Efficient identification of tubby-binding proteins by an improved system of T7 phage display.
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.
Caberoy
Nora B
NB
Bascom Palmer Eye Institute, Department of Ophthalmology, University of Miami Miller School of Medicine, Miami, Florida 33136, USA.
Zhou
Yixiong
Y
Jiang
Xiaoyu
X
Alvarado
Gabriela
G
Li
Wei
W
D048868:Adaptor Proteins, Signal Transducing; D000595:Amino Acid Sequence; D017123:Bacteriophage T7 / Q000378:metabolism*; D001483:Base Sequence; D005136:Eye Proteins / Q000737:chemistry / Q000378:metabolism; D015723:Gene Library; D005822:Genetic Vectors; D006801:Humans; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D019151:Peptide Library; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000737:chemistry / Q000378:metabolism*; D020798:Two-Hybrid System Techniques
D048868:Adaptor Proteins, Signal Transducing; D005136:Eye Proteins; D019151:Peptide Library; D011506:Proteins; C508878:TUB protein, human; C105443:TULP1 protein, human