Abstract
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.
Highlights
Protein–protein interactions are involved in all biological processes
Expression vectors (Figures 3E, 3F). These results demonstrated that specific protein interactions between p53 and SV40 large T-antigen (SV40LT) or orfA and E2F5 reconstituted FokI and ZFP as an efficient zinc-finger nucleases (ZFNs) to target binding sites, and functional Gal4 was restored by single strand annealing (SSA) repair to drive the expression of His3 and Ade2 reporter genes
With traditional Y2H screening methods, some detected interactions are unlikely to be biologically relevant because of the non-specific interactions that are introduced by Gal4 fusion bait and prey proteins
Summary
Protein–protein interactions are involved in all biological processes. The exploration of these interactions aims to characterize intricate protein networks and functions in living cells. Various Y2H methods have been developed to investigate protein interactions, and they have shown success in screening novel protein interactions [2,3,4,5]. In the presence of fusion protein interactions, Gal is reconstituted, which drives reporter gene expression [6]. This classical Y2H system was widely used in cDNA library screens. Various Y2H systems are expected to screen for novel protein–protein interactions, to explore global or targeted interactomics
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