Selective Binding of Inhibitor-Assisted Surface-Imprinted Core/Shell Microbeads in Protein Mixtures

An innovative approach for molecularly imprinting proteins, i.e., inhibitor-assisted imprinting was used for addressing the major challenge of protein purification from biotechnologically relevant media. Pepstatin-assisted pepsin surface-imprinted microbeads were synthesized as a selective sorbent for solid phase extraction (SPE). The amino-functionalized porous core was prepared by co-condensation and post-grafting strategies. The immobilized inhibitor pre-organizes the template pepsin with a defined orientation prior to imprinting ensuring enhanced efficiency of the imprinting process. 3-aminophenylboronic acid (APBA) was used as a functional monomer establishing organic nanoscale films comprising poly(3-aminophenylboronic acid) (pAPBA) within the pores of the beads in the presence (MIP) and absence (NIP) of pepsin. Next to a detailed characterization of these advanced functional hybrid materials it was demonstrated that such porous surface-imprinted core/shell beads feature exquisite selectivity for pepsin during individual protein rebinding studies, and more importantly, during competitive rebinding studies in protein mixtures.