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

Nucleic Acids Res. 2018 Feb 16;46(3):e17. doi: 10.1093/nar/gkx1173.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Autophagy-Related Proteins / classification
  • Autophagy-Related Proteins / genetics*
  • Autophagy-Related Proteins / metabolism
  • Bacterial Proteins / classification
  • Bacterial Proteins / genetics*
  • Bacterial Proteins / metabolism
  • CRISPR-Cas Systems
  • Deoxyribonucleases, Type II Site-Specific / chemistry
  • Gene Editing / methods
  • Gene Library*
  • Genes, Reporter
  • High-Throughput Nucleotide Sequencing
  • High-Throughput Screening Assays*
  • Host-Pathogen Interactions / genetics*
  • Integrases / genetics
  • Integrases / metabolism
  • Luminescent Proteins / genetics
  • Luminescent Proteins / metabolism
  • Mice
  • Mycobacterium tuberculosis / genetics*
  • Mycobacterium tuberculosis / metabolism
  • Phagosomes / metabolism
  • Phagosomes / microbiology
  • Plasmids / chemistry
  • Plasmids / metabolism
  • Protein Interaction Mapping / methods
  • RAW 264.7 Cells
  • Recombination, Genetic
  • Siphoviridae / chemistry
  • Two-Hybrid System Techniques

Substances

  • Autophagy-Related Proteins
  • Bacterial Proteins
  • Luminescent Proteins
  • red fluorescent protein
  • Integrases
  • endodeoxyribonuclease MmeI
  • Deoxyribonucleases, Type II Site-Specific

Supplementary concepts

  • Streptomyces virus phiC31