The Cloning And Expression Of RPTPα-D1 And The Preparation Of RPTPα-2D Polyclonal Antibody And Their Renaturation

Protein tyrosine dephosphorylation by protein tyrosine phosphatases (PTPs) and phosphorylation by protein tyrosine kinases (PTKs) both play an important role in regulating cell growth, cell development and cell signal transduction, which are involved in many physiological and pathological phenomena.In the quantities of regulation mechanisms of cell signal transduction, the phosphorylation and dephosphorylation of proteins are of importance.Especially, phosphorylation of specific cellular proteins on tyrosyl residues is one of the most fundamental regulatory mechanisms in signal transduction that dictates cell-cell communication, cell growth and proliferation, regulation of cell cycle and differentiation, tumorogenesis and tumor metastasis, nerve conduction, angiogenesis, embryogeny, and kinds of hereditary and non-hereditary diseases. The research of protein tyrosine phophorylation has been a hot spot in the field of biological science.RPTPs have a single membrane-spanning domain and most RPTPs contain two cytoplasmic catalytic domains.The N-terminal membrane-proximal domain,RPTP-D1, containsmost of the catalytic activity, while the C-terminal membrane-distal PTP domain, RPTP-D2, appears to play a regulatory role.RPTP activity may be regulated by dimerization,analogous to their enzymatic counterparts, the receptor PTKs (RPTKs). The Frst evidence for dimerization as a regulatory mechanism of RPTP activity came from studies with a chimeric protein consisting of the extracellular domain of the epidermal growth factor receptor (EGFR,a proto-typical RPTK) and the intracellular domain of the RPTPCD45.Ligand-induced dimerization of EGFR/CD45 leads to functional inactivation of CD45, suggesting that dimerization inhibits CD45 activity. The crystal structure of the N-terminal membrane-proximal PTP domain of RPTP (RPTPα-D1) provides structural support for dimerization-induced inhibition of RPTP activity . A helix–turn–helix wedge-like segment to the N-terminal side of RPTP-D1 that is conserved in RPTPs, but not in cytoplasmic PTPs, interacts with the dyad-related monomer, thus forming a dimer in which both catalytic sites are occluded.Forced dimerization of RPTP by insertion of a cysteine at position 137 in the extracellular domain, resulting in the formation of a disulphide bridge between two monomers, leads to inactivation of RPTP catalytic activity,which is dependent on an intact wedge. However, introduction of cysteines at other positions in the extracellular domain (135, 139, and 141) leads to constitutive dimerization but not to inactivation of RPTPα, demonstrating that dimerization does not lead to inactivation.The positions of the disulphide bonds suggest thatrotational coupling between the two monomers in a dimer is an important determinant for dimer activity.Evidence is accumulating that RPTPs can be regulated by dimerization, as discussed above. It has been found that RPTPs dimerize constitutively in living cells . Different domains in RPTP contribute to dimerization.Since RPTP dimerizes constitutively on the cell surface and since the position of the two monomers in the dimer relative to each other determines catalytic activity, it is tempting to speculate that RPTP activity is regulated by small changes in rotational coupling as a result of ligand binding or post-translational modiWcation.Oxidative stress, known to inhibit PTPs by oxidation of the catalytic cysteine,induces a conformational change in the C-terminal PTP domain (RPTP-D2) and leads to stabilization of the dimer, resulting in prolonged inactivation of RPTP after removing oxidative stress.c-Src is a kind of protein tyrosine kinase, the whole cell division cycle follows the activation of onco-gene c-Src. Overexpression of c-Src leads to tyrosine phosphorylation and cell transformation of many kinds of protein substrates. In interphase Src proteins fold themselves to form inactive state.RPTPαcan dephosphorylate at Tyr529 of c-Src, then c-Src unfold and become active state. This activity exists in the whole cell mitosis. RPTPαcan activate Src family kinases by dephosphorylation and promote transformation.RPTPαmaybe play an important role in inhibiting tumors related with the high activity of Src. What we plan to do is: First, we want to find the tissue of high RPTPαexpression from many kinds of tumors with antibodies of RPTPαand obtain the cells which have high RPTPαexpression by primary culture. Second, we will screen the specific inhibitor of RPTPα. At last it will be identified that the low expression of RPTPαcan decrease the activity of Src, so we can obtain a new method of indirect therapy or prophylaxis to cancers. RPTPα-1 Ddomain was cloned and constructed into protein expression vector .PT7-7-RPTPα-D1 was high expressed in E.coli BL21 Codon plus. PT7-7-RPTPα-2D is maintained in the laboratory. Then we obtained RPTPα-2D protein by preparative electrophoresis. Then we prepared polyclonal antibodies of RPTPα-2D for the next step.1. Clone and expression of RPTPα-D1 and RPTPα-2D genesRPTPα-D1 was cloned by means of gene engineering and constructed into soluble expression vector PT7-7- RPTPα-D1, which was then sequenced and verified. Then it was used to transform E.coli BL21 Codon plus, RPTPα-D1 was high expressed and RPTPα-D1 was high expressed with the same method.2. Preparation polyclonal antibody of RPTPα-2DRabbit were immuned with puritified RPTPα-2D. Valence of antibodies were detected by ECL, the result was consistent with the next study.It establish the foundation of tissular distribution and disease associativity of RPTPα3. Renaturation of RPTPα-D1 and RPTPα-2DAt the same time, the renaturation phosphatase conditions were explored in order to lay a foundation for future research by use of dilution, dialysis, G-100 column refolding.