Cloning And Expression Of The Catalytic Domain Of Protein Tyrosine Phosphatase SHP-1 And Screening Of Its Inhibitors

Protein tyrosine dephosphorylation by protein tyrosine phosphatase (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. A lot of studies have shown that human diseases such as cancers, diabetes, leukemia, immuno-deficiency diseases and result from mutations or abnormality of PTPs genes. Therefore, more and more studies are focusing on PTPs.SHP-1(SH2-containing tyrosine phosphatase 1), also called HCP, SHPTP1 and PTP1C, is an important member of protein tyrosine phosphotase subfamily with highly conserved sequence. SHP-1 is a kind of cytosolic protein containing two SH2 domains at the N-terminal and one catalytic domain at the C-terminal. It was reported that increased SHP-1 activity led to immuno-deficient syndrome andn leukemia. The most recent studies have shown that SHP-1 is related to type II diabetes and high level of blood sugar is due to elevated SHP-1 activity which means that endogenous SHP-1 plays a critical role in regulating blood sugar metabolism and SHP-1 may be a potential target for reducing the blood sugar of diabetes patients by inhibiting its activity.The catalytic domainâ–³SHP-1 of SHP-1 was amplified from human SHP-1 cDNA and inserted into pT7 vector which is Amp resistant. High expression of the protein products was performed using E. coli DE3 strain and the enzymatic characteristics ofâ–³SHP-1 were determined after isolation and purification through ion-exchange chromatography. Also, purifiedâ–³SHP-1 was used to stimulate rabbit for polyclonal antibody preparation.Many traditional Chinese herbs have significant effect on diabetes through different mechanisms. The results of our in vitro assays show that among hundreds of traditional Chinese herbs screened, the water-extracts of herb A and herb B inhibit the activity of ?SHP-1 effectively. The animal assays provide further evidence that both of these two herbs significantly reduce the blood sugar of type II diabetes mice.1. The Cloning and Expression of Catalytic Domain of SHP-1The catalytic domain encoding sequence was amplified from human SHP-1 cDNA, and two sites for restriction enzyme Nde I and Hind III were introduced into N-terminal and C-terminal, respectively.ΔSHP-1 in the PCR products investigated by agar electrophoresis is ligated into the PKS clone vector which is digested by EcoRV. After that, PKS-ΔSHP-1 is digested by Nde I and Hind III ,check out then constructed into pT7 expression vector. pT7-ΔSHP-1 is delt with T4 DNA ligase, the recombinant plasmid was subsequently transformed into E.coli DE3 and high expression ofΔSHP-1 protein was achieved.2. Isolation, Purification and enzyme characterization ofΔSHP-1Two steps of ion-exchange chromatography, Q-Sepharose Fast Flow and SP-Sephadex, were used to isolate and purifyΔSHP-1 protein obtained from E.coli DE3 strain. The purity of target protein was more than 90% determined by SDS-PAGE and HPLC analysis. The results of enzymatic characteristics determination indicated that when p-NPP was added as substrate, the optimal pH forΔSHP-1 activity was 5.0, optimal temperature 36℃and ion concentration 0. The enzyme was most stable at pH 7.5, 15℃and without ions. The kinetics analysis of its catalytic reaction showed that the Km was 23.75mM and Vmax was 8.882μmol/min.3. The Preparation and Purification of Polyclonal Antibody ofΔSHP-1The purifiedΔSHP-1 was used to stimulate rabbit, and anti-ΔSHP-1 polyclonal antibody was gained by applying PVDF column. Before and after purification of antibody, by means of ECL , the both efficency were 1:10000, the sensitivity is 0.01μg and 1 ng respectively which is increased to 10 fold. The purity of antibody is over 90% by the investigation of SDS-GAGE and western blot. These results indicated the antibody match to and surpass the standard for further experiments. The purified antibody is dealt with at 56℃for 30 min ,added by azide sodium with final concentration 1/1000(W/V) then fractioned which was stored under -80 oC.4. Screening Traditional Chinese Herbs for Inhibitors of Protein Tyrosine Phosphatase SHP-14.1 Determine the inhibition ratios of Traditional Chinese Herbs20g herb was added to 200ml distilled water, soaked for 1 hour, heated until boiling and held for 30min. The mixture was filtered before another 150ml distilled water was added to the residue and boiled for 20 more minutes. Then the mixture was filtered again. The liquors from these two steps were combined and centrifuged at 4℃and the supernate was collected as water extract of herbs. p–NPP was used as substrate for measuring the inhibitory rate of herb extract onΔSHP-1. The reaction system contained 20mM p-NPP, Mops-NaOH buffer (25mM,pH7.0,1mM DTT,0.1M NaCl,1mM EDTA,1mg/ml BSA),ΔSHP-1(10μg/ml)and 10ul extract. The reaction condition was designed as incubation at 37℃for 20min, and then the absorbance was measured at 405nm. Each experiment was performed in duplicate and the inhibitory rate of each extract was calculated.Two herbs which exhibited more than 95% inhibition onΔSHP-1 were screened from about 160 kins of traditional Chinese herbs, herb A 96.77% and herb B 95.10%, respectively.4.2 Determination of IC₅₀:IC₅₀ was analyzed with 20mM p-NPP as a substrate in a buffer (pH=7.0) containing 20mM Mops, 1mM DTT, 0.1M NaCl, 1mM EDTA, and 1 mg/ml BSA for 20mins at 37℃. 10μg/mlΔSHP-1 and varying concentrations of herb A or herb B extract were added to the buffer. Then the absorbance of p-NPP at 405nm was analyzed after the termination of the reaction with NaOH. IC₅₀ values are calculated by constructing dose-response curves, and, as a result, the IC₅₀ of P herb A and herb B are 2μg/ml and 4μg/ml, respectively.5. Herb A and Herb B decrease the blood glucose of Typeâ…¡Diabetes Mice5.1 The model of Typeâ…¡Diabetes Mice40 mice were selected for establishing type II diabete animal model which were injected with 50mgSTZ/kg per day for 5 days. The control group were 10 mice which were injected with pt aequ citrate buffer solution. After the treatment, each mouse was weighed every day and the blood sugar was detected every week using Blood Glucose Kit. The mouse with more than 11.1mmol/L blood sugar was considered as established model.Our result showed that after 14 days of treatment, 37 mice out of 40 had significantly increased blood sugar level with average 28.3mmol/L, which meant that the type II diabetes model was well established.5.2 Experiment of administerThe mice were randomly grouped into control group, herb-treated group, and negative control group. Both the control group (healthy mice) and the negative control group (type II diabetic mice) were administrated with 25ml/kg water according to body weights, while the herb-treated group (type II diabetic mice) was administrated with 10g/kg herbs according to body weights for 25days. Body weight, food intake, water intake, and miction variation were observed everyday. Blood glucose levels were analyzed every five days after mice'being abrosia for 16hrs. The blood glucose values were calculated according to the standard preparation.Our animal experiments provided strong evidence that both of these two herbs, herb A and herb B significantly reduced the blood sugar of type II diabetes animal model with 19.6mmol/L and19.4mmol/L, respectively.Screening effective drugs based on their direct targets is one of the methods for new drug discovery, which has multiple advantages including precise efficacy, clear mechanism as well as reliable quality. Besides its specificity and accuracy, this method can be utilized to find out the novel potency of existing drugs, which may lead to the secondary development of Chinese traditianal herbs.