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Y. Zhou, N. B. Caberoy, W. Li; Identification and Characterization of Tubby-Binding Proteins by ORF Phage Display. Invest. Ophthalmol. Vis. Sci. 2009;50(13):4492.
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© ARVO (1962-2015); The Authors (2016-present)
Tubby mutation causes adult-onset obesity, progressive retinal and cochlear degeneration with unknown mechanism. Tubby is a cytoplasmic protein that translocates into the nucleus upon receptor-mediated activation of G protein. The purpose of this study is to elucidate its molecular mechanism in retinal degeneration by identifying its unknown binding proteins.
A novel high throughput screening technology of open-reading-frame (ORF) phage display has been developed to rapidly identify unknown proteins binding to tubby N-terminal region (tubby-N). Recombinant tubby-N was expressed in E. coli, affinity purified, and immobilized in ELISA plates. ORF phage display was performed with cDNA library generated from mouse eye. Identified phage clones encoding tubby-N-binding proteins were independently verified by yeast two-hybrid system (Y2H). Tubby-N-binding phage clones were characterized for their binding specificity to full-length tubby, tubby-like protein (Tulp) 1, 2 and 3.
A total of 28 tubby-N-binding phage clones were identified. Twenty six clones matched to 15 different protein sequences as novel tubby-binding proteins. Among them were 10 nuclear proteins, suggesting that tubby may function as an important transcription factor. The remaining tubby-N-binding proteins were cytoplasmic proteins. Tubby-N interaction with these binding proteins was individually verified by Y2H or co-immunoprecipitation. They exhibited different binding specificities to tubby, Tulp1, 2 and 3.
Tubby N-terminal region is capable of binding to multiple partners both in the cytoplasm and nucleus.This study will help us to understand the mechanism in retinal degeneration.Our study also demonstrated that ORF phage display is a powerful and convenient technology to identify unknown protein-protein interactions.
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