Amelioration Of Expression Of V-Src Tyrosine Kinase And Activity Analysis

Src is a kind of non-receptor protein tyrosine kinase of molecular weight (Mr) 60,000 that has two categories. One is virus-Src (v-Src) and the other is cell-Src (c-Src). Abnormal expression and activity of c-Src is one of the reasons of tumor; v-Src itself is capable of inducing tumors in the host animal, and cell transformation in tissue culture. Both are cooperate with many receptor and nonreceptor protein tyrosine kinases to diversify signals that regulate cell behaviors.Previous researches mostly focused on the structure, regulation and function of c-Src. The cloning of v-src and researches on its product were relatively few. Our experiment is to construct the recombinant plasmid pGEX-KT/v-src and analyze the characterizations of the product. This paper focuses on the study below:Part one: To obtain the optimal expression conditions, we investigate the effects of cell stain, growth temperature, Aeoo, medium composition, IPTG concentration, induction temperature and induction time on the GST-Src fusion protein yield. Also, we carried out purity and protein yield of this product. The Escherichia coli were inoculated at different conditions according to the orthogonal design, and compare the GST-Src fusion proteins using SDS-PAGE. It showed that medium composition and induction temperature affected the GST-Src fusion protein yield obviously. The procedure for GST-Src expression at the optimal condition was: the overnight culture was diluted 1:100 into fresh TY medium and growed at 30 with shaking ( 250r / min ) until the ASOO reaches 0.6-0.7. Then added IPTG to a final concentration of 0.1 mM and continued incubation for 3h. GST-Src reached 30% in relation to the whole bacterial proteins. Approximately 13.5 mg of GST-Src inclusion body with 98% purity and 2mg of the affinity - purified GST-Src fusion protein with 85% purity were obtained from 1L BL21(DE3)pLysS culture.Part two: The optimal extract condition of cells and solubilization conditions of inclusion body were obtained: E. coli cells were disrupted by ultrasonic in 4 minutes (120X2s), alternation time was 4s; the inclusion body was washed at 4, thewashing concentration of urea was 2.0 mol/L, and the washing time was 8h. After solubilizing by 8 mol/L urea in lysate buffer, the inclusion concentration was about 0.181 mg/ml.Part three: The optimal purification condition of cell culture lysates was obtained: eluted overnight at 4 and collected No.2 -No.4 ml elutions.Part four: The tyrosine kinase activities of the affinity-purified GST-Src and the inclusion solution were measured using ELIS A technique with poly Glu:Tyr(4:l). The specific activity of the affinity-purified GST-Src was about 0.38 nmol/min/mg and the specific activity of the inclusion solution was about 1.13 nmol/min/mg.Our experiment developed the optimizing method to yield a high level of GST-Src expression and get the preferable effects of purification and solubilization of the inclusion body. Comparing with the other researches, our experiment had 6 characteristics:(1) The full-length v-src cDNA was cloned into the pGEX-KT vector and the product could be used for the whole structure analysis.(2) The vector gave rise to GST-Src fusion protein that could be purified in a one-step procedure using GSH Sepharose 4B. Moreover, the GST protein didn't affect the enzymatic activity greatly.(3) The production and tyrosine kinase activity were relatively high.(4) The tyrosine kinase activities were quantitatively analyzed with ELISA technique that had no isotope hazards.The effective screening of v-Src targets and / or inhibitors depended on the immunity and tyrosine kinase activity. The active preparation would provide a good PTK source for screening of v-Src targets and / or inhibitors and for researching of structure, antibody and biochemistry and biophysical properties.