Development of a new platform technology for the recognition and validation of peptide-protein interactions

Global functional genome and proteome research aims at a complete description of the network of protein interactions within a cell or organ(ism) that is diagnostic for a specific cellular state. Many proteins are built from smaller domains, which are stably folded structural modules still displaying their specific functional properties. Most protein domains recognize and bind linear epitopes, which can be effectively represented by small peptide fragments that are readily available through simultaneous and parallel chemical synthesis. Arrays of peptides are synthesized in situ by SPOT-synthesis on a planar substrate. This doctoral work aimed to establish a convenient methodology for the genome-wide mapping of interactions between protein domains and peptide ligands. A combination of three well characterized methodologies led to an effective process, entirely based on high-throughput biochip technologies. A phage library displaying protein domains from a randomly fragmented and cloned cDNA library will be screened on an array of synthetic peptides. After multiplexed affinity enrichment, peptide-specific phage populations will be eluted, propagated and phage DNA inserts will be labeled and hybridized to a DNA microarray in order to identify peptide bound protein domain/s. This process was successfully implemented for isolation of three protein domains, used as model in these studies, from complex phage mixtures, using their synthetic peptide-ligands as targets. Application of this process for screening of a phage-displayed human brain cDNA library, succeeded in selection and identification of WW domain containing proteins (AIP4, AIP5), which interacted with proline-rich peptides immobilized on a cellulose membrane. The developed process can be applied to diverse types of libraries, constructed from DNA or cDNA preparations of tissues or organisms of healthy or pathogenic origin, to be screened against any type of dedicated or non-dedicated peptide repertoires.