Gene Recombination, Expression And Activity Analysis Of V-Src

Protein tyrosine kinase (PTK) which has tyrosine kinase activity, can transfer theβ-phosphate group of ATP to other important protein tyrosine residues, make them phosphorylated,and also mediate cell growth, cell division, signaling and a series of physiological and biochemical reactions and so on. Src family kinases (SFKs) include eight proteins besides Src, belonging to PTK. Src protein mainly exists in two forms:protein expressed by oncogene of Rous sarcoma virus and protein expressed by proto-oncogene of eukaryocytes. c-Src protein involves in intracellular signal transduction, closely relevant to cell growth, differentiation, and apoptosis and so on. At present, the domestic and foreign research of Src family are focused on the c-Src protein, whose structure, physical and chemical properties cannot be further studied. c-Src protein's homologue, the v-Src protein, both the structure and function of which are quite similar to the former, seldom shows any change in the activity of in vitro in contrast to in vivo, while c-Src protein in vitro shows little activity. Therefore, the physical and chemical properties of v-Src protein can be easily studied through constructing restructured v-src gene and expressing the v-Src protein in E. coli in great quantities. Therefore, the physical and chemical properties of v-Src protein can be well studied by expressing the v-Src protein in E.coli in great quantities, and a model system for screening of Src tyrosine kinase inhibitors is estabilished.First, through gene engineering technology,v-src gene was cloned into the middle cloning vector pUCm-T and then into the expression vector pGEX-KT, then the recombined expression plasmid pGEX-KT/v-src was completed. PCR, double digests and sequencing identify the successfully constructed expression plasmid. Secondly, by orthogonal experiment optimization, the optical condition for expressing was determined:37℃,1 mM IPTG and 5 hours induced expressing. The v-Src protein was bacterially expressed and collected in the form of inclusion-body under that condition, and then, was washed,dissolved,affinity chromatographied,digested by thrombin and renatured by continuous gradient of urea. Again, screening the extract of Scutellaria barba(ESB), the ethanol extract is found to have inhibitory effect on v-Src protein, and the inhibition is relevant to dose. Cell experiments show that the composent also has inhibition over mitochondria activity.This research has obtained active v-Src protein by E.coli expression system, and selected the anti-tumor composent of ESB. It can provide a basis for further study on the structure and function of Src protein, and lay the foundation of the anti-tumor drug screening system in vitro.