Study On Expression, Purification Of Endostatin And Its Biological Activity Identification

Tumors do great harm to human ,in common radiotherapy, chemo-therapy and immuntherapy are used in clinic,but side effect is easy to emerge and patients often develop resistence .Therefore those traditional theraphy is limited in clinic .The growth and metastases of tumors depend on neovascularization,thus by inhibiting the neovascularization to prevent the tumor growth and metastasis is a strategy for tumor therapy. Endostatin is a C-terminal fragment of collagen XVIII.,which can inhibit the endothelial cell proliferation and inhibit growth and metastasis of tumor.Most importmently ,it has no side effect related to dosage ,and it brings no resistence.Antiangiogenesis maybe combine traditional therapy to enhance effect of antitumor. Poduction of enough endostatin benefit various clinic trials to develop new method to cure tumors. The sequence encoding endostatin was amplified from a human fetal kidney cDNA library through PCR,the amplified fragment was inserted to expressive plasmid pPICZ〆A, Pichia pastoris was served as host strain.The constructed gene-engineering Pichia pastoris to produce endostatin can use methanol as exclusive carbon source.The expressive plasmid including 〆factor signal sequence facilitates secretion of the expressed protein into the medium.The yeast strain secretes only very low levels of endogenous host protein, which further simplifies the purification. Soluble recombinant protein has natural activity needless of renaturation and refold process, which can improve recovery rate of extraction. The strain used in this experiment is selected and the peak lever of expression was about 60mg/L. Ferment of Pichia pastoris can be separated to two phases: one is proliferate number of yeast ,in which glycerol was used as carbon source .the other is to express protein, in which methanol was used as carbon source. Excessive quantity of Glycerol or methanol can inhibit growth , so they were supplied slowly. Induced phase can be started after Glycerol was depleted. It was proved induction phase of 48 hours can give best expression. After fermention, the supernatant was collected by centrifugation, and then concentrated by tangential flow ultra-filtration system with 10kDa NMWL membrane to remove the substances of small molecular weight and to reduce the concentration of salts,balance buffer is 0.5M Tris-HCI,0.1MNaCl ,pH 7.4. Utilizing the heparin-binding characteristic of endostatin heparin-Sepharose CL-6B are selected at first. The final optimized condition was: after loading of the sample, the column was washed with buffer of 0.5M Tris-HCI,0.1MNaCl ,pH 7.4 to baseline and then elute with buffer of 0.5M Tris-HCI,0.3MNaCl pH 7.4 to baseline to get rid of contamination . Then buffer of 0.5M Tris-HCI,0.6MNaCl pH 7.4 is used to elute target protein-endostatin. Finally strongly combined contaminations to column are removed with buffer of 0.5M Tris-HCI,1MNaCl pH 7.4.After the first purification step ,the purity is great than 90% with only a little contamination of high molecular weight. The target peak after heparin-binding column is concentrate to about 25mg/ml with tangential flow ultra-filtration system with 10kDa NMWL membrane . Condensate is applied to Sephacryl S-200 to get final product, in which the buffer is 0.2M PB, pH7.0.