CONSTRUCTION OF TRANSFORMING-GROWTH-FACTOR-ALPHA (TGF-ALPHA) PHAGE LIBRARY AND IDENTIFICATION OF HIGH BINDERS OF EPIDERMAL GROWTH-FACTOR RECEPTOR (EGFR) BY PHAGE DISPLAY

TGF-alpha, a 50 amino acid growth factor containing 3 disulfide bonds, was fused to the N-terminal domain of the pill protein of fusN, a der ivative of phagemid fd-tet, to form a TGF-alpha phage. The fusion phag e showed binding activity to epidermal growth factor receptor (EGFR). A library of approximately 4 x 10(7) variants of TGF-alpha was generat ed with substitutions of total of 10 amino acids located in the C-loop region. This C-loop subdomain of TGF-alpha consists of a small antipa rallel double hairpin structure involving interactions between intra-p olypeptide segments. Mutants isolated from the phage library with grea tly increased binding affinity were selected through panning with A431 cells (a cell line expressing an elevated number of EGFRs), Following two rounds of stringent selection, variant phages with higher binding affinity than wild type TGF-alpha were identified and the phage DNAs were sequenced for the alignment analysis, Absolute selection at posit ion 42 as Arg, preferential selection at position 38 and 45 as Tyr or Phe with aromatic side chain and selection at position 41 with acidic residues, were obtained, Although an amino acid residue with smaller s ide chain at position 35 and one with larger side chain at position 36 were preferred, the steric hindering of the structure in side chains was minimized between these adjacent amino acids.