CONICET | Buscador de Institutos y Recursos Humanos

The alginic acid, a constituent of marine algae, is a copolymer of β-3-D-mannuronic and α-L-guluronic acid. Yersinia outer protein P (YopP), a virulence factor of Yersinia enterocolitica (Ye), causes suppression of pro-inflammatory cytokines and induces apoptosis both in macrophages and in dendritic cells. Galectin-1 (Gal-1) is a ?proto-type? β-galactoside-binding lectin widely distributed in host tissues with an important immunomodulatory role. We previously demonstrated that Ye-induced apoptosis of macrophages depends on both YopP and Gal-1 and that Gal-1 binds in vitro to YopP preventing its auto-degradation. The aim of this study was to establish the optimal parameters for non-covalent peptides immobilization on sodium alginate beads to allow the purification of YopP-Gal-1 complex. In this study, we used alginate beads like solid support for non-covalent adsorption for proteins purification. The beads were prepared beginning with sodium alginate in the presence of calcium ions. Then, we immobilized polyclonal antibodies on alginate beads in water-acetonitrile medium. After sequential binding of YopP and Gal-1 on the bead, we performed enzyme-linked immunosorbent assay (ELISA), in order to evaluate bead capacity of immobilization and the level of non-specific interactions. The time and temperature of incubations and washing-equilibration procedures were optimized. The surface of the beads was blocked to avoid non-specific binding. The presence of Gal-1 and YopP was confirmed by Western blot. We successfully obtained the isolated Gal-1-YopP complex using immobilization of polyclonal antibodies anti-YopP adsorbed on alginate beads surface. We conclude that sodium alginate beads can be used for purification of YopP-Gal-1 complex.