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

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

Yang F1,2, Lei Y3,4, Zhou M3,4, Yao Q3,4, Han Y5, Wu X3,4, Zhong W1,2, Zhu C3,4, Xu W3,4, Tao R3,4, Chen X3,4, Lin D3,4, Rahman K3,4, Tyagi R3,4, Habib Z3,4, Xiao S3,4, Wang D3,4, Yu Y6, Chen H3,4, Fu Z3,4,7, Cao G3,4,5,8,9.

Author information

1
National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University, Wuhan 430070, China.
2
College of Plant Science and Technology, Huazhong Agricultural University, Wuhan 430070, China.
3
State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan 430070, China.
4
College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070, China.
5
College of Informatics, Huazhong Agricultural University, Wuhan 430070, China.
6
Key Laboratory of RNA Biology, Institute of Biophysics, CAS, Beijing 100101, China.
7
Departments of Pathology, College of Veterinary Medicine, University of Georgia, Athens, GA 30602, USA.
8
Bio-Medical Center, Huazhong Agricultural University, Wuhan 430070, China.
9
Key Laboratory of Development of Veterinary Diagnostic Products, Ministry of Agriculture, Huazhong Agricultural University, Wuhan 430070, China.

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.

PMID:
29165646
PMCID:
PMC5815087
DOI:
10.1093/nar/gkx1173
[Indexed for MEDLINE]
Free PMC Article

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