Homodimeric peptides displayed by the major coat protein of filamentous phage

Peptide libraries displayed by filamentous bacteriophage have proven a powe rful tool for the discovery of novel peptide agonists, antagonists and epit ope mimics. Most phage-displayed peptides are fused to the N terminus of ei ther the minor coat protein, pill, or the major coat protein, pVIII. We rep ort here that peptides containing cysteine residues, displayed as N-termina l fusions to pVIII, can form disulfide-bridged homodimers on the phage coat . Phage clones were randomly selected from libraries containing one or two fixed Cys residues, and surveyed for the presence of peptide-pVIII homodime rs by SDS-PAGE analysis that involved pretreatment of the phage with reduci ng or thiol-modifying agents. For all phage whose recombinant peptide conta ined a single Cys residue, a significant fraction of the peptide-pVIII mole cules were displayed as dimers on the phage coat. The dimeric form was in g reater abundance than the monomer in almost all cases in which both forms c ould be reliably observed. Occasionally, peptides containing two Cys residu es also formed dimers. These results indicate that, for a given pVIII-displ ayed peptide bearing a single Cys residue, a significant fraction of the pe ptide (>40%) will dimerize regardless of its sequence; however, sequence co nstraints probably determine whether all of the peptide will dimerize. Simi larly, only occasionally do peptides bearing two Cys residues form intermol ecular disulfide bridges instead of intramolecular ones; this indicates tha t sequence constraints may also determine dimerization versus cyclization. Sucrose-gradient analysis of membranes from cells expressing pVIII fused to a peptide containing a single Cys residue showed that dimeric pVIII is pre sent in the cell prior to its assembly onto phage. A model of the peptide-p VIII homodimer is discussed in light of existing models of the structure an d assembly of the phage coat. The unique secondary structures created by th e covalent association of peptides on the phage surface suggest a role for homo- and heterodimeric peptide libraries as novel sources of bioactive pep tides. (C) 2000 Academic Press.