Studies On The Cloning, Expression And Biological Function Of Protein Tyrosine Phosphatases MEG2

Protein tyrosine phosphatases (PTPs) play an important role in cell signal transduction,and closely associated with numerous physiologic and pathologic processes in organism. They have a strict regulation on cell growth and development, cell metabolism,cell cycle,cell migration,cell-cell communication and gene transcription and so on .Among Protein tyrosine phosphatases (PTPs),PTP-MEG2 is an intracellular enzyme of 593 amino acid residues with a putative lipid-binding domain at the N-terminus. In the present study,we cloned the full-length form of the enzyme and expressed it in E. coli cells as a 6xHis-tagged protein. The majority of the expressed enzyme was found in the inclusion body of E. coli cell extracts. Upon extraction with a buffer containing urea,the recombinant enzyme was purified to near homogeneity by using a single Ni-NTA-agarose column. This procedure resulted in the production of over 100 mg purified recombinant PTP-MEG2 from one liter of E. coli cell culture. The purified protein displayed a single polypeptide band with expected molecular size on SDS-polyacrylamide gel electrophoresis under reducing conditions. Isolated under denatured conditions in urea,the purified enzyme was re-natured by dialyzing against a refolding buffer. The re-natured enzyme effectively dephosphorylates the common PTPs substrate para-nitrophenylphosphate with a specific activity of 2000 units/mg. Meanwhile,the denatured enzyme was used to immunize a rabbit to produce antibodies. The resulting anti-serum had extremely high sensitivity and specificity. When used for Western Blotting analyses,the anti-serum revealed a wide expression of PTP-MEG2 in many tissues of mice. Together,we developed a highly effective way to purify a large amount of PTP-MEG2 and generated a highly sensitive antibody that can specifically detect endogenous expression of the enzyme in tissues.Protein tyrosine phosphatase 1B (PTP1B) is the first credit PTP. In insulin signal pathways, PTP1B plays an important role of negative regulation. Recently research shows that PTP1B use the insulin receptor and insulin receptor substrate to obstruct insulin signal pathways,and this will cut down the transportation of glucose from blood to intra-cellular and blood glucose will stet up at the end.TCPTP is a kind of intra-cellular non-receptor PTP,and it wide expressed in mature cells and embryo.The express dose in lymphatic system and hematopoietic tissucellscane is very high.TCPTP have a regulation of cytokine response and erythropoiesis. This enzyme has a negative regulation on the cancer generate process, but has a active regulation on cell growth.SHP1 is a non-receptor PTP which contains an SH2 domain.Recent research confirmed that SHP1 plays a role of negative regulation in insulin signal pathways. It utilizes the inhibition of IRs-PI3K-Akt signal transduction to finish this function. So inhibit the activity of SHP1 will find a new way to cure diabetes.SHP2 is a kind of non-receptor PTP which contains two SH2 domains on it N termination. It is a downstream signaling molecules,and can combine with many kinds of growth factor receptors directly,it also can combine with mang kinds of signal transduction medium vector,and participate in regulating cell growth and development. Besides,the mutation of SHP2 has been detected in various clinical diseases. The recent research showed that SHP2 is a negative regulator in insulin signal pathways,and its target may be insulin receptor (IR) and insulin receptor substrates (IRs).HePTP is a kind of intra-cellular non-receptor PTP whose molecular weight is 38kDa. It expressed in spleen, thymus and mainly leukemic cell line. HePTP missing will strengthen cell growth. Treat T cells with IL-2, heptp gene transcription will be sensitized, which suggest that HePTP has correlation with lymphocyte proliferation.YOPH is the only one Yersinia outer membrane proteins which has protein tyrosine phosphatase activity. It can dephosphorylation some kinds of proteins in eukaryotic cells. Not only can it destroy cytoskeleton and reject phagolysis,but also can restrain PI-3 pathway,and accordingly,restrain T lymphocytic proliferation. So takeYOPH as the target to select high performance single inhibitor will provid a new way to prevent and treat plague.To sum up,some human serious disease (such as tumor,hematologic disease and diabetes) occur just because of abnormal expression and genetic mutation of PTPs. So PTPs as a new target of curing disease,providing a new way to research and development new medicine.In order to select effective and selective inhibitors,we also employed the catalytic domain of the PTP1B,TCPTP,SHP1,SHP2,YOPH and fusion protein GST-HePTP. Using ion exchange chromatography and affinity chromatography,we obtained high-purity△P TP1B,△TCPTP,△SHP1,△SHP2,△YOPH andΔHePTP fusion protein. We find specific inhibition of 1 # natural product to MEG2. Its inhibition ratio can reach above 95% and its IC50 is 4μg/ml. We also do some researches on HepG2 cells to prove that 1 # natural product can enter into the cells,and have positive regulation on insulin signal pathway.In recent years,researches show that PTP1B,SHP1,SHP2 and MEG2 negatively modulate glucose homeostasis through the insulin signal pathway. Inhibiting thses enzymes'activity may help cure diabetes. Some of the natural products in our test have obviously effects on reducing the blood glucose,but their therapeutical effection through inhibiting PTP1B,SHP1,SHP2 or MEG2 still need more evidences to be proved.We get several natural products which have selective and conspicuous inhibition onΔSHP1 andΔSHP2. It may provide theory basis for the development of novel drugs which treat PTPs related diseases.