Prokaryotic Expression And Purification Of N-terminal Domain And Central Domain Of MDM2

MDM2 is a mouse double minute gene and ubiquitin E3-ligase of the RING family. It has a key role in enhancing viability of the cells and prolongs the cell survival rate. Studies have shown that MDM2 is involved in the development of human tumors and plays important role in cell cycle regulation and it makes one of the interests in cancer research.While the tumor suppressor protein p53 works importantly in maintaining the integrity of the genome by inducing cell cycle arrest and apoptosis in response to stress. Under normal physiological conditions p53 exhibited a low level, when the cells were subjected to external stimuli, p53 exhibit high levels and enhance stabilization, thus involved in a series of important life processes such as cellular aging, DNA repair or apoptosis. Both over expression and inappropriate activation of p53 will lead cells to death and decreased expression or inactivation cause tumors. p53 protein expresses in most human cancers for mutation or functional inactivation, which provides an attractive therapeutic strategies based on the activation of p53, although clinically has been approved but the development of p53 activator still fail to achieve results.The mechanism of tumor suppressor MDM2 is one of the important factors as negative regulator of p53 by binding tumor suppressor gene p53 and mediate degradation pathway of ubiquitylation proteolytic enzyme. Whether the N-terminal domain, zinc finger domain and a central domain(zinc finger domain & acidic domain) of MDM2 is the main binding site of p53 has no relevant reports, therefore focus on biological research of these domains of MDM2 has important implications for cancer prevention.The purpose of our experiment is to construct a recombinant plasmid containing the His-sumo tag to obtain fusion proteins. The method we use PCR technology to amplify N-terminal domain, zinc finger domain and central domain of the coding sequence of MDM2 from MDM2 gene bank. After getting recombinant plasmid vector by insert pp SUMO, transformation of E.coli followed by pick a small amount of induced recombinant bacteria to induce and optimize expression condition. Using SDS-PAGE for protein purity and molecular weight analysis, based on the former transfer film and hybridization step, namely by Western blot to analyze the expression of the target protein. Analyze whether it’s monomeric state by sieve exclusion or ion exchange.The recombinant plasmid was successfully transformed into E.coli BL21 and after effective induction, obtained relative molecular mass of approximately 26 k Da N-terminal domain of the MDM2, about 21 k Da fusion zinc finger protein and relative molecular mass of approximately 31 k Da central domain fusion protein. We purify ULP1 protein to cut SUMO tag. Conclusion: This study successfully obtained the portion N-terminal domain of MDM2, zinc finger domain protein and central domain protein, to lay the foundation for the study of protein crystallization and threedimensional structure.