A Novel Genetic System Based on Zinc Finger Nucleases for the Identification of Interactions between Proteins In Vivo

作者：Wang, Ling; Xu, Kun; Lin, Juan; Shao, Simin; Zhang, Tingting; Xu, Huarong; Wei, Zehui; Zhang, Zhiying^*

来源：PLos One, 2013, 8(12): e85650.

DOI：10.1371/journal.pone.0085650

• YEAST 2-HYBRID
• DOMAIN
• DROSOPHILA
• NETWORK
• CELLS
• MAP

Yeast two-hybrid (Y2H) methods are powerful tools for detecting protein-protein interactions. The traditional Y2H method has been widely applied to screen novel protein interactions since it was established two decades ago. The high false-positive rate of the traditional method drove the development of modified Y2H systems. Here, we describe a novel Y2H system using zinc-finger nucleases (ZFNs). ZFNs contain two functional domains, a zinc-finger DNA-binding domain (ZFP) and a non-specific nuclease domain (FokI). In this system, the bait is expressed as a fusion protein with a specific ZFP, and the prey is fused to the FokI. A reporter vector is designed such that the ZFN target site disrupts the Gal4 open reading frame. By transforming the three plasmids into a yeast strain (AH109), the interaction between the bait and prey proteins reconstitutes ZFN function and generates the double-strand break (DSB) on its target site. The DNA DSB repair restores Gal4 function, which activates the expression of the four reporter genes. We used p53-SV40LT interacting proteins to prove the concept. In addition, 80% positive rate was observed in a cDNA screening test against WDSV orfA protein. Our results strongly suggested that this Y2H system could increase screening reliability and reproducibility, and provide a novel approach for interactomics research.

• 出版日期2013-12-31
• 单位西北农林科技大学