Expression and characterization of Rv2430c, a novel immunodominant antigen of Mycobacterium tuberculosis.
About 10% of the coding sequence of Mycobacterium tuberculosis corresponds to hitherto unknown members of the PE and PPE protein families which display significant sequence and length variation at their C-terminal region. It has been suggested that this could possibly represent a rich source of antigenic variation within the pathogen. We describe the purification and biophysical characterization of the recombinant PPE protein coded by hypothetical ORF Rv2430c, a member of the PPE gene family that was earlier shown to induce a strong B cell response. Expression of the recombinant PPE protein in Escherichia coli led to its localization in inclusion bodies and subsequent refolding using dialysis after its extraction from the same resulted in extensive precipitation. Therefore, an on-column refolding strategy was used, after which the protein was found to be in the soluble form. CD spectrum of the recombinant protein displayed predominantly alpha helical content (81%) which matched significantly with in silico and web-based secondary structure predictions. Furthermore, fluorescence emission spectra revealed that aromatic amino acids are buried inside the protein, which are exposed to aqueous environment under 8M urea. These results, for the first time, provide evidence on the structural features of PPE family protein which, viewed with its reported immunodominant characteristics, have implications for other proteins of the PE/PPE family.
Choudhary
Rakesh K
RK
Centre for DNA Fingerprinting and Diagnostics, Hyderabad, India.
Pullakhandam
Raghu
R
Ehtesham
Nasreen Z
NZ
Hasnain
Seyed E
SE
D020816:Amino Acid Motifs / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification*; D002853:Chromatography, Liquid; D002942:Circular Dichroism; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics; D016056:Immunodominant Epitopes / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification*; D009169:Mycobacterium tuberculosis / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D016366:Open Reading Frames / Q000235:genetics / Q000276:immunology; D017510:Protein Folding; D017433:Protein Structure, Secondary; D014508:Urea / Q000737:chemistry
D001426:Bacterial Proteins; D016056:Immunodominant Epitopes; C495699:Rv2430c protein, Mycobacterium tuberculosis; D014508:Urea