FIG. 4.

GST-LcrG binds to LcrV. (A) Y. enterocolitica MC2 (lcrG)/pVL48 (gst-lcrG) was grown at 37°C and induced for type III secretion by the chelation of calcium ions. Expression of GST-LcrG was induced by the addition of 1 mM 1PTG to the culture medium. Cells (10¹² CFU) were harvested by centrifugation and lysed in a French pressure cell, insoluble material was removed by centrifugation at 100,000 × g, and the supernatant (L, load) was subjected to affinity chromatography on glutathione-Sepharose. Flowthrough (FT), wash (W), and eluate (E) fractions of 1 ml were collected and analyzed by silver-stained SDS-PAGE. Arrowheads indicate the positions of GST-LcrG (Black) and LcrV (white). The asterisk indicates the migration of an unknown Yersinia protein that binds to glutathione-Sepharose. (B) The load (L) and eluate (E; fraction 1) of the affinity chromatography analyses were analyzed by immunoblotting with antisera (LcrG, LcrV, LcrH, YopB, YopD, and CAT) and quantified. Results are reported as the ratio of signal intensity observed for the eluate fraction divided by that of the load fraction (E/L). The migration of molecular mass markers (M) is indicated in kilodaltons. The positions of GST-LcrG (filled arrowhead) and LcrV (open arrowhead) are indicated. As a control for the specific binding of LcrV to GST-LcrG, Y. enterocolitica expressing GST alone was subjected to affinity chromatography and analyzed by silver-stained SDS-PAGE (D) and immunoblotting (C).