High Density Culture of P. pastoris GS115(pPIC9K-EDN)Habouring Endostatin Gene and Purification and Bioactivity Analysis of Product
WU Jiang xue, FU Wu zhao, ZHANG Tian yuan, LUO Jin xian\+*, WU Qun yue (Key Laboratory of Gene Engineering of Ministry of Education, Department of Biochemistry, Zhongshan University, Guangzhou 510275, China)
The high cell density of P. pastoris GS115(pPIC9K EDN ) strain harboring human endostatin (EDN) was cultured in a 30 liter fermentor and the effect of media, pH and induction time on cell growth and gene expression were determined. By fed batch fermentation and methanol induction for 48 hours,the biomass A 600 of GS115(pPIC9K EDN ) reached 250, the secreted EDN was 150 mg/L. The fermentation product was purified to 97% by Streamline SP and Sepharose SP F F cation exchanger column and Sepharose\|Heparin Hi Trap affinity chromatography. The total recovery was 60%. Western blot analysis demonstrated that the purified product reacted with anti mEDN antibody. The purified product specifically inhibited the proliferation of human umbilical vein endothelial cells(ECV304) by 68.9% at 2 ��g/ml. The purified endostatin also suppressed in vivo growth of B16 murine melanoma by 94 2% at a dose of 10 mg/(kg��d).
.High Density Culture of P. pastoris GS115(pPIC9K-EDN)Habouring Endostatin Gene and Purification and Bioactivity Analysis of Product [J] Chinese Journal of Biochemistry and Molecular Biol, 2004,V20(02 ): 224-224