FIG. 5.

Nickel chromatography, under native conditions, of recombinant amino-terminally-tagged ExoU. (A) SDS-PAGE (13.5% polyacrylamide stained with Coomassie blue R250) analysis of the cell lysate from host strain E. coli BL21(DE3) pLysS with pETexoU (N-terminal 10-histidine tag). Protein profiles of the cell lysate are shown as the material loaded onto a nickel column (Load), the flowthrough from the column (FT), wash fractions 1 and 2, and the elution fractions 1 to 5 in the presence of high concentrations of imidazole. The arrow denotes the position of His-ExoU. (B) SDS-PAGE analysis of peak eluate fractions from nickel chromatography of two expression constructs. Lane 1 contains the peak eluate fraction from an expression experiment using the pETexoUspcU construct to produce recombinant protein (His-ExoU and SpcU). Lane 2 contains a similar fraction from the expression of pETexoU (His-ExoU). Molecular weight (MW) standards (left [thousands]) and recombinant proteins (arrows) are indicated.