Angiogenesis Inhibitors Gene Recombinant Soluble Type Kringle5 Chromatographic Separation And Quality Standards Of Research

Cancer, known medically as a malignant neoplasm, is a broad group of various diseases involved unregulated cell growth. At present, they have not invented a drug that can treat cancer efficiently. Human Plasminogen Kringle5(referred to as K5) act as a tumor suppressor protein, which can inhibit cell proliferation and migration, and it is the most powerful endogenous angiogenesis inhibitor with high efficiency, low toxicity and less drug resistance. K5also can treat other angiogenesis-related diseases efficiently, such as diabetes, sickle cell retinopathy and rheumatoid arthritis. It is prospective that angiogenesis inhibitor K5developed to become the drug of treating angiogenesis-related diseases.In the study, on the foundation of successful clone and expression of recombinant soluble and non-fusion angiogenesis inhibitor K5, a two-step chromatographic method, including SP Sepharose Fast Flow and Sephacryl S-100HR in sequence, was established to separate and purify angiogenesis inhibitor K5. On SP Sepharose Fast Flow column, the buffer A was consisted of50.0mmol/L acetic acid-sodium acetate (pH5.2), and the buffer B was consisted of buffer A with the addition of0.5mol/L sodium chloride (pH5.2); on Sephacryl S-100HR column, the elution buffer was5.0mmol/L phosphate solution (pH7.0).Through the two-step chromatographic purification process, the molecular weight of the obtained K5was about28000, which was about twice of that of single K5, and the purity of obtained K5was more than98%. In addition, it was found that the obtained K5could inhibit the blood vessel growth of chick embryo chorioallantoic membrane effectively.The denaturation temperature of the purified K5with different protective agents was tested by differential scanning calorimetry (DSC). The results show that the denaturation temperature of the purified K5ranges from70℃to80℃. With the protective agent that dextran40is twice of trehalose in quality, the denaturation temperature of K5was the highest, which is78.974℃. There is a close relationship between denaturation temperature and the protective agents. F410kit detected that the E.coli host protein is0.0359%of the purified protein, which is in line with the relevant provisions of the national protein products. These studies lay an important foundation for the further medical research of K5.