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

J Mol Recognit. Jan-Feb 2010;23(1):74-83. doi: 10.1002/jmr.983.

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 approximately 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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Validation Study

MeSH terms

  • Adaptor Proteins, Signal Transducing
  • Amino Acid Sequence
  • Bacteriophage T7 / metabolism*
  • Base Sequence
  • Eye Proteins / chemistry
  • Eye Proteins / metabolism
  • Gene Library
  • Genetic Vectors
  • Humans
  • Molecular Sequence Data
  • Open Reading Frames
  • Peptide Library*
  • Protein Structure, Tertiary
  • Proteins / chemistry
  • Proteins / metabolism*
  • Two-Hybrid System Techniques

Substances

  • Adaptor Proteins, Signal Transducing
  • Eye Proteins
  • Peptide Library
  • Proteins
  • TUB protein, human
  • TULP1 protein, human