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

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 3 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.

Unraveling the Molecular Mystery of Retinal Pigment Epithelium Phagocytosis

“…Owing to uncontrollable protein reading frame, phage display with conventional cDNA library identified high percentage of non-ORF clones encoding short unnatural peptides (Li and Caberoy 2010), which have minimal implication in protein interaction networks. To address this problem, we constructed an ORF phage display cDNA library from adult mouse eye with minimal reading frame problem (Caberoy et al 2010b). We designed a strategy of phagocytosis-based functional cloning for unbiased identification of RPE phagocytosis ligands (Fig.…”

“…This further led to identification of its receptor MerTK and characterization of the related intra-cellular signaling cascade. The C-terminal domain of Tulp1 with phagocytosis prey binding activity could be used as a bait to further identify its binding partners on apoptotic cells and POS vesicles in the future by yeast two hybrid system, mass spectrometry or even ORF phage display (Caberoy et al 2010b). In summary, this study illustrated that the combination of ORF phage display and phagocytosis-based functional selection is capable of identifying phagocytosis ligands in the absence of receptor information (Fig.…”

Synergistic Interaction of Tubby and Tubby-Like Protein 1 (Tulp1)

“…Hence, the lack of accumulation of unphagocytosed POS debris in Tubby mice or Tulp1 −/− mice could be due to functional compensation by other MerTK ligands. A body of evidence, including previously described intracellular functions of tubby and Tulp1, 16 new tubby-binding proteins identified by our ORF phage display [10] and 4 distinct clinical manifestations of retinal degeneration associated with 23 different hTulp1 mutations [3], suggest that tubby and Tulp1 are proteins with diverse functions. Stimulation of RPE phagocytosis is only one of their important functions.…”

“…Protein was purified using anti-FLAG mAb affinity columns (Sigma), eluted with FLAG peptide (100 mg/ml), and dialyzed against PBS. Purified tubby-F and GFP were immobilized onto the ELISA plates (5 mg/ml, 100 ml/well) and used for phage binding using open reading frame (ORF) phage display [10]. After three rounds of phage selection, individual clones were randomly picked from plates of enriched phages and analyzed for their binding to purified Tubby.…”

The Phage Display Technique: Advantages and Recent Patents

“…The engineered ORF phage display system was developed from the T7 phage vector with a biotinylation tag in the C-terminus of cDNA library proteins Fig. (3) [137]. A disadvantage of this technique is is that proteins displayed on phage surface lack appropriate post-translational modifications, such as glycosylation (glycosylation-dependent protein-protein interactions may not be identified by ORF phage display).…”