Proteins are the building blocks of life, and a vast array of cellular processes is handled by protein-protein interactions (PPIs). The protein complexes formed via PPIs lead to tangled networks that, with their continuous remodeling, build up systematic functional units. Over the years, PPIs have become an area of interest for many researchers, leading to the development of multiple in vitro and in vivo methods to reveal these interactions. The yeast-two-hybrid (Y2H) system is a potent genetic way to map PPIs in both a micro- and high-throughput manner. Y2H is a technique that involves using modified yeast cells to identify protein-protein interactions. For Y2H, the yeast cells are engineered only to grow when there is a significant interaction between a specific protein with its interacting partner. PPIs are identified in the Y2H system by stimulating reporter genes in response to a restored transcription factor. However, Y2H results may be constrained by stringency requirements, as the limited number of colony screenings through this technique could result in the possible elimination of numerous genuine interactions. Therefore, DEEPN (dynamic enrichment for evaluation of protein networks) can be used, offering the potential to study the multiple static and transient protein interactions in a single Y2H experiment. DEEPN utilizes next-generation DNA sequencing (NGS) data in a high-throughput manner and subsequently applies computational analysis and statistical modeling to identify interacting partners. This protocol describes customized reagents and protocols through which DEEPN analysis can be utilized efficiently and cost-effectively.

• PMID: 37450148
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Journal Article | Research Support, U.S. Gov't, Non-P.H.S.

Authors

Fakhar AZ, Liu J, Pajerowska-Mukhtar KM

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