FIGURE 8.

Definition of the Galα1–4Gal-binding region of SadP and its binding kinetics. A, the N-terminal fragment of SadP, N(31–328), excluding the C-terminal tandem repeat region, was cloned and expressed in E. coli. B, SDS-PAGE of N(31–328). For ligand blotting, full-length SadP and N(31–328) were separated with native PAGE and transferred onto nitrocellulose membrane and probed with biotinylated pigeon ovomucoid (1 and 0.1 μg). C, pigeon ovomucoid was coated on a Biacore sensor chip, the binding of SadP or N(31–328) to the coated surface was recorded, and the association and dissociation constants were calculated. For clarity, only binding curves of a 10 μg/ml concentration of the adhesins are shown. D, SadP or N(31–328) was mixed with synthetic galabiose inhibitor (Galα1–4Galβ1-OMe) at the concentrations indicated and allowed to adhere to ovomucoid-coated sensor chip. Binding is expressed as percentage of binding in the absence of inhibitor.