title: Rational design and synthesis of affinity matrices based on proteases immobilized onto cellulose membranes.
abstract: Discovery of new protease inhibitors may result in potential therapeutic agents or useful biotechnological tools. Obtainment of these molecules from natural sources requires simple, economic, and highly efficient purification protocols. The aim of this work was the obtainment of affinity matrices by the covalent immobilization of dipeptidyl peptidase IV (DPP-IV) and papain onto cellulose membranes, previously activated with formyl (FCM) or glyoxyl groups (GCM). GCM showed the highest activation grade (10.2 µmol aldehyde/cm2). We implemented our strategy for the rational design of immobilized derivatives (RDID) to optimize the immobilization. pH 9.0 was the optimum for the immobilization through the terminal α-NH2, configuration predicted as catalytically competent. However, our data suggest that protein immobilization may occur via clusters of few reactive groups. DPP-IV-GCM showed the highest maximal immobilized protein load (2.1 µg/cm2), immobilization percentage (91%), and probability of multipoint covalent attachment. The four enzyme-support systems were able to bind at least 80% of the reversible competitive inhibitors bacitracin/cystatin, compared with the available active sites in the immobilized derivatives. Our results show the potentialities of the synthesized matrices for affinity purification of protease inhibitors and confirm the robustness of the RDID strategy to optimize protein immobilization processes with further practical applications.
authors.affiliation: a Center for Protein Studies, Faculty of Biology , University of Havana , Havana , Cuba.
authors.forename: Alberto
authors.initials: A
authors.lastname: Del Monte-Martínez
authors.affiliation: a Center for Protein Studies, Faculty of Biology , University of Havana , Havana , Cuba.
authors.forename: Jorge
authors.initials: J
authors.lastname: González-Bacerio
authors.affiliation: a Center for Protein Studies, Faculty of Biology , University of Havana , Havana , Cuba.
authors.forename: Bessy
authors.initials: B
authors.lastname: Cutiño-Avila
authors.affiliation: b Center for Process Engineering , Higher Polytechnic Institute "José Antonio Echeverría" , Havana , Cuba.
authors.forename: Jorge
authors.initials: J
authors.lastname: Rojas
authors.affiliation: a Center for Protein Studies, Faculty of Biology , University of Havana , Havana , Cuba.
authors.forename: Mae
authors.initials: M
authors.lastname: Chappé
authors.affiliation: a Center for Protein Studies, Faculty of Biology , University of Havana , Havana , Cuba.
authors.forename: Emir
authors.initials: E
authors.lastname: Salas-Sarduy
authors.affiliation: a Center for Protein Studies, Faculty of Biology , University of Havana , Havana , Cuba.
authors.forename: Isel
authors.initials: I
authors.lastname: Pascual
authors.affiliation: c Department of Biocatalysis , Institute for Catalysis, Higher Council for Scientific Research, Campus Cantoblanco , Madrid , Spain.
authors.forename: José M
authors.identifier: http://orcid.org/0000-0003-1627-6522
authors.initials: JM
authors.lastname: Guisán
mesh_terms: D000327:Adsorption; D000818:Animals; D029441:Carica; D002482:Cellulose / Q000737:chemistry*; D002645:Chickens; D018819:Dipeptidyl Peptidase 4 / Q000737:chemistry*; D004800:Enzymes, Immobilized / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D008567:Membranes, Artificial; D008958:Models, Molecular; D010084:Oxidation-Reduction; D010206:Papain / Q000737:chemistry*; D012685:Sepharose; D013552:Swine
chemical_list: D004800:Enzymes, Immobilized; D008567:Membranes, Artificial; D002482:Cellulose; D012685:Sepharose; D018819:Dipeptidyl Peptidase 4; D010206:Papain
keywords: Affinity matrix; cellulose membranes; dipeptidyl peptidase IV; papain; protein immobilization; rational design of immobilized derivatives