Bacteriophage M13 has been adapted for the expression of diverse populations of peptides in a manner that affords the rapid purification of active peptides by affinity selection. Libraries of combinatorial peptides can be expressed as N-terminal fusions to the M13 minor coat protein pIII. Such peptides are encoded by a DNA insert, which has been assembled from synthetic, degenerate oligonucleotides, and cloned into the gene for protein III. Using standard molecular biology techniques, it is possible to construct libraries comprised of billions of different peptide sequences in as little as one month. Peptides also have been expressed as fusions to the other five capsid proteins of bacteriophage M13, using either phage or phagemid vectors. 

With libraries of phage particles that display libraries of combinatorial peptide on their surface, it is possible to isolate peptides that bind to target proteins. With as few as four rounds of affinity selection over two weeks) and a modest amount of protein (100 micrograms), one can identify peptide ligands to a wide variety of targets, such as antibodies, calmodulin, caveolin, double minute 2 protein, E-selectin, erythropoietin receptor, estrogen receptor, Gal80 repressor, the N-methyl-D-aspartate receptor, protein interaction modules, such as PDZ, SH3, and WW domains, thrombopoietin receptor, troponin C, and vascular endothelial growth factor. It has also been possible to isolate peptides that bind to small molecules other than proteins, such as polyglutamine and taxol. Thus, if a peptide ligand exists in a phage-displayed combinatorial peptide library, one can readily isolate the “needle in the molecular haystack”.

In most instances, the peptide ligands that one isolates bind to a biologically relevant pocket in the protein targets. In our experience, for M13 phage displaying peptides at the N-terminus of mature pIII to withstand the vigorous washing steps during affinity selection they must bind with a dissociation constant of 50 micromolar or better, which is typical of many types of protein-protein interaction. Weaker binding peptides, isolated from screens of peptides displayed at the N-terminus of another capsid protein, pVIII, may have lower affinities (i.e., mM), but the binding of such peptides can be improved through a round of molecular evolution. Consequently, one can use the peptide ligands identified from the phage-displayed combinatorial peptide libraries to provide insight into the molecular basis of the specific interactions between particular proteins and between proteins and small molecules. For example, the primary structures of the selected peptides often resemble those within known or potential interacting proteins, a phenomenon we have termed “convergent evolution”. Thus, phage-displayed combinatorial peptides have been very useful in mapping protein-protein interactions.