The Preliminary Research Of The NDRG2 Protein

The human ndrgl (N-myc downstream regulated gene 2) gene was first discovered and cloned from normal human whole brain cDNA library in our lab in 1999. This gene locates on human chromosome 14qll.2, including 16 exons and 15 introns, and its full mRNA is 2,024 nt in length, encoding the 357 amino acid> 40 kD NDRG2 protein. The first member be found ofndrg gene family is ndrgl, which was isolated as a new gene upregulated in N-myc mutant mouse embryos and ft is repressed by N-myc and c-myc.Ndrg gene tamily consists of 4 members: ndrg],ndrg2,ndrg3 and ndrg4, which has high similarity in nuclear acid or amino acid sequences, but their expression levels are distinct in spatio-temporal distribution and periods of cells and tissues development. The precise molecular and cellular functions of members of this family are still unknow so far.The expression of NDRG protein family is regulated by lots of intracellular and extracelluar factors. NDRG1 protein expression is upregulated by homocystein > tunicamycin and P53. In addition, the expression is also activated when differentiation is induced by chemicals such as retinoic acid or when cells are under oxidative stress conditions caused by hypoxia> nickel and other reductive agents. On the other hand, the expression is repressed by the proto-oncogenes N-myc and c-myc, And in some cases the expression is also repressed by testosterone or dihydrotestosterone (androgen) at mRNA level Infering from all the references that can be obtained, the functions of NDRG family may be involved in cellular differentiation events > maintaining the balance of cell redox potential (in oxidative stress condition or biological conversion process), or counteracting cell malignant transformation, and so on. Therefore, to identify the roles of NDRG family and to investigate the possible sigaling pathway which are involved in will be very significant.The high titer specific NDRG2 antibody is indispensable to reseach deeply the functions or the tissue and subcellular distribution features of NDRG2, ha order to prepare NDRG2 antibody, the whole ndrgl sequence was cloned into pRset-A vector and two truncated sequences of ndrg2 were cloned into pGEX-4T-l vector. After induced by IPTG, the fusion proteinswere expressed in E.coli; Rabbits immunized with the whole length NDRG2 protein were reinforced with two shortened fragments of NDRG2; after immunization, rabbits produced high titer antiserum against NDRG2. Then antisemm was absorbed using NDRG2 antigen immobilized on NC filters, the purified product of antiserum shows high special to NDRG2 protein, and the separated inclusion body of 6His-NDRG2 will be useful for the further reseach.The yeast recombinant expression vector pPlC3.5K-ndrg2 was constructed, then the NDRG2 protein was expressed in soluable form in pichia. After purification by metal chelate affinity chromatography, we get 6His-NDRG2 fusion which be nature in structure and will be useful to reseach its functions in future. NDRG2 protein has no homology region with any other proteins whose functions are clear either in nucleic acid or in amino acid sequences. In order to get some functional clues from their structures, the upstream regulation region of ndrgl gene and second structure of NDRG2 protein are performed bioinformatics analysis; we found that there are several binding sequences of some diffirent transcription factors, their functions include regulating tissue-specific gene expression, regulating expression of genes related to growth and early development of cells, besides this, regulating expression of genes under some stimulated conditions,and so on. Predict in protein fold classification shows that NDRG2 belongs to alpha/beta hydrolase fold family, and there are high similarity between NDRG2 and epoxide hydrolase from bacteria, this suggests that NDRG2 protein may has enzymatic functions associated with resisting the oxidative stress, maintaining the balance of cell redox potential,involving in the metabolism process of xenobiotics or intracellular toxic...