The Studies Of The Catalytic Domain Of Protein Tyrosine Phosphatase MEG1 On Cloning, Soluble Expression And Enzymatic Properties

Protein tyrosine phosphorylation is a reversible protein post-translational modification,which regulated by protein tyrosine kinases(PTKs)and protein tyrosine phosphatases(PTPs).It plays an extremely important role in the process of cell signal transduction,which almost regulates all biological life activities.There are 38 kinds of classical PTPs in the human body and each one has a specific function.The protein encoded by the PTPN4 gene is PTP-MEG1,which is an important member of the PTPs family.Studies have shown that PTP-MEG1,which is overexpressed in eukaryotic cells,exerts a negative regulatory effect by the dephosphorylation of substrate tyrosine residues.It inhibits cell proliferation and reduces the degree of cell enrichment.Although research on PTP-MEG1 has made some progress in recent years,we still know little about its biological function.One of the main obstacles is that PTP-MEG1 cannot be expressed in prokaryotic expression systems.In order to solve the problem,an expression vector containing the His-tagged PTP-MEG1 catalytic domain(?PTP-MEG1)was constructed,and a peptidyl-prolyl cis-transisomerase FKBP-C was introduced into the vector.It helps the target protein fold correctly in E.coli and increase its solubility.At the same time,we used E.coli strain C43(DE3)as an expression host,which has the ability to express toxic proteins.The experimental results show that this expression system greatly increases the soluble expression rate of ?PTP-MEG1.The bacteria disruption solution was purified by Ni chromatography and we got the high-purity ?PTP-MEG1.In order to examine whether the target protein has the properties of PTPs,we performed a series of enzymatic characterizations of ?PTP-MEG1 and determined the optimum temperature,the optimum pH,the optimum ion intensity and the Km value.The results show that ?PTP-MEG1 has similar enzymatic properties as other non-receptor PTPs,which lays the foundation for further study of the physiological functions of PTP-MEG1.At the same time,we also provide a prokaryotic expression systems which can increase the soluble expression rate of the target protein.In addition,we also used this expression system to induce the protein tyrosine kinase cKIT.Although the target protein was not correctly expressed,this is still a meaningful attempt.We will continue to improve the prokaryotic expression system in order to make a greater contribution on improving the soluble expression rate of eukaryotic proteins in prokaryotic expression systems.