CLONING, EXPRESSION, AND AFFINITY PURIFICATION OF RECOMBINANT SHIGELLA-FLEXNERI INVASION PLASMID ANTIGENS IPAB AND IPAC

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 shigello sis stems from S. flexneri invasion of colonic epithelial cells, leadi ng to localized cell death and inflammation. Invasion plasmid antigens (Ipa) B, C, and D are three secreted proteins encoded by the large vi rulence plasmid of S. flexneri that have been implicated as essential effecters 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 Ip a invasins has been complicated by the fact they have not been purifie d in the quantities needed for detailed in vitro analysis. Here we des cribe the first cloning, expression, and extensive purification of Ipa B 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 prote ins in their soluble forms, including the use of different host cell l ines, modification of bacterial growth conditions, and the use of alte rnative plasmid expression vectors. Now that these Ipa proteins are av ailable in a highly pure form, it will be possible to initiate studies on their important biological and immunological properties as well as their recruitment into high-molecular-weight protein complexes. Toget her with IpaD (purified as part of a previous study), these purified p roteins will be useful for: (a) exploring properties of the host immun e response to S. flexneri invasion, (b) elucidating the specific bioch emical properties that lead to pathogen internalization, (c) analyzing the importance of specific Ipa protein complexes in host cell invasio n, and (d) monitoring, or perhaps even augmenting, the efficacy of liv e oral vaccines in human trials. (C) 1996 Academic Press, Inc.