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G. S. Alvarado, N. Caberoy, W. Li; Elucidation of Protein Interaction Network for Tubby-Like Protein 1. Invest. Ophthalmol. Vis. Sci. 2010;51(13):4037. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Mutations in tubby-like protein 1 (Tulp1) cause progressive retinitis pigmentosa. However, delineation of its pathological mechanisms is hindered by its poorly defined protein interaction networks. The purpose of this study is to elucidate its binding partners by a newly developed technology of functional proteomics.
Tulp1-binding proteins were identified by open reading frame (ORF) phage display technology as the following. ORF phage display cDNA library of mouse eye was incubated with immobilized Tulp1 protein on ELISA plates, washed, eluted, amplified and reselected. A total of three rounds of affinity selection were performed. Enriched individual phage clones were re-verified for their binding activity to Tulp1 and identified by sequencing. Their binding specificity to Tulp1 and other proteins in the same family were analyzed. Identified Tulp1-binding proteins were independently validated by yeast two-hybrid system or protein pull-down assay.
Several new Tulp1-binding proteins were efficiently identified by ORF phage display technology, including acidic nuclear phosphoprotein 32 A (Anp32a), cyclic nucleotide gated channel beta 1 (Cng1), cell division cycle 2-like 1 (cdc2l1), PRP38 pre-mRNA processing factor 38B (Prpf38b), DnaJ (Hsp40) homolog A1 (Cnaja1) and D4 zinc and double PHD fingers 3 (Dpf3). They exhibited different binding specificities to Tulp1 and other proteins in the same family. Furthermore, their interactions with Tulp1 were independently verified.
These data revealed that Tulp1 has multiple binding partners and may play multiple intracellular and extracellular roles. The elucidation of these new binding partners will facilitate the delineation of their functional roles and disease mechanisms. This study also demonstrates that ORF phage display is a powerful technology of functional proteomics with efficiency, sensitivity and convenience for re-verification and analysis of their binding specificity.
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