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 pIII protein of fusN, a derivative of phagemid fd-tet, to form a TGF-alpha phage. The fusion phage showed binding activity to epidermal growth factor receptor (EGFR). A library of approximately 4 x 10(7) variants of TGF-alpha was generated 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 antiparallel double hairpin structure involving interactions between intra-polypeptide segments. Mutants isolated from the phage library with greatly 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 position 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 side 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.
Tang
X B
XB
Department of Biochemistry, University of Alberta, Edmonton, Canada. xiaotang@pop.srv.ualberta.ca
Dallaire
P
P
Hoyt
D W
DW
Sykes
B D
BD
O'Connor-McCourt
M
M
Malcolm
B A
BA
D000595:Amino Acid Sequence; D000818:Animals; D001435:Bacteriophages / Q000235:genetics*; D001483:Base Sequence; D002460:Cell Line; D003001:Cloning, Molecular; D066246:ErbB Receptors / Q000378:metabolism*; D006801:Humans; D016296:Mutagenesis; D010957:Plasmids; D011485:Protein Binding; D017386:Sequence Homology, Amino Acid; D016211:Transforming Growth Factor alpha / Q000235:genetics* / Q000378:metabolism
D016211:Transforming Growth Factor alpha; D066246:ErbB Receptors