Abstract
A comprehensive examination of protein-protein interactions (PPIs) is fundamental for the understanding of cellular machineries. However, limitations in current methodologies often prevent the detection of PPIs with low abundance proteins. To overcome this challenge, we develop a mRNA display with library of even-distribution (md-LED) method that facilitates the detection of low abundance binders with high specificity and sensitivity. As a proof-of-principle, we apply md-LED to IAV NS1 protein. Complementary to AP-MS, md-LED enables us to validate previously described PPIs as well as to identify novel NS1 interactors. We show that interacting with FASN allows NS1 to directly regulate the synthesis of cellular fatty acids. We also use md-LED to identify a mutant of NS1, D92Y, results in a loss of interaction with CPSF1. The use of high-throughput sequencing as the readout for md-LED enables sensitive quantification of interactions, ultimately enabling massively parallel experimentation for the investigation of PPIs.
Highlights
A comprehensive examination of protein-protein interactions (PPIs) is fundamental for the understanding of cellular machineries
MRNA display using cDNA libraries has been reported for identifying binders of target proteins, which can be released from beads under specific elution conditions after multiple rounds of selection[32,33]
An initial round of FLAG purification was used on the resultant protein library to remove untranslated mRNAs, prematurely stopped proteins, nonspecific binders, and precipitated peptides. mRNA/ cDNA duplexes were generated through reverse transcription to remove secondary structures of the mRNAs, prevent mRNA degradation during selection, and enable PCR amplification post selection
Summary
A comprehensive examination of protein-protein interactions (PPIs) is fundamental for the understanding of cellular machineries. In parallel with AP-MS, yeast-two-hybrid[12,13] and several additional display-based methods are being widely used, including phage display, yeast display, ribosome display and mRNA display[14,15,16,17,18,19,20,21,22,23,24,25,26] These methods effectively link each protein to its genotype, allowing for multiple rounds of selection and easy downstream analysis by deep sequencing. We have recently improved the efficiency of mRNA display even further to enable single-round selection of highaffinity binders by optimizing the complexity and uniformity of the library[30,31] These improvements offer the potential to increase the scale of experiments and allow for multiple experiments to be run in parallel. We hypothesize that a more evenly distributed input library with an unbiased representation of the proteome will facilitate efficient detection of PPIs, especially those with low abundance proteins
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