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W. Li, N. Caberoy; Orf Phage Display: The Fastest Technology to Identify Protein-Protein Interactions. Invest. Ophthalmol. Vis. Sci. 2009;50(13):1870.
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© ARVO (1962-2015); The Authors (2016-present)
Elucidation of protein-protein interactions is critical for exploration of global or pathway-specific protein interaction networks. Current technologies, such as yeast two-hybrid system (Y2H) or mass spectrometry (MS)-based functional proteomics, are limited by technical complexity, instrument requirement, enormous labor and time commitment. The purpose of this study is to develop an efficient, convenient and versatile technology to identify unknown protein-protein interactions.
An open-reading-frame (ORF) phage display cDNA library was generated from mouse eye. A high throughput screening technology of ORF phage display was developed to screen and identify unknown proteins binding to the N-terminal region of tubby, whose mutation caused retinal degeneration with unknown mechanism, or to the extracellular domains of Axl receptor tyrosine kinase that plays a role in retinal pigment epithelium (REP) cell phagocytosis.
Our high throughput ORF phage display technology can rapidly screen 1011 library clones and identify large number of unknown bait-binding proteins only 4-7 days, where as Y2H will take minimal 4-5 months. ORF phage display rapidly identified 15 different tubby-N-binding proteins. Several putative Axl-binding ligands were identified, including the well-characterized Axl ligand of growth arrest-specific gene 6 (Gas6). These protein-protein interactions were independently verified by Y2H and co-immunoprecipitation.
These data demonstrated that ORF phage display is a powerful, convenient and versatile new technology that can be used to investigate unknown protein-protein interactions. It is much fast than Y2H and much more sensitive than MS-based functional proteomics. With an ELISA-like procedure, ORF phage display can be conveniently adapted by individual laboratories with minimal requirement of technical experience.
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