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Gabriela S. Alvarado, Hui Wang, Nora B. Caberoy, Rui Chen, Wei Li; Global Mapping Of Tulp1-binding Proteins By Next Generation DNA Sequencing. Invest. Ophthalmol. Vis. Sci. 2011;52(14):5381.
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© ARVO (1962-2015); The Authors (2016-present)
Mutations in tubby-like protein 1 (Tulp1) cause retinal degeneration with undefined mechanisms. Global identification of Tulp1-binding proteins will help define its underlying molecular mechanisms in the maintenance of retinal homeostasis. The purpose of this study is to globally identify Tulp1-binding proteins by a new technology of open reading frame phage display and next generation DNA sequencing (OPD-NGS).
Glutathione S-transferase (GST) and GST-Tulp1 fusion protein were expressed, purified and used as baits for multi-round phage selection with an open reading frame (ORF) cDNA library of adult mouse eye. Total binding phages at each round were quantified by plaque assay. After 3 rounds of selection, cDNA inserts of enriched phages were amplified by PCR and analyzed by NGS. The sequencing data were blasted against NCBI GenBank database to identify all enriched proteins. Tulp1-binding proteins were globally identified by comparing the entire OPD-NGS datasets for GST-Tulp1 and GST. Identified Tulp1-binding proteins were independently validated by yeast two-hybrid assay.
Three rounds of phage selection resulted in ~18.7-fold and ~6.6-fold increase in total bound phages for GST-Tulp1 and GST control, respectively. NGS sequenced all enriched phage clones in each sample. Data analysis identified two large sets of binding proteins for GST-Tulp1 and GST control. Comparative analysis globally identified a long list of Tulp1-binding proteins, which were selectively enriched by GST-Tulp1, but not by GST. All non-specifically enriched proteins with GST and GST-Tulp1 were either GST-binding proteins or plate-binding phages, and were eliminated by the comparative analysis. The enrichment frequencies of identified Tulp1-binding proteins in NGS analysis were the equivalents of their relative Tulp1-binding activities. Several identified Tulp1-binding proteins were validated by yeast two-hybrid assay.
These results showed that OPD-NGS is a powerful technology of functional proteomics for global identification of binding proteins. OPD-NGS hybrid technology is much more efficient and sensitive than yeast two-hybrid system and mass spectrometry. This new technology is applicable to all other bait proteins and non-protein bait molecules and will revolutionize our capability to globally identify binding proteins.
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