Phage display combinatorial libraries of short peptides: ligand selection for protein purification

A library of heptapeptides displayed on the surface of filamentous phage M1 3 was evaluated as a potential source of affinity ligands for the purificat ion of Rhizomucor miehei lipase. Two independent selection (biopanning) pro tocols were employed: the enzyme was either physically adsorbed on polystyr ene or chemically immobilized on small magnetic bends. From screening with the polystyrene-adsorbed lipase it was found that there was a rapid enrichm ent of the library with "doublet" clones i.e. the phage species which carri ed two consecutive sequences of heptapeptides, whilst no such clones were o bserved from the screening using lipase attached to magnetic beads. The bin ding of the best clones to the enzyme was unambiguously confirmed by ELISA. However the synthetic heptapeptide of identical sequence to the best "mono meric" clone did not act as a satisfactory affinity ligand after immobiliza tion on Sepharose. This indicated that the interaction with lipase was due to both the heptapeptide and the presence of a part of the phage coat prote in. This conclusion was further verified by immobilizing the whole phage on the surface of magnetic beads and using the resulting conjugate as an affi nity adsorbent. The scope of application of this methodology and the possib ility of preparing phage-based affinity materials are briefly discussed. (C ) 2001 Elsevier Science Inc. All rights reserved.