Overexpression, purification and validation of antigenic Salmonella enterica serovar Typhi proteins identified from LC-MS/MS

Appl Biochem Biotechnol. 2014 Nov;174(5):1897-906. doi: 10.1007/s12010-014-1173-y. Epub 2014 Aug 23.

Abstract

In our earlier study, an immunoblot analysis using sera from febrile patients revealed that a 50-kDa band from an outer membrane protein fraction of Salmonella enterica serovar Typhi was specifically recognized only by typhoid sera and not sera from other febrile illnesses. Here, we investigated the identities of the proteins contained in the immunogenic 50-kDa band to pinpoint antigens responsible for its immunogenicity. We first used LC-MS/MS for protein identification, then used the online tool ANTIGENpro for antigenicity prediction and produced recombinant proteins of the lead antigens for validation in an enzyme-linked immunosorbent assay (ELISA). We found that proteins TolC, GlpK and SucB were specific to typhoid sera but react to antibodies differently under native and denatured conditions. This difference suggests the presence of linear and conformational epitopes on these proteins.

Publication types

  • Research Support, Non-U.S. Gov't
  • Validation Study

MeSH terms

  • Bacterial Outer Membrane Proteins / chemistry*
  • Bacterial Outer Membrane Proteins / immunology*
  • Bacterial Outer Membrane Proteins / isolation & purification
  • Chromatography, Liquid
  • Epitope Mapping
  • Epitopes / chemistry*
  • Epitopes / immunology*
  • Humans
  • Immunoblotting
  • Mass Spectrometry
  • Salmonella typhi / chemistry*
  • Salmonella typhi / immunology*
  • Typhoid Fever / immunology*
  • Typhoid Fever / microbiology
  • Up-Regulation

Substances

  • Bacterial Outer Membrane Proteins
  • Epitopes