FIG 6.
PorZ binds PorU sortase with an affinity that is enhanced by A-LPS. (A) In a pull-out assay, purified recombinant proteins 6His-PorU and PorZ (right two lanes) were mixed and incubated with cobalt-based magnetic beads. The beads were washed, and the bound protein was eluted. The resulting fractions were analyzed by SDS-PAGE and Coomassie blue staining. TS, sample before loading on beads; FT, flowthrough; W, bead wash; E, eluted proteins. (B to D) Fluorescently-labeled PorZ from P. gingivalis (PorZPg) was titrated with increasing concentrations of PorU from P. gingivalis (PorUPg) without A-LPS (B) in the presence of 1 × 105 EU/ml A-LPS from W83 (C) or titrated with increasing concentrations of PorU from T. forsythia (PorUTf). In a reciprocal experiment, fluorescently-labeled PorZ from T. forsythia (PorZTf) was titrated with increasing concentrations of PorU from P. gingivalis (PorUPg). (E) Binding data were curve-fitted and used to determine Kd values. The results are presented as the means ± the SD of three experiments.