Genetic Optimization, Protein Expression, Purification And Preliminary Application Of Human Hexastatin

Angiogenesis is the basis for many important physiological and pathological processes. Angiogenesis not only plays a very important role in normal physiological processes such as embryogenesis and placental development, but also is the key process of tumor occurrence, development and metastasis. In1971, Folkman proposed that tumor growth and metastasis are angiogenesis-dependent. The famous theory of "Tumor Hungry Therapeutics" declared that if tumor angiogenesis is inhibited, tumor cells will apoptosis for the lack of oxygen and nutrients supplies, which provided a novel strategy for tumor biotherapy. Since the endogenous angiogenesis inhibitor showed highly selective in inhibiting the proliferation of the tumor bed, it was reasonably to be considered the most potential protein drug for anti-angiogenic therapy. Searching for new endogenesis angiogenesis inhibitors become the hotspot in recent years.Hexastatin is a novel angiogenesis inhibitors, and derived from NCI domain of the a6chain of type IV collagen. It is composed by229amino acids with molecular weight of25-KD. In recent years it has been found that Hexastatin can inhibit the tumor angiogenesis and anti-tumor activity. Experimental studies have shown that abnormal expression of the COL4A6gene encoding the a6chain are directly related with tumor formation; while the other studies found Hexastatins level is lower in cancer tissues which can be used as an important indicator of tumor-infiltrating, and all of these are early warning signs.This study optimized and synthesized human Hexastatin gene, and then connected to the pET28a expression vector, induced to express by IPTG under the optimized conditions. After being processed by ultra-sonication and inclusion bodies and purified with Ni-NTA chromatographic column, it analyzed and identified by methods of SDS-PAGE and Western blot, conducted activity test of tumor cells and HMEC in vitro by MTT. Results:pET28a-seq702expression plasmid was constructed and expressed in E. coli BL21. The expression level of soluble Hexastatin protein accounted for45.1%of the total protein. The purity of human Hexastatin protein reached at least90%, and the density was160.24μg/ml after purification. Human Hexastatin protein could significantly inhibit the growth of C6, MCF-7and HMEC cells, and inhibition ratio was72.95%±3.6,48.8%±2.9,52.7%±2.5respectively, with significant difference from those of controls (P<0.05). It was confirmed that the optimized human Hexastatin protein was effective in the growth inhibition of tumor cell and HMEC, provided a new way for tumor anti-angiogenesis therapy.