( luciferase gene transcriptio...)New Mammalian Two-Hybrid System Detects Protein-Protein Interactions

luciferase gene transcription. Expression of the GAL4 BD-p53GAL4 BD-CD40 fusion proteins and the NF-kB AD-SV40TNF-kB AD-TRAF2 fusion proteins were confirmed by Western blot analysis using anti-GAL4 DNA-binding domain monoclonal antibody (Santa Cruz Biotech) and anti-NF-kB polyclonal antibody (Santa Cruz Biotech), respectively (data not shown). These results demonstrated that the mammalian two-hybrid assay kit detects known interaction between p53 and SV40 large T-antigen.

In addition to the reporter plasmid pFR-Luc, other PathDetect reporter plasmids such as pFR-bGal (b-galactosidase) and pFR-SEAP (secreted alkaline phosphatase) were also used in the mammalian two-hybrid assays. Pairs of plasmids were cotransfected with reporter plasmids pFR-bGal or pFR-SEAP into CHO cells. Forty-eight hours after transfection, cells lysates were collected and assayed for reporter gene expression (b-galactosidase and secreted alkaline phosphatase).

Conclusions

We demonstrated our systems ability to detect known protein-protein interactions, such as p53 and SV40 large T-antigen in vivo in mammalian cells. In addition, we showed that various reporter genes, such as luciferase, b-galactosidase, and secreted alkaline phosphatase can be used to detect interactions. The mammalian two-hybrid assay kit is especially useful in confirming suspected interactions between two proteins or interactions identified by yeast two-hybrid screens. This system is the most practical method for studying interactions between mammalian proteins in their native environment because they are more likely to represent biologically significant interactions
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