Identification of functional interaction sites on proteins using bacteriophage-displayed random epitope libraries

Gene. 1995 Dec 29;167(1-2):49-52. doi: 10.1016/0378-1119(95)00614-1.

Abstract

We describe a phage-display-based method to identify epitopes or interaction sites on proteins. DNA encoding the protein of interest is partially degraded with DNase I to generate random fragments of 50-200 bp. These fragments are then cloned into a phagemid vector that has been modified to allow the expression of the random fragments and the construction of a (bacterio)phage-displayed random epitope library. Phages displaying functional epitopes can be selected from these libraries by affinity selection or panning. To test this method we have constructed a random-epitope library for human plasminogen-activator inhibitor 1 and used this library to map the epitope of a monoclonal antibody (mAb) directed against this protein. By alignment of the selected overlapping epitope-containing fragments, we were able to locate the epitope of the mAb on a stretch of 39 amino acids spanning from E128 to V166. The approach may also be applied to more complex systems than single-protein genes, such as viral genomes or complete cDNA libraries.

MeSH terms

  • Amino Acid Sequence
  • Antibodies, Monoclonal / immunology
  • Antigen-Antibody Reactions
  • Base Sequence
  • Coliphages / genetics*
  • DNA Primers / chemistry
  • Epitope Mapping / methods*
  • Genetic Vectors*
  • Humans
  • Molecular Sequence Data
  • Plasminogen Activator Inhibitor 1 / chemistry
  • Plasminogen Activator Inhibitor 1 / genetics
  • Plasminogen Activator Inhibitor 1 / immunology*
  • Protein Binding
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / metabolism

Substances

  • Antibodies, Monoclonal
  • DNA Primers
  • Plasminogen Activator Inhibitor 1
  • Recombinant Proteins

Associated data

  • SWISSPROT/P05121