Cloning And Expressing Of Human Canstatin And Investigating It's Anti-Angiogenic Activity

When the solid cancer grew up to 2 mm3, they need for the supplement of nutrition and oxygen, or there will not be further growth of cancer cells due to starvation. This property provided the feasibility for the treatment of cancer by inhibiting of angiogenesis. Endostatin, angiogenestatin, tumstatin, have all been demonstrated to effectively inhibit angiogenesis and the growth of solid cancer.Canstatin is the 25kD C-terminal fragment of α2 chain of type IV collagen. Its pI is 6.7, which consists of 227 amino acid residues. It induced apoptosis in cultured endothelial cells and inhibited angiogenesis in vitro and in vivo. Canstatin-induced apoptosis is associated with phosphatidylinositol 3-kinase/Akt inhibitions and is dependent upon signaling events through death receptors. In thymidine incorporation assays of endothelial cell proliferation, mean effective dose value of canstatin is 0.5?μg/ml while that of endostatin is 1.5ug/ml, this indicated that canstatin is more powerful than endostatin in cure of solid tumors and diseases associated with angiogenesis. We cloned hCS cDNA, and expressed the protein in prokaryote cells and pichia pastoris cells and tested it`s anti-angiogenesic biological activity for further research of the structure and function of hCS and to pave a way to cure human tumors.First, Total RNA was extracted from Chinese placenta tissue. The canstatin cDNA was amplified from total RNA by RT-PCR, then cloned into pTYB1 and sequenced. Thus, the Prokaryotic expression vector, pTYB1-hCS was constructed, and the fusion protein was expressed after IPTG induction. We renatured canstatin inclusion body by density gradual dialysis, and purified canstatin by using chitin affinity chromatography. The biological activity of rhCS was arrayed by the chick embryo chorioallantoic membrane (CAM).We obtained hCS cDNA from pTYB1-hCS and constructed the shuttle vector pPIC9K-hCS by subcloning. Escherichia coli was transformed by pPIC9K-hCS for the amplification. pPIC9K-hCS was extracted and linearized by SacI, then introduced into pichia pastoris GS115 and SMD1168 activated by PEG1000. When the canstatin cDNA was integrated into the genome of the host. The recombinant pichia strains were selected by G418 and direct PCR. The transformant containing multicopies of canstatin cDNA were selected with increasing concentration of G418. The expression was induced with 1% methanol, and the canstatin protein in the culture supernatant was assayed with 12% SDS-PAGE and Western blot.We have cloned and expressed human canstatin cDNA. The hCS-Intein fusion protein expressed in Escherichia coli ER2566 was insoluble inclusion body, the fusion protein accounted for 37.2 % of total protein in the transformed bacteria. Soluble fusion protein was obtained by renaturing and chitin affinity chromatography. The molecular weight was about 25kDa. The purity of the product was up to 91.6 percent. The expressed and purified canstatin product could suppress the formation of nascent vessel in CAM assay, and indicated its potent in treat of solid tumors and diseases associated with angiogenesis. The recombinant pichia pastoris GS115 and SMD1168 successfully expressed the canstatin in the first time, whose molecular weight analysed by SDS-PAGE is consistent with that expected. The expressed canstatin could be specifically recognized by His-Tag monoclonal antibodies. The successful expression of human canstatin laid the foundation for the anti-angiogenesis therapy.