Selection of ligands by panning of domain libraries displayed on phage lambda reveals new potential partners of synaptojanin 1.
One of the goals of functional genomics is the description of reliable and complete protein interaction networks. To facilitate ligand discovery from complex protein mixtures, we have developed an improved approach that is affected by a negligible fraction of false positives. We have combined a novel technique based on the display of cDNA libraries on the capsid of bacteriophage lambda and an efficient plaque assay to reveal phage displaying ligands that are enriched after only a couple of affinity purification steps. We show that the lambda display system has a unique ability to display, at high density, proteins ranging in size from a few to at least 300 amino acid residues. This characteristic permits attenuation of the size bias in the selection procedure and, at the same time, offers a sensitive plaque assay that permits us to do away with the ligand background without unduly increasing the number of selection cycles. By using a proline-rich fragment of the synaptojanin 1 protein as a bait, we have identified, in a brain cDNA display library, seven ligands all containing either SH3 or WW domains. Four of these correspond to proteins that have already been validated as physiological partners, while the remaining three are new partners, whose physiological relevance remains to be established. Two different proline-rich regions of the p21-activated protein kinase 1 (Pak1) and WAVE/SCAR2 protein retrieve from the library different proteins containing SH3 or WW domains.
Zucconi
A
A
Department of Biology, University of Rome Tor Vergata, Rome, Via della Ricerca Scientifica, 00133, Italy.
Dente
L
L
Santonico
E
E
Castagnoli
L
L
Cesareni
G
G
D000595:Amino Acid Sequence; D017123:Bacteriophage T7 / Q000235:genetics; D010582:Bacteriophage lambda / Q000235:genetics*; D001665:Binding Sites; D001921:Brain / Q000378:metabolism; D015723:Gene Library; D006801:Humans; D008024:Ligands; D008840:Microfilament Proteins / Q000737:chemistry / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D009419:Nerve Tissue Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D010446:Peptide Fragments; D019151:Peptide Library; D010744:Phosphoric Monoester Hydrolases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011392:Proline / Q000378:metabolism; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity; D010948:Viral Plaque Assay; D051300:Wiskott-Aldrich Syndrome Protein Family; D054462:p21-Activated Kinases; D018909:src Homology Domains
D008024:Ligands; D008840:Microfilament Proteins; D009419:Nerve Tissue Proteins; D010446:Peptide Fragments; D019151:Peptide Library; D011993:Recombinant Fusion Proteins; D051300:Wiskott-Aldrich Syndrome Protein Family; D011392:Proline; C515903:PAK1 protein, human; D017346:Protein Serine-Threonine Kinases; D054462:p21-Activated Kinases; C097702:synaptojanin; D010744:Phosphoric Monoester Hydrolases