Two-hybrid systems can be used for investigating protein-protein interactions and may provide important information about gene products with unknown function. Despite their success in mapping protein interactions, two-hybrid systems have remained mostly untouched by improvements in next-generation DNA sequencing. The two-hybrid systems rely on one-versus-all methods in which each bait is sequentially screened against an entire library. Here, we developed a screening method that joins both bait and prey as a convergent fusion into one bacterial plasmid vector that can then be amplified and paired-end sequencing by next-generation sequencing (NGS). Our method enables all-versus-all sequencing (AVA-Seq) and utilizes NGS to remove multiple bottlenecks of the two-hybrid system. AVA-Seq allows for high-resolution protein-protein interaction mapping of a small set of proteins and has the potential for lower-resolution mapping of entire proteomes. Features of the system include ORF selection to improve efficiency, high bacterial transformation efficiency, a convergent fusion vector to allow paired-end sequencing of interactors, and the use of protein fragments rather than full-length proteins to better resolve specific protein contact points. We demonstrate the system's strengths and limitations on a set of proteins known to interact in humans and provide a framework for future large-scale projects.

• PMID: 31182485
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Journal Article | Research Support, Non-U.S. Gov't

Authors

Andrews SS, Schaefer-Ramadan S, Al-Thani NM, Ahmed I, Mohamoud YA, Malek JA

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