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�й����ﻯѧ���������ѧ�� 2004, Vol. 20 Issue (02 ):224-224     DOI:
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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)

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ժҪ �� 30��͹��о�����Ƥϸ�������� (EDN)��ĸ���̾�P .pastorisGS115 (pPIC9K EDN)�ĸ��ܶȷ��͹��� ,��������������� ,pH ,�յ�ʱ��ȶԾ�����ͻ������Ӱ�� .�����ȷ���������������з��� ,ͨ���Ϻͼ״��յ� 4 8h�� ,���̾�GS115 (pPIC9K EDN)��������A60 0 ֵ�ﵽ 2 5 0 ,������Ϊ 15 0mg L .����Һ��StreamlineSP ,SepharoseSPFF���ӽ������Sepharose HeparinHiTrap�׺��� ,���﴿�ȴ� 97%���� ,������Ϊ 6 0 % .Westernӡ�������� ,��ĸ���̾�GS115 (pPIC9K EDN)������������Ƥϸ�������ؾ�����ȻEDN������ԭ�� .MTT�����ְ�ʵ������ʾ :�����������������꾲����Ƥϸ�����ֳ����������ֲ��С��ĺ�ɫ�������� ,��ƽ���������� 94 2 % .
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Abstract�� 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).
Keywords�� human endostatin   Pichia pastoris   high density culture   purification   bioactivity      
Received 2003-05-29;
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�⽭ѩ.��Ƥϸ�������ؽ�ĸ���̾�ĸ��ܶȷ��ͼ����﴿���ͻ��Է��� [J]  �й����ﻯѧ���������ѧ��, 2004,V20(02 ): 224-224
.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
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http://cjbmb.bjmu.edu.cn/CN/     ��     http://cjbmb.bjmu.edu.cn/CN/Y2004/V20/I02 /224