title: Affinity chromatography based on a combinatorial strategy for rerythropoietin purification.
abstract: Small peptides containing fewer than 10 amino acids are promising ligand candidates with which to build affinity chromatographic systems for industrial protein purification. The application of combinatorial peptide synthesis strategies greatly facilitates the discovery of suitable ligands for any given protein of interest. Here we sought to identify peptide ligands with affinity for recombinant human erythropoietin (rhEPO), which is used for the treatment of anemia. A combinatorial library containing the octapeptides X-X-X-Phe-X-X-Ala-Gly, where X = Ala, Asp, Glu, Phe, His, Leu, Asn, Pro, Ser, or Thr, was synthesized on HMBA-ChemMatrix resin by the divide-couple-recombine method. For the library screening, rhEPO was coupled to either Texas Red or biotin. Fluorescent beads or beads showing a positive reaction with streptavidin-peroxidase were isolated. After cleavage, peptides were sequenced by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Fifty-seven beads showed a positive reaction. Peptides showing more consensuses were synthesized, and their affinity to rhEPO was assessed using a plasma resonance biosensor. Dissociation constant values in the range of 1-18 μM were obtained. The best two peptides were immobilized on Sepharose, and the resultant chromatographic matrixes showed affinity for rhEPO with dissociation constant values between 1.8 and 2.7 μM. Chinese hamster ovary (CHO) cell culture supernatant was spiked with rhEPO, and the artificial mixture was loaded on Peptide-Sepharose columns. The rhEPO was recovered in the elution fraction with a yield of 90% and a purity of 95% and 97% for P1-Sepharose and P2-Sepharose, respectively.
authors.affiliation: Cátedra de Microbiología Industrial y Biotecnología, Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, Junín 956, (1113) Buenos Aires, Argentina.
authors.forename: María C
authors.initials: MC
authors.lastname: Martínez-Ceron
authors.forename: Mariela M
authors.initials: MM
authors.lastname: Marani
authors.forename: Marta
authors.initials: M
authors.lastname: Taulés
authors.forename: Marina
authors.initials: M
authors.lastname: Etcheverrigaray
authors.forename: Fernando
authors.initials: F
authors.lastname: Albericio
authors.forename: Osvaldo
authors.initials: O
authors.lastname: Cascone
authors.forename: Silvia A
authors.initials: SA
authors.lastname: Camperi
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D002846:Chromatography, Affinity / Q000379:methods*; D020650:Combinatorial Chemistry Techniques; D006224:Cricetinae; D003412:Cricetulus; D004921:Erythropoietin / Q000737:chemistry / Q000302:isolation & purification*; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D011994:Recombinant Proteins; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013816:Thermodynamics
chemical_list: D010446:Peptide Fragments; D011994:Recombinant Proteins; D004921:Erythropoietin