Expr Purif. 2006 Aug;48(2):274-80. doi: 10.1016/j.pep.2006.03.010. Epub 2006 Mar 30.

Abstract

Secreted proteins encoded by different regions of difference (RDs) from the genome of Mycobacterium tuberculosis have been considered as attractive candidates for vaccination against tuberculosis owing to their absence in most BCG strains. In this study, the structural gene for the RD2 locus encoding protein CFP21 was PCR amplified and expressed as a fusion protein with hexahistidine residues in Escherichia coli. Expression of CFP21 in E. coli under transcriptional regulation of the T7 promoter yielded a protein located within inclusion bodies. The inclusion bodies were solubilized in the presence of 8M urea and the protein was purified to homogeneity under denaturing conditions at low pH using nitrilotriacetic acid (Ni-NTA) affinity chromatography. The denatured protein was renatured by gradient dialysis against a decreasing concentration of urea. The purified protein was shown to have esterase activity. CFP21 protein was evaluated for immunogenicity in C57BL/6J mice. We observed an elevated T cell proliferative response and production of IFN-gamma and IL-12 (p40). CFP21 also induced an optimum level of cytotoxic T cell activity and induced a strong humoral response as indicated by higher levels of specific IgG1 and IgG2a antibody isotypes. In addition, a moderate level of protection was observed against experimental tuberculosis. This is the first report describing esterase activity of the M. tuberculosis complex-restricted protein CFP21 and its protective potential against experimental tuberculosis.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Antigens, Bacterial / biosynthesis
  • Antigens, Bacterial / genetics
  • Antigens, Bacterial / immunology*
  • Antigens, Bacterial / isolation & purification*
  • Disease Models, Animal
  • Escherichia coli / genetics
  • Gene Expression*
  • Mice
  • Mice, Inbred C57BL
  • Models, Animal*
  • Models, Immunological
  • Mycobacterium tuberculosis / genetics
  • Mycobacterium tuberculosis / immunology*
  • Phylogeny
  • Recombinant Fusion Proteins / biosynthesis
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / immunology
  • Recombinant Fusion Proteins / isolation & purification
  • Tuberculosis / immunology
  • Tuberculosis / prevention & control

Substances

  • Antigens, Bacterial
  • Recombinant Fusion Proteins