Fig 4.
Affinity chromatography of Strep-tagged LcrV. (A) Cleared lysates of Y. enterocolitica CT1 (ΔlcrV) harboring plcrVW22703 or Strep-tagged LcrV (plcrVS1, plcrVS20, plcrVS55, plcrVS170, plcrVS228, plcrVS270, and plcrVS324) were derived from cultures grown for 3 h at 37°C in TSB supplemented with 5 mM EGTA to chelate calcium and 1 mM IPTG to induce the expression of plasmid-borne lcrV alleles. Cleared lysates (L) were subjected to affinity chromatography on Strep-Tactin resin and eluted with desthiobiotin (E). Proteins in both samples were analyzed by Coomassie blue-stained SDS-PAGE. The migratory positions were proteins with known molecular mass (in kDa, indicated on the left). (B) Samples were subjected to immunoblotting with rabbit antisera raised against LcrV (αLcrV), LcrG (αLcrG), and RpoA (αRpoA) or with monoclonal antibody against the Strep tag (αStrep).