FIGURE 4.

The intact anchor peptide is required for binding of TbpB to a TbpA-Tf complex. Purified, polyhistidine-tagged, recombinant TbpA from A. pleuropneumoniae strain H49 was mixed with recombinant porcine Tf (left panel) or detergent-containing buffer alone (right panel) and incubated overnight at 4 °C. Buffer without (lanes 1 and 4) or with TbpB with a truncated (37–528) or intact (2–528) N-terminal anchor peptide region was added, and the mixtures were incubated overnight at 4 °C. Ni-NTA resin was added to the incubation mixtures, collected by centrifugation, and washed twice prior to the addition of SDS-PAGE buffer to elute bound proteins.