Octapeptide ligands with affinity for recombinant erythropoietin derived from the screening of combinatorial libraries

M. C. MARTÍNEZ CERON; M. TAULÉS; M. M. MARANI; M. ETCHEVERRIGARAY; F. ALBERICIO; O. CASCONE; S. A. CAMPERI

Peptides. Copenhagen 2010: Tales of peptides

European Peptide Society

Lugar: Copenhagen; Año: 2010; p. 194 - 195

Recombinant erythropoietin (rhEPO) is used for therapeutics of anemia associated with chronic renal failure and for AZT-induced anemia of AIDS.  Monoclonal antibody (mAb) affinity chromatography and dye affinity chromatography are alternative techniques nowadays in use for its purification. MAb are expensive, thus increasing the cost of the final product. Affinity chromatography with Cibacron Blue as the ligand is widely used, but the selectivity is not high. The use of short peptides would result in a more economic process than with mAb and a more selective process than with dyes. Divide-couple-recombine (DCR) method allows obtaining a library with all possible combinations of the amino acids in the form of "one bead-one peptide". Peptide ligands can be selected from library screening. We have developed a rapid and non-expensive strategy for the identification of peptides contained on positive beads, by using matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF-MS), and 4-hydroxymethylbenzoic acid (HMBA). In this work the method designed was applied for the identification of peptide ligands for rhEPO purification. In previous works we have obtained low affinity tetrapeptide ligands for rhEPO. Taking into account our previous results, a combinatorial library containing the octapeptides XXXFXXAG where X=A, D, E, F, H, L, N, P, S or T was synthesised on HMBA-ChemMatrix resin by the DAR method using Fmoc chemistry. Side-chain deprotection was carried out with TFA. For the solid-supported library screening the rhEPO was coupled with either Texas Red or Biotin. Fluorescent beads or beads showing a positive reaction with streptavidin-peroxidase were isolated. After peptides cleavage with NH4OH, they were sequenced by MALDI-TOF-MS. 50 peptides beads showed positive reactions. Those peptides that showed more consensus were resynthesised and theirs affinity to rhEPO were evaluated using a plasma resonance biosensor and values of Kd between 10-5-10-6 M were obtained.  Peptides with the highest affinities were immobilised on agarose. All the peptide-agarose matrices showed affinity for rhEPO. Also we evaluated the affinity of the peptide-agarose matrices for bovine seroalbumin, usually present in the culture supernatants. Those peptides with the highest selectivity were selected for future development of a rhEPO purification method.