Basic Work Of CUEDC2 Functional Study

We demonstrated that CUEDC2 represses the transcription of PR target genes through promotion of progesterone-induced PR degradation by the ubiquitin-proteasome pathway, and then inhibites estrogen-mediated proliferation of breast cancer cells. Moreover, it inhibits the activationof NF-?B as an important adaptor protein of protein phosphatase 1(PP1). However, its biological function has not been explained in detail, it is revealed by bioinformatics analysis as a CUE domain-contained protein, which is a small, moderately-conserved domain of about 40 amino acid residues that are found in a variety of eukaryotic proteins, where it interacts with both mono- and poly-ubiquitin, and has a dual role in mono- and poly-ubiquitin recognition. In addition, we found that CUEDC2 can be phosphorylated during mitosis (M phase) in recent research, which may be closely related to its function.In order to clarify the function of CUEDC2 in M phase, At first, I established a serious of CUEDC2 mutants by molecular biology methods including CUE domain deletion mutantâ–³CUE, phosphorylated and non-phosphorylated analog in 110 serine mutants S110E and S110A. The second, a variety of CUEDC2 mutants were cloned into pDisRed-N1 vector and expressed in Hela cells which were synchronized at M phase when CUEDC2 might have specific location. The third, these mutants were cloned into a prokaryotic expression vector and insect expression system, expressed and purified by molecular biological methods ultimatly to obtain a variety of CUEDC2 mutant proteins, which is ready for the function study of CUEDC2. Finally, CUEDC2 mutant stable expression NIH3T3 cell lines were created by retroviral packaging systems to clarify the transformation ability of CUEDC2.Location and function are closely related, therefore, that of CUEDC2 is very important for its function study. In this paper, we found that both Exogenous and endogenous CUEDC2 has specific location in centrisome and midbody in M phase Hela cells using two fixation methods, the standard and mild fixation methods, furthermore, it does not depend on the phosphorylation of 110Ser and CUE domain, which suggests that it may play a role in M phase.The method of protein expression in prokaryotic system is widely used, readily available, simple, timeshort, easy purification and low cost,yet it does not have an advantage with functional research on some Post-translational modification of the protein, for which the eukaryotic expression system to make up. Insect baculovirus expression system is the one of the commonly used eukaryotic expression systems, which is the trend of structural and functional research of proteins. For further research, we have to clone CUEDC2 mutants to prokaryotic and insect expression vectors and explore the expression and purification methods.To generate a serious of CUEDC2 expression mutants, the primers were designed according to the CDS of human CUEDC2. A serious of CUEDC2 mutants were cloned to expression vectors such as pFastBac1-Flag-N, pFastBac-HTA and pET28a and transformed into the host cells E.coli BL21(DE3) and DH10Bac respectively. The expressed CUEDC2 mutant proteins in BL21(DE3) were purified by the His-tag affinity chromatography and tested by SDS-PAGE. After isolation of recombinant bacmid DNA and transfection of Sf9 cells with them, the recombinant proteins were testified by Western-Blot. The SDS-PAGE and Western-Blot tests revealed that the recombinant proteins were expressed successfully in E.coli and Sf9 cells, which would be useful for the functional and structural research of CUEDC2.Using calcium phosphate transfection method to package retrovirus and infect NIH3T3 cells to establish a variety of CUEDC2 mutants overexpressing cell lines after Flow cytometry sorting, which is ready for the cytological study of Transforming and tumorigenic capacity of CUEDC2.