Figure 1.
Schematic diagram describing the zinc finger nuclease yeast two-hybrid (ZFN-Y2H) system to identify protein–protein interactions.
(A) Traditional ZFNs are composed of a fused protein of a zinc finger DNA-binding domain (ZFP) and a non-specific nuclease domain (FokI), in which ZFPs recognize binding sites and recruit dimers of FokI to cleave DNA specifically and highly efficiently. (B) A known protein partner X is expressed as a fusion protein with BCR-ZFP for the bait, and another known protein Y or cDNA library is fused with FokI as the prey. When proteins X and Y interact with each other, FokI is recruited as a dimer to cleave a target site within Gal4 and generate double-strand breaks (DSBs), which could be repaired by single-strand annealing (SSA). Thus, a functional Gal4 DNA-binding domain (DBD) and activation domain (AD) are restored, which drives the expression of reporter genes. (C) Illustration of p53-SV40LT and WDSV orfA-E2F5 interactions in this Y2H system.
Figure 2.
Detection of the function of ZFNs with different distances between the ZFP and FokI domains.
(A) Detection of auxotrophic selection pressure. Yeast strain AH109 was transformed with a reporter plasmid and different ZFNs. Transformed yeast colonies were spotted? on non-selective medium and selective medium, and they were grown at 30°C for 3 days. (B) Relative β-galactosidase activity assay. Three independent transformants were assayed in each group. The transformants of pADH-Gal4 were the positive control, and β-galactosidase activity was set to 100%. Error bars represent the standard deviation from 3 independent experiments.
Figure 3.
Confirmation of the ZFN-mediated protein identification system.
Upon the interaction of well-known proteins fused with BCRZFP and FokI, ZFNs were functional and successfully cleaved DNA at target sites. (A) Transformants with the reporter vector only cannot survive on plates lacking histidine and adenine. In contrast, colonies were observed on plates with histidine, adenine, and G418. (B) Co-transformants pRS-ZFP and pYE-FokI empty expression vectors cannot survive on plates lacking histidine and adenine. (C) Transformants of the ZFP-SV40LT fusion protein and FokI-p53 fusion protein. Upon the interaction of p53 and SV40LT, ZFNs were reconstituted and bound the target binding site. Subsequently, colonies grew on plates lacking histidine and adenine upon the restoration of functional Gal4. (D) Co-transformation of BCRZFP-orfA and FokI-E2F5 expression vectors into reporter yeast cells. Transformants survived on plates lacking histidine and adenine. (E) Co-transformation of ZFP-SV40LT and FokI-E2F5 expression vectors into reporter yeast cells. (F) Co-transformation of ZFP-orfA and p53 expression vectors into reporter yeast cells. No colonies survived on selective plates when yeast co-expression of non-cognate proteins. No cross reactivity was observed between either SV40LT/E2F5 or orfA/p53. L, leucine; T, tryptophan; H, histidine; A, adenine; and G, G418.
Figure 4.
Further investigations of protein interactions.
(A) Serial dilutions of yeast transformant colonies that were spotted on plates with histidine and adenine all showed robust growth. Under the auxotrophic selection pressure, colonies containing reporter vectors and empty expression vectors did not grow. In contrast, colonies containing reporter vectors and ZFP-p53 + FokI-SV40LT or ZFP-orfA + FokI-E2F5 protein pair expression vectors grew well. (B) Relative β-galactosidase activity assay. Three independent transformants were assayed in each group. Error bars represent the standard deviation from 3 independent experiments. (C) Western blot was performed to validate ZFP- and FokI- fusion proteins expression in yeast.
Figure 5.
Specificity test for library screening via this system.
(A) Performance of orfA screening in a library. Hybrid products were plated on non-selective and selective plates respectively. (B) Double digestion of library plasmids recovered from candidate yeast colonies. To determine the insert size in prey vectors, extracted plasmids from positive colonies were digested by BamHI and XhoI.