Of C-src Kinase Domain Gene Cloning And Its Expression In Escherichia Coli

c-src is a proto-oncogene, belonging to the non-receptor protein kinases family, which plays a central role in a variety of cell signaling pathways that regulate cell growth, differentiation, apoptosis, and other important cellular processes. Growing evidence suggests that elevated expression of c-Src contributes to the development of a variety of human cancers.Researches about c-Src are mostly focused on its molecular mechanism of signaling pathways and cell events regulation. Also, inhibition of deregulated protein tyrosine kinases represents an attractive strategy for controlling cancer growth, this could constitute a new therapeutic target in human cancer. Expression and purification of c-Src by genic technology provide a practical way to target specificity c-Src inhibitor and to generate its detailed mechanistic information.In this paper, we describe the clone and expression of the gene of kinase domain of human c-src from a bacterial system. The kinase gene sequence is obtained from plasmid pP_roEX HTC/c-src by PCR, after cloned in pUCm-T vector, it was inserted into the expression vector, such as pET-22b(+). The target protein was expressed by the induction of IPTG Analyze the result by SDS-PAGE and get the best induction condition. Purification the protein by Ni²⁺-NTA affinity gel and renaturation it through the Sephadex G-25. the PTK activity of the target protein was examined by ELISA.SDS-PAGE analysis of the IPTG induced expression of pET-22b(+)/c-src (BL21) showed that the weight of the target protein is correct, and the best induction condition is 0.1mmol/L IPTG for 5h on 30℃. ELISA test of the renatured protein showed that the PTK activity of target protein is not very high. The target protein being translated is without the SH2 domain and SH3 domain, so the tyrosine 527 at the C-terminal lost the binding domain, this result the protein in an active conformation constantly in theory , and provide the foundation for c-Src inhibitionstudies. The protein after well renaturation can be used in aim specificity c-Src inhibitor.