Efficient identification of tubby-binding proteins by an improved system of T7 phage display

Nora B. Caberoy, Yixiong Zhou, Xiaoyu Jiang, Gabriela Alvarado, Wei Li

Research output: Contribution to journalArticle

33 Scopus citations

Abstract

Mutation in the tubby gene causes adult-onset obesity, progressive retinal, and cochlear degeneration with unknown mechanism. In contrast, mutations in tubby-like protein 1 (Tulp1), whose C-terminus is highly homologous to tubby, only lead to retinal degeneration. We speculate that their diverse N-terminus may define their distinct disease profile. To elucidate the binding partners of tubby, we used tubby N-terminus (tubby-N) as bait to identify unknown binding proteins with open-reading-frame (ORF) phage display. T7 phage display was engineered with three improvements: high-quality ORF phage display cDNA library, specific phage elution by protease cleavage, and dual phage display for sensitive high throughput screening. The new system is capable of identifying unknown bait-binding proteins in as fast as ~4-7 days. While phage display with conventional cDNA libraries identifies high percentage of out-of-frame unnatural short peptides, all 28 tubby-N-binding clones identified by ORF phage display were ORFs. They encode 16 proteins, including 8 nuclear proteins. Fourteen proteins were analyzed by yeast two-hybrid assay and protein pull-down assay with ten of them independently verified. Comparative binding analyses revealed several proteins binding to both tubby and Tulp1 as well as one tubby-specific binding protein. These data suggest that tubby-N is capable of interacting with multiple nuclear and cytoplasmic protein binding partners. These results demonstrated that the newly-engineered ORF phage display is a powerful technology to identify unknown protein-protein interactions.

Original languageEnglish (US)
Pages (from-to)74-83
Number of pages10
JournalJournal of Molecular Recognition
Volume23
Issue number1
DOIs
StatePublished - Jan 1 2010

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Keywords

  • cDNA library
  • ORF phage display
  • Protein-protein interaction
  • T7 phage
  • Tubby
  • Tubby-binding proteins

ASJC Scopus subject areas

  • Molecular Biology
  • Structural Biology

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