Prokaryotic Expression And Purification Of Fusion Protein Of PTD-SH2

Hepatocellular carcinoma(HCC) is a serious cancer to humanbeing. The overall incidence is estimated to be 1 million cases per year with a wide geographic variability. China is a country with high risk of HCC, 45 percent of the new cases of the world are occurred in China approximately. Because of hepatic cirrhosis and fast living of tumor cell the prognosis of HCC is not good. Until now, the main treatments include surgical resection, liver transplantation, cryoablation, radiofrequency(RF), microwave therapy, etc. But the effect of single therapy mode is usually not good enough, so one treatment always combine with others. It's high time to take some efficent measures to solve the problem of hepatoma.The thought of including tumor cell apoptosis will lead to the emergence of novel antitumor therapy with high efficacy and low toxicity and inflammatory reaction.Accumulating research indicates that protein tyrosine kinases are an especially important target as they play an important role in the modulation of growth factor signaling. The over expression of PTKs and abnormal signal transduction induced by them are obviously related with HCC. Src is discovered as the first tumor protein with tyrosine kinases activation. The oncogene c-Src can participate in a wide variety of many physiological functions. Moreover, the protein production of c-Src are high activation and/or over expression in many tumor cells. The over expression and activation of Src-protein exists in many cancers such as colon carcinoma, mammary cancer, pancreatic carcinoma and adenocarcinoma of lung. SH2 domain plays many different roles in cellular protein tyrosine kinase(PTK) signaling pathways. Therefore the activation of oncogenes like src, abl, ras and raf are mediated directly or indirectly by SH2 domain following PTKs'increased activition. So SH2 domain becomes a new interference target in abnormal signal transduction pathway of malignant tumor. Protein transduction domain(PTD)can translocate across cell membrane and get into cytoplasma directly effiently and promptly. When fused with some effectors it can lead these factors into cytoplasma, which can improve the cytotoxity of them. In sumary, we construct the recombinant expression vector containing PTD and SH2 fusion gene and express PTD-SH2 fusion protein in E.Coli and efficientily purify the fusion proteins. Therefore the present study probably provides laboratory data for the application of PTD-SH2 fusion protein into gene therapy of hepatocellular carcinoma.Objectives: To construct the recombinant expression vector containing PTD and SH2 fusion gene and express PTD-SH2 fusion protein in E. Coli and purify the fusion proteins .Methods: A 297 bp of human SH2 gene fragment was amplified by PCR method and cloned into pET-16b vector immediately downstream of the PTD fragment, an E.coli expression vector, to construct a recombinant plasmid pET-16b-PTD- SH2. The plasmid was transformed into E. coli BL21 (DE3) and induced to express fusion protein PTD- SH2 with IPTG. The expression of PTD- SH2 was detected by SDS-PAGE electrophoresis and Western blot.Results: SH2 was identical to what reported by Genebank. A novel protein with expected molecular mass (about Mr 15×103 )was expressed upon induction with IPTG. The expressed product showed good reactivity to anti-His tag antibody , and was mostly in the form of inclusion bodies. The expressed proteins could be purified via Ni-NTA affinity chromatography in denatured condition.Conclusion: Our successful cloning and efficient expression of SH2 gene and purification of SH2 protein lay a basis for further study of SH2 functions.