Cloning, Expression, and Affinity Purification of RecombinantShigella flexneriInvasion Plasmid Antigens IpaB and IpaC

Combination of Two Separate Binding Domains Defines Stoichiometry between Type III Secretion System Chaperone IpgC and Translocator Protein IpaB

“…Inactivation of ipgC led to loss of virulence in HeLa cells, possibly due to decreased stability of IpaB and IpaC (13). We and others have shown that heterologous expression of ipaB leads to the death of E. coli and IpaB is observable only by immunoblot while IpaC accumulates in inclusion bodies (32). However, improved expression of ipaB and better solubility of IpaC is achieved in E. coli when co-expressed with ipgC.…”

“…1B), and this correlates with low protein levels of IpaB evaluated by immunodetection assays (32). IpaC, though expressed in significant amounts, remained mostly insoluble when standard expression conditions were employed (32). Although heterologous co-expression of the ipaB/ipaC could be observed (Fig.…”

“…Individually, expression of ipaB led to death of E. coli as evidenced by decreasing cell density (Fig. 1B), and this correlates with low protein levels of IpaB evaluated by immunodetection assays (32). IpaC, though expressed in significant amounts, remained mostly insoluble when standard expression conditions were employed (32).…”

Production of IFN-γ and IL-10 to Shigella Invasins by Mononuclear Cells from Volunteers Orally Inoculated with a Shiga Toxin-Deleted Shigella dysenteriae Type 1 Strain

“…The coding sequence of ipaC was amplified from the virulence plasmid of S. flexneri 2a strain 2457T using PCR and inserted into the plasmid vector pET15b (Novagen, Madison, WI) to give pWPC15 for expression in Escherichia coli BL21(DE3) as described previously (25,26). The coding sequence of ipaD, also from S. flexneri, was cloned into the pET15b vector to give pWPD10 (27).…”

“…Synthesis of IpaC or IpaD was induced in 400 ml mid-log phase cultures with 1 mM isopropylthio-␤-D-galactoside. Recombinant IpaC and IpaD were purified by virtue of a short (20-aa) leader sequence that contained six tandem histidine residues (His-Tag) using nickel chelation affinity chromatography as previously described (25,27). Briefly, after induction of protein synthesis, the cells were harvested by centrifugation, and the pellets were resuspended in 20 mM Tris-HCl (pH 7.9), 0.5 M NaCl, and 5 mM imidazole (binding buffer).…”