title: Overexpression, refolding, and purification of the major immunodominant outer membrane porin OmpC from Salmonella typhi: characterization of refolded OmpC.
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.
authors.affiliation: Department of Genetic Engineering, School of Biotechnology, Madurai Kamaraj University, Madurai 625 021, India.
authors.forename: P D
authors.initials: PD
authors.lastname: Kumar
authors.forename: S
authors.initials: S
authors.lastname: Krishnaswamy
mesh_terms: D004795:Enzyme Stability; D006358:Hot Temperature; D016056:Immunodominant Epitopes / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D018272:Porins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics* / Q000276:immunology*; D011489:Protein Denaturation; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification; D012485:Salmonella typhi / Q000737:chemistry / Q000276:immunology*; D015854:Up-Regulation
chemical_list: D016056:Immunodominant Epitopes; C113724:OmpC protein; D018272:Porins; D011994:Recombinant Proteins