Development and application of a recombination-based library versus library high- throughput yeast two-hybrid (RLL-Y2H) screening system

Abstract

Protein-protein interaction (PPI) network maintains proper function of all organisms. Simple high-throughput technologies are desperately needed to delineate the landscape of PPI networks. While recent state-of-the-art yeast two-hybrid (Y2H) systems improved screening efficiency, either individual colony isolation, library preparation arrays, gene barcoding or massive sequencing are still required. Here, we developed a recombination-based ‘library vs library’ Y2H system (RLL-Y2H), by which multi-library screening can be accomplished in a single pool without any individual treatment. This system is based on the phiC31 integrase-mediated integration between bait and prey plasmids. The integrated fragments were digested by MmeI and subjected to deep sequencing to decode the interaction matrix. We applied this system to decipher the trans-kingdom interactome between Mycobacterium tuberculosis and host cells and further identified Rv2427c interfering with the phagosome–lysosome fusion. This concept can also be applied to other systems to screen protein–RNA and protein–DNA interactions and delineate signaling landscape in cells.

Construction and characterization of a high-quality cDNA library of Cymbidium faberi suitable for yeast one- and two-hybrid assays

“…Moreover, in recent studies two cDNA libraries were respectively used as bait library and prey library, followed by mating and screening, enabling the screening of multiple libraries in one pool. Examples include BFG-Y2H [20], CrY2H [21], and RLL-Y2H [22]. The continuous improvement of the construction methods makes the information included in the library more comprehensive and more integral.…”

A Multiplexed Bacterial Two-Hybrid for Rapid Characterization of Protein-Protein Interactions and Iterative Protein Design

“…By uniting gene synthesis with a mapping step and a barcode readout, our system allows high throughput characterization of any binary PPI. Previous high-throughput studies used highly constrained libraries--either the ORFome [21][22][23][24] of one of a handful of reference genomes, targeted single residue mutations which only explore a sliver of sequence space around a primary sequence 25,26 or several randomly sheared coding sequences 27 . Using the capabilities of DNA synthesis broadens the testable sequence space which facilitates investigations of a variety of areas such as protein domains, extant genetic variation, evolutionary trajectories or epistatic effects.…”

An Interactome Map of Maize (Zea mays L.)

“…An interactome covering all layers of genetic information flow would further promote the precise genetic improvement of this crop. Here, we leveraged advances in high-throughput sequencing techniques to measure genomic interactions by ChIA-PET 9 ; transcriptomic interactions for all detectable elements by strand-specific RNA-seq, circRNA-seq, and small RNA-seq; translatomic interactions by Ribo-seq; and protein-protein binary interactions (PPIs) by recombination-based library vs. library yeast-2-hybrid (RLL-Y2H) 8 analysis throughout the lifecycle of the maize inbred line B73 (Fig. 1a).…”

“…Modified mAD and mBD vectors were used to construct a maize cDNA yeast library according to the Clontech Y2H transformation method 8 at 30°C. Each library mating was repeated at least 10 times.…”