Long-effect Endostatin Expression In Pichia Pastoris And Expression, Purification, Activity Of Heparinase

Endostatin is known as one of tumor angiogenesis inhibitor, and be widely used for the treatment of solid tumors. However,the serumhalf-life of endostatin is very short, so it’s necessary to develop the long acting endostatin. Human Serum Albumin (HSA) is a single-chain and free-glycosylation protein,composed of 585 amino acids. The molecular weight is approximately 68kDa and is asingle protein of the highest level in blood. HSA can maintain blood pressure in thebody, transport nutrients and plays other important role of biological material,withnon-immunogenicity,fine human compatibility,and longer serum half-life (nearly 19days). It can be used as the biological activity carriers. The technique of the albuminfusion don’t need additional chemical modification, the production of technology issimple, the substrate is uniform,the quality control is relatively easy,the amount ofthe expressed protein can increase after fuse with albumin,and the effect of prolongthe half-life of drug is well,so the researcher pay close attention to the technique. In this study, we constructed the endostatin-human serum albumin by albuminfusion to extend the serum half-life of endostatin. This study will be helpful for thepre-clinical research of long acting endostatin. Pichia pastoris is a methanol yeast expressed system. It’s a widely used eukaryotic expression system and developed rapidly in recent years.Heparanase (heparanase, Hep) is a class of D glucuronidase enzyme (endo-D-glucuronidase) researched widely, both present in normal tissues such as placenta and lymphoid organs. It plays a normal physiological function on the formation of embryo, nervous system development, angiogenesis and inflammation; which is also expressed in a variety of cancer cell surface, promotes cell invasion and metastasis, induces angiogenesis and reduces tumor survival. Heparin acting as treatment and prevention of thrombosis has a long history, but studies have shown that heparin has some side effects, such as those affecting the stability of platelets which leading to post-operative bleeding. The molecular weight of 4000 to 8000 (11-14 monosaccharides) of low molecular weight heparin (LMWH) can be effective in treatment of thrombosis, but no significant side effects. At present, the need of LMWH in China most relied on imports or produced by the joint venture production, so the production of LMWH study has important industrial applications. One of the most important uses of Heparanase is the production of LMWH.To solve the above problems, this research built an efficient expression of soluble heparin, recombinant in E. coli enzyme system, to obtain comparatively high purity heparanase.