The Expression, Purification And Identification Of Recombinant Human Oncoprotein MDM2

MDM2(Murine double minute 2) is one of the important members of ubiquitin protein ligase. Its main physiological function is to negative regulate the tumor suppressor protein p53, MDM2 can promote p53 ubiquitin modification, degradation and nuclear translocation through directly combine with p53 and restrain the activity of p53 transcription factors. MDM2 is the most important suppressor of p53, through a variety of molecular mechanisms regulating the stability and biological function of p53, the process and the specific mechanism is various and complex. MDM2 protein is mainly composed of four functional areas, including 19-110 amino acids area I, which can make MDM2 closely combining with p53 gene; 200-280 amino acids area highly acidic region II, which can mutually combine with protein 15 and 5 srrna; The III area in 300-330 amino acid area containis a zinc finger structure; 438-491 amino acids of IV area contains a ring structure, which participate in a variety of proteins function.MDM2 can not only bind with p53, but also participates in p53-independent pathways, often by dimerizing through its C-terminus, to develop its carcinogenesis. Studies have found that under the condition of lacking of p53, mdm2 transgenic mice were also detected spontaneous tumor formation. In at least seven percent of the tumor, the p53 gene is normal and mdm2 gene is abnormal amplified. At the same time, in the tumor and normal tissue, people has identified at least 40 different mdm2 spliceosome, most of which most does not contain p53 area, showing that mdm2 cancer gene activity exist some other way that does not depend on the p53. MDM2 protein stability control based on a series of a substrate molecule; participating in regulating genome stability, cell cycle, cell differentiation and a variety of biological processes such as DNA damage repair. MDM2 as a contact tumorigenesis and ubiquitin proteasome system, plays an important role in protein function, is undoubtedly one of the promising target for drug development in the future.In this paper, we use E. coli to express MDM2 and study on the purification and identification of the protein. We found that one-step purification can make the purity of MDM2 protein up to 90 %; also, we found that the MDM2 protein amino acid sequence aa368- aa429, including six mutations of MDM2 protein phosphorylation site, play an important role on the MDM2 function rather than protein expression. MDM2 C terminal domain plays an important role on MDM2 structure function. In this paper, we creatively restructure MDM2 C terminal domain(aa 300-aa 491, CTD, including Zinc Finger Domain and RING Domain) using E. coli, through Ni-NTA affinity purification and ULP1 digestion, we got free CTD protein. CHAPS can efficiently remove lipid which may combine in protein. The protein can be further purified by ammonium sulfate precipitation and remained stable. Ion exchange show CTD protein remains the DNA binding ability, therefore, adding DNASE in the cell suspension can effectively remove the DNA and increase protein yield during affinity purification steps. We finally USE size exclusion(Superdex75) to identify target protein, the volume of protein elution corresponds to the molecular weight between 35-43 kDa, which is consistent with the molecular weight on SDS-PAGE, meaning CTD protein is monomer structure.This paper creatively express recombinant MDM2 protein and its important CTD domain, and find a way to efficiently purify the target protein, laying a solid foundation for future structure and crystal study. Also, previous studies have found that MDM2 c-terminal domain RING forms as dimers and is difficult to purify. Therefore, getting the monomer CTD protein will provide a new avenue for studying MDM2’s p53-independent roles.