Expression And Purification Of Soluble Recombinant Hexastatin In E.coli

Angiogenesis, the formation of new capillaries from pre-existing blood vessels, is an event critical to many normal physiological processes. In 1971, Folkman proposed that tumor growth and metastasis are angiogenesis-dependent. The diffusion capacity for oxygen and nutrients in tissues is limited to about 150μm. If tumor angiogenesis is inhibited, tumor cells will apoptosis for the lack of oxygen and nutrients supplies. This is the famous "Tumor Hungry Therapeutics". The propose of this theory brings many new strategies for the biotherapy of tumor, makes the effort to search many new endogenesis angiogenesis inhibitor become the hotspot in recent years.Hexastatin composed of 229 amino acids, is a 25-kDa NCI domain of the a6 chain of typeⅣcollagen, which derives from extracellular matrix. The recent researches show that Hexastatin binds to endothelial cells viaαvβ3 integrin, and theαvβ3 integrin binding is facilitated by an RGD-independent mechanism. It regulates the adhesion and migration of endothelial cells, inhibites their proliferation in a dose-dependent way. Furthermore, solid tumor studies show that Hexastatin can potently inhibites growth of tumor cells. Its level is always lower in cancer tissues, which may be an important symbol for tumor progression.The Hexastatin gene was cloned from EC9706 cells and Hexastatin protein was recombinantly produced in E.coli BL21. Then the soluble recombinant protein was purified with Amylose Resin Heads. The results show that RT-PCR product was about 687 bp, its sequence was the same as that of Hexastatin reported. The recombinant protein was expressed in E.coli BL21 with high level, the soluble protein accounted for 24.8% of the total bacterial protein. The purity of recombinant protein purified by Amylose Resin Heads reached more than 90%. The cloning, expression and purification of human Hexastatin has laid a foundation for its anti-angiogenesis theraphy for tumor.