Cloning, expression, and affinity purification of recombinant Shigella flexneri invasion plasmid antigens IpaB and IpaC

Protein Expression and Purification
William D PickingWendy L Picking

Abstract

Shigella flexneri and related enteropathogenic bacteria are important agents of bacillary dysentery, a potentially life-threatening illness for children in underdeveloped regions of the world. Onset of shigellosis stems from S. flexneri invasion of colonic epithelial cells, leading to localized cell death and inflammation. Invasion plasmid antigens (Ipa) B, C, and D are three secreted proteins encoded by the large virulence plasmid of S. flexneri that have been implicated as essential effectors of this cell invasion process. These proteins are expressed as part of the ipa operon and are among the major targets of the host immune response to shigellosis. Biochemical characterization of the Ipa invasins has been complicated by the fact they have not been purified in the quantities needed for detailed in vitro analysis. Here we describe the first cloning, expression, and extensive purification of IpaB and IpaC fusion proteins from Escherichia coli for use in dissecting of the protein biochemistry of S. flexneri pathogenesis. A variety of approaches were used to prepare significant quantities of these proteins in their soluble forms, including the use of different host cell lines, modification of bacterial growth conditions, and th...Continue Reading

Citations

Feb 22, 2003·Infection and Immunity·Amanda T HarringtonWilliam D Picking
Jul 29, 2008·Microbial Pathogenesis·Christina M TerryWilliam D Picking
Aug 18, 2004·Journal of Biotechnology·Amy F MacRaeRobert I Bolla
Apr 25, 2006·Journal of Molecular Biology·Lingling ZhangRoberto N De Guzman
Jan 10, 2014·Clinical and Vaccine Immunology : CVI·R W KaminskiE V Oaks
Nov 13, 2002·The Journal of Biological Chemistry·Lisa A KueltzoC Russell Middaugh
Mar 15, 2018·Journal of Translational Medicine·Franklin R ToapantaMarcelo B Sztein
Feb 5, 2000·The Journal of Immunology : Official Journal of the American Association of Immunologists·T SamandariM B Sztein
May 18, 2001·Analytical Biochemistry·S O HaticW D Picking

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