OPTIMIZATION OF NON-COVALENT PEPTIDE IMMOBILIZATION ON SODIUM ALGINATE BEADS FOR PROTEIN PURIFICATION PROCESS

JOFRÉ B; DAVICINO RC; DI GENARO M.S

San Luis

Congreso; SAMIGE; 2018

SAMIGE

The alginic acid, a constituent of marine algae, is a copolymer of b-3-D-mannuronic and a-L-guluronicacid. Yersinia outer protein P (YopP), a virulence factor of Yersinia enterocolitica (Ye), causessuppression of pro-inflammatory cytokines and induces apoptosis both in macrophages and indendritic cells. Galectin-1 (Gal-1) is a ?proto-type? b-galactoside-binding lectin widely distributed in hosttissues with an important immunomodulatory role. We previously demonstrated that Ye-inducedapoptosis of macrophages depends on both YopP and Gal-1 and that Gal-1 binds in vitro to YopPpreventing its auto-degradation. The aim of this study was to establish the optimal parameters fornon-covalent peptides immobilization on sodium alginate beads to allow the purification of YopP-Gal-1complex. In this study, we used alginate beads like solid support for non-covalent adsorption forproteins purification. The beads were prepared beginning with sodium alginate in the presence ofcalcium ions. Then, we immobilized polyclonal antibodies on alginate beads in water-acetonitrilemedium. After sequential binding of YopP and Gal-1 on the bead, we performed enzyme-linkedimmunosorbent assay (ELISA), in order to evaluate bead capacity of immobilization and the level ofnon-specific interactions. The time and temperature of incubations and washing-equilibrationprocedures were optimized. The surface of the beads was blocked to avoid non-specific binding. Thepresence of Gal-1 and YopP was confirmed by Western blot. We successfully obtained the isolatedGal-1-YopP complex using immobilization of polyclonal antibodies anti-YopP adsorbed on alginatebeads surface. We conclude that sodium alginate beads can be used for purification of YopP-Gal-1complex.