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Proceedings of the 31 st European Peptide SymposiumMichal Lebl, Morten Meldal, Knud J. Jensen, Thomas Hoeg-Jensen (Editors)European Peptide Society, 2010Octapeptide Ligands with Affinity for RecombinantErythropoietin Derived From the Screening ofCombinatorial LibrariesMaría C. Martínez-Ceron 1 , Mariela M. Marani 1 , Marta Taulés 2 ,Marina Etcheverrigaray 3 , Fernando Albericio 4 , Osvaldo Cascone 1 ,and Silvia A. Camperi 11 Cátedra de Microbiología Industrial y Biotecnología, Facultad de Farmacia yBioquímica, Universidad de Buenos Aires, Junin 956, Bs. As., 1113, Argentina; 2 ServeisCientificotècnics, Universitat de Barcelona, Barcelona, 08028, Spain; 3 Laboratorio deCultivos Celulares, Facultad de Bioquímica y Ciencias Biológicas, UNL. CiudadUniversitaria, Santa Fe, 3000, Argentina; 4 IRB Barcelona and CIBER-BBN,Parc Científic de Barcelona, Barcelona, 08028, SpainIntroductionErythropoietin (Epo), glycoprotein hormone produced in mammalian kidney and liver, isthe main inducer and regulator of red cell proliferation and differentiation in bone marrow.Recombinant erythropoietin (rhEpo) is used for therapeutics of anemia associated withchronic renal disease, AZT-induced anemia of AIDS and for the treatment of cancerpatients on chemotherapy and surgical patients to avoid the need for a red blood celltransfusion [1]. Affinity Chromatography (AC) is ideally suited for the purification oftherapeutic proteins as it is the most effective method for the direct isolation andpurification of biomolecules from complex mixtures. Its good selectivity minimizescontamination and yields samples of high purity in a single step [2]. Successful separationby AC requires the availability of a ligand with satisfactory affinity and selectivity. Smallpeptides consisting of a few amino acids represent promising affinity ligand candidates forindustrial separations. Peptide ligands are much more physically and chemically stable thanantibody ligands and are very resistant to proteolytic cleavage. They can be readilysynthesized in bulk amounts at a lower cost under good manufacturing practices (GMP) bystandard chemistry. Also, peptides can be easily modified by chemical methods to facilitateproduct elution under mild conditions. Furthermore, peptides allow site-directedimmobilization and high ligand density and the matrices are more robust during elution andregeneration than protein-based affinity matrices such as monoclonal antibodies. Moreover,in the case of leakage into the product, peptides have low toxicity and generate lowimmune responses compared with proteins, dyes and transition metal ion ligands [3]. Whenleakage does occur, small peptide molecules can be easily removed from a macromolecularproduct. The combinatorial synthesis of peptide libraries allows the production of millionsof peptides, thus greatly facilitating the discovery of suitable ligands for a given protein ofinterest. The preparation of combinatorial libraries by the divide–couple–recombine (DCR)or mix and split solid-phase method assures a theoretically even representation of thelibrary members and a one-bead-one-compound distribution [4,5].In previous studies, we developed a rapid and inexpensive strategy for theidentification of peptides contained on positive beads, using matrix-assisted laserdesorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS), and4-hydroxymethylbenzoic acid (HMBA) immobilized in ChemMatrix resin [6-8].The aim of this study was to apply that strategy for the identification of peptideligands with affinity for rhEpo and to attach these peptides to agarose in order to purifyrhEPO by AC.Results and DiscussionBy the screening of a one-bead-one peptide library composed of 132,000 tetrapeptides withthe combination of all the natural amino acids except Cys, we obtained low-affinitytetrapeptide ligands for rhEpo. Cys was omitted in order to prevent disulfide bridgeformation. Afterward, a one-bead-one peptide combinatorial library containing 100,000octapeptides XXXFXXAG where X=A, D, E, F, H, L, N, P, S or T was synthesized onHMBA-ChemMatrix resin by the DCR method using Fmoc chemistry. Side-chaindeprotection was carried out with TFA. For the library screening, the rhEpo was coupled194