Screening Of P53 Interacted Proteins By Yeast Two-Hybrid System

The tumor suppressor p53 gene locates in human chromosome 17 short arm. Its encoding wild-type p53 protein is composed of four parts: N-terminal activation domain, DNA-binding domain, oligomerization domain, and C-terminal regulation domain. The mutated p53 gene is discovered in over 50% of all human tumors. Several dozen downstream genes are controlled directly by p53, and the activity of p53 falls into four categories: cell cycle inhibition, apoptosis, genetic stability, and inhibition of blood vessel formation.P53 protein is a tumor suppressor, playing a key role as a factor in transcript network. Ubiquitination, phosphorylation, acetylation, sumoylation and methylation are post-translational modifications to p53 that affect its overall appearance and activity. We used a yeast-based genetic assay, the two-hybrid system, to screen the interacted proteins of the p53 protein. Yeast two hybridization is one of the methods to detect protein- protein interactions. This assay relies on the reconstitution of the function of a transcriptional activator: the yeast GAL4 protein, via the interaction of a protein fused to the DNA-binding domain of GAL4.We used human breast cDNA as library. Since the p53 protein contains a transcriptional activation domain at its N-terminus, we then used the p53 protein that has been truncated at its N-terminus (1-62aa), as the bait. The interaction between p53 and sv40-antigen was evaluated as a positive control, sv40-antigen and laminc as a negative control. As the results show:1. We gained the targeting fragment of p53 (62-393aa), and inserted it into the PGBKT7 vector correctly.2. PGBKT7-p53 (62-393aa) was transformed into the yeast AH109, and was extracted the protein. By western blotting, the protein was identified correctly.3. By testing the self-transcriptional activity, it has no self-transcriptional activity.4. By lots of screening, PIAS3, protein inhibitor of activated STAT3, was identified to interact with p53 (62-393aa). Their interaction may suggest a new mechanism to reflect the function of p53.5. The gene PIAS3 was fished from human breast cDNA library, and sequenced correctly.6. The gene PIAS3 was inserted into the PGEX-KG vector, and expressed the relevant protein successfully.