Bio-layer interferometry using optic-based biosensor for screening of affinity ligand for rhEPO

MC MARTÍNEZ CERON; MM MARANI; M ETCHEVERRIGARAY; M TAULÉS; F. ALBERICIO; O CASCONE; SA CAMPERI

Puerto Madryn, Chubut, Argentina

Otro; XLVI Reunión Anual- Sociedad Argentina de Investigación en Bioquímica y Biología Molecular; 2010

Sociedad Argentina de Investigación en Bioquímica y Biología Molecular

Bio-layer interferometry is used in optic-based biosensors (ForteBio OCTET RED)for protein-ligands interactions analysis.Short peptides are ideal to be used as affinity ligands for protein purification.Divide-couple-recombine (DCR) method allows obtaining a library with allpossible combinations of the amino acids in the form of "one bead-one peptide".An octapeptide library XXXOXXOO (O=fixed positions and X =Ala, Asp, Glu,Phe, His, Leu, Asn, Pro, Ser y Thr) was synthesised.After the library screening 60 peptides with affintiy for rhEPO were selected. Tostudy the peptide affinity for the rhEPO we used a BLI biosensor. rhEPO waslabeled with biotin and it was immobilised on a super streptavidin biosensor tip(SSA). The running buffer was phosphates 20 mM, pH 7.0, Tween 20, 0.05%. Thereference biosensor tip was a SSA with immobilised biocitin.Peptides with the highest affinities were immobilised on agarose. All the peptideagarosematrices showed affinity for rhEPO. Also we evaluated the affinity of thepeptide-agarose matrices for bovine seroalbumin, usually present in the culturesupernatant. Those peptides with the highest selectivity were selected for futuredevelopment of a rhEPO purification method.