Identification of functional interaction sites on proteins using bacteriophage-displayed random epitope libraries.
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.
van Zonneveld
A J
AJ
Department of Biochemistry, Academic Medical Center, University of Amsterdam, The Netherlands.
van den Berg
B M
BM
van Meijer
M
M
Pannekoek
H
H
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000276:immunology; D000937:Antigen-Antibody Reactions; D001483:Base Sequence; D003090:Coliphages / Q000235:genetics*; D017931:DNA Primers / Q000737:chemistry; D018604:Epitope Mapping / Q000379:methods*; D005822:Genetic Vectors; D006801:Humans; D008969:Molecular Sequence Data; D017395:Plasminogen Activator Inhibitor 1 / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism
D000911:Antibodies, Monoclonal; D017931:DNA Primers; D017395:Plasminogen Activator Inhibitor 1; D011994:Recombinant Proteins