C-src, Full-length Gene Cloning And Expression In Escherichia Coli

The nonreceptor tyrosine kinase c-Src, first identified as an oncogenic protein, has two categories, including virus-Src (v-Src) and cell-Src (c-Src), Both of which are highly homologous with similar structures and protein tyrosine kinase (PTK) activity. In eukaryote, the proto-oncogene c-src plays an important role in many signal transduction pathways, which expressed with a broad spectrum to regulate the cell physiological process of growth, proliferation, differentiation, migration and apoptosis. Besides, overexpression and abnormal activition of c-Src is closely implicated in the development of many tumorigenesis.For the moment, most of reports about c-Src are focused on its molecular regulation to cellular function and its relation with human diseases. Furthermore, owing to oncogenic protein, people forecast it should have fine foreground of development and application to produce c-Src inhibitors or immunogenic antibodies as anti-cancer agents by gene engineering technology.In this paper, it is studied in clone and expression of the human c-src full gene from a bacterial system. The human c-src gene was obtained by PCR techniques from the mRNA of human pulmonary cancer cells and plasmid pcDNA3.1/Hygro contained the c-src sequence. After verifying the PCR sequence through cloned into pUCm-T vector, c-src gene was inserted into some IPTG-inducible expression vectors, such as pET-28a(+) pET-22b(+) and pPRoEX HTc plasmid, and these recombinant plasmid were expressed in BL21(DE3) bacterial cells. SDS-PAGE analysis showed that pProEX HTc was a highly effective expression vector to c-Src. and pET victors were not contrarily. High-yield expression of c-Src protein can reached upon 20% in relation to the whole bacterial peoteins in a most suitable condition of 0.1mM 1PTG for5h on 30℃. Using a Ni~2+affinity chromatography, (HisVc-Src fusion protein was purified effectively, and subsequently, the PTK activity of the c-Src was determined with poly-Glu:Tyr(4:l) by ELISA technique. The result showed that protein has PTK activity.