Overexpression, refolding, and purification of the major immunodominant outer membrane porin OmpC from Salmonella typhi: characterization of refolded OmpC

Protein Expr Purif. 2005 Mar;40(1):126-33. doi: 10.1016/j.pep.2004.12.023.

Abstract

The major immunodominant integral outer membrane protein C (OmpC) from Salmonella typhi Ty21a was overexpressed, without the signal peptide, in Escherichia coli. The protein aggregates as inclusion bodies (IBs) in the cytoplasm. OmpC from IBs was solubilized with 4 M urea and refolded. This involved rapid dilution of unfolded OmpC into a refolding buffer containing polyoxyethylene-9-lauryl ether (C(12)E(9)) and glycerol. The refolded OmpC (rfOmpC) was shown to be structurally similar to the native OmpC by SDS-PAGE, Western blotting, tryptic digestion, ultrafiltration, circular dichroism, and fluorescence spectroscopic techniques. Crystals of rfOmpC were obtained in preliminary crystallization trials. The rfOmpC also sets a stage for rational design by recombinant DNA technology for vaccine design and high resolution structure determination.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Enzyme Stability
  • Hot Temperature
  • Immunodominant Epitopes / chemistry
  • Immunodominant Epitopes / genetics
  • Immunodominant Epitopes / isolation & purification
  • Porins / biosynthesis
  • Porins / chemistry
  • Porins / genetics*
  • Porins / immunology*
  • Protein Denaturation
  • Protein Folding
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / isolation & purification
  • Salmonella typhi / chemistry
  • Salmonella typhi / immunology*
  • Up-Regulation

Substances

  • Immunodominant Epitopes
  • OmpC protein
  • Porins
  • Recombinant Proteins