Constitutive expression of human angiostatin in Pichia pastoris by high-density cell culture
文献类型： 外文期刊
作者： Zhang, A. L. 1 ; Zhang, T. Y. 2 ; Luo, J. X. 2 ; Chen, S. C. 3 ; Guan, W. J.; Fu, C. Y.; Peng, S. Q.; Li, H. L.;
作者机构： 1.CATAS, Inst Trop Biosci & Biotechnol, Natl Key Biotechnol Lab Trop Crops, Haikou Hainan 571101, Peoples R China
2.CATAS, Inst Trop Biosci & Biotechnol, Natl Key Biotechnol Lab Trop Crops, Haikou Hainan 571101, Peoples R China; Sun Yat Sen Univ, Key Lab Gene Engn, Minist Educ, Guangzhou 510275, Guangdong, Peoples R China; Sun Yat Sen Univ, Dept Biochem, Guangzhou 510275, Guangdong, Peoples R China; Hainan Prov Inst Drug Control, Haikou Hainan 570216, Peoples R China
3.CATAS, Inst Trop Biosci & Biotechnol, Natl Key Biotechnol Lab Trop Crops, Haikou Hainan 571101, Peoples R China; Sun Yat Sen Univ, Key Lab Gene Engn, Minist Educ, Guangzhou 510275, Guangdong, Peopl
期刊名称：JOURNAL OF INDUSTRIAL MICROBIOLOGY & BIOTECHNOLOGY （ 影响因子：3.346； 五年影响因子：3.426 ）
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收录情况： SCI
摘要： A high-density cell culture method to produce human angiostatin has been successfully established by constitutive expression of the protein in Pichia pastoris. The fermentation was carried out in a 20 l bioreactor with a 10 l working volume, using a high-density cell culture method by continuously feeding with 50% glycerol-0.8% PTM4 to the growing culture for 60 h at 30 degrees C. Dissolved oxygen level was maintained at 25-30% and pH was controlled at 5 by the addition of 7 M NH(4)OH. Angiostatin was constitutively expressed during the fermentation by linking its expression to the P. pastoris constitutive GAP promoter (pGAP). But after 36 h of fermentation, the peak biomass growth was 305 as measured by absorption of 600 nm, while the peak angiostatin expression was 176 mg/l. Similar to the product expressed from inducible system [24], angiostatin produced from constitutive system also inhibited the angiogenesis on the CAM and suppressed the growth of B16 melanoma in C57BL/6J mouse. The above results suggest that GAP promoter is more efficient than AOX1 promoter for the expression of angiostatin in P. pastoris by shake flask culture or high-density cell fermentation and is likely to be an alternative to AOX1 promoter in large-scale expression of angiostatin and other heterologous proteins.
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