The Expression And Purification Of Protein Tyrosine Phosphatases And Screening Of Their Inhibitors

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 .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 in the final.TC-PTP 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. TC-PTP 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. SHP-1 is a non-receptor PTP which contains an SH2 domain. Recent research confirmed that SHP-1 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 SHP-1 will find a new to cure diabetic.SHP-2 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 SHP-2 has been detected in various clinical diseases. The recent research showed that SHP-2 is a negative regulator in insulin signal pathways, and its target may be insulin receptor (IR) and insulin receptor substrates (IRS).He-PTP is a kind of intra-cellular non-receptor PTP whose molecular weight is 38kDa. It expressed in spleen, thymus and mainly leukemic cell line. He-PTP missing will strengthen cell growth.Treat T cells with IL-2, He-PTP gene transcription will be sensitized, suggest that He-PTP 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 onl can destroy cytoskeleton, reject phagolysis, but also can restrain PI-3 pathway, accordingly,restrain T lymphocytic proliferation.So take YopH as the target to select high performance single inhibitor will provid a new way to prevent and treat plague.PTP-MEG-2 is an intracellular enzyme of 593 amino acid residues with a putative lipid-binding domain at the N-terminus. When PTPMEG-2 over expessed in cancer cells, it causes accumulation of gluconeogenesis transfer factor in cell nucleus; PTPMEG-2 can regulate the balance of blood glucose,it may provide a new way to treat diabetes.To sum up, some human serious disease(such as tumor, hematologic diseases, diabetes)just because of abnormaly express 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 MEG-2,PTP1B,TC-PTP,SHP-1,SHP-2,YopH and fusion protein GST-He-PTP.Using ion exchange chromatography and affinity chromatography, we obtained high-purity△MEG-2,△P TP1B,△TC-PTP,△SHP-1,△SHP-2,△Y opH and GST-He-PTP fusion protein. There are nine traditional Chinese medicines'inhibition ratio above 90%, 5#, 6#, 10#, 17# and21# traditional Chinese medicines have high inhibition onΔSHP-1. 1# and 16# traditional Chinese medicines have strong inhibition both onΔSHP-1 andΔSHP-2. In the teas, we find 2 tea powder and 12 teas'inhibition ratio above 90% on 7 PTPs.In recent years, researches show that PTP1B, SHP-1, SHP-2 and MEG-2 negatively modulate glucose homeostasis through the insulin signal pathway. Inhibiting thses enzymes'activity may help cure diabetes. Some of the traditional Chinese medicine herbs in our test have obviously effects on reducing the blood glucose, but whether their therapeutical effection through inhibiting PTP1B, SHP-1, SHP-2 orMEG-2 still need more evidences to be proved. In our research, some kinds of teas and tea powder show selective and conspicuous inhibition onΔSHP-1.We get several traditional Chinese medicine herbs, teas and tea powder which have selective and conspicuous inhibition onΔSHP-1 andΔSHP-2. It may provide theory basis for the development of novel drugs which treat PTPs related diseases.