Figure 4

Copurification of LcrG variants with His6-tagged LcrV. Cells of Y. pestis KIM8-3002.8 (ΔlcrGV2) containing plasmids pAra-HT-V and pAraG18K (lanes 1 to 3), pJM131 (LcrG S23R) (lanes 4 to 6), pJM99 (LcrG L30R) (lanes 7 to 9), or pJM89 (LcrG A16R) (lanes 10 to12) were grown in TMH with calcium and induced with 0.2% (wt/vol) arabinose prior to temperature shift to 37°C. Cultures were harvested after 4 h of growth at 37°C, and cellular extracts were disintegrated using a French press (20,000 lb/in2). Unbroken cells and large debris were removed by centrifugation (14,000 × g) for 10 min and the cleared extracts (lanes 1, 4, 7, and 10) were applied to a Talon column. Proteins that did not bind were collected as the flowthrough fraction (lanes 2, 5, 8, and 11). Proteins were eluted from the column with 50 mM imidazole and collected (lanes 3, 6, 9, and 12). All protein samples were resolved by SDS-PAGE in a 12.5% polyacrylamide gel after dilution in 2X SDS sample buffer and analyzed by immunoblotting with α-LcrG and α-LcrV. Proteins were visualized by probing with alkaline phosphatase-conjugated secondary antibodies and developing with NBT-BCIP.