Inducible expression of human angiostatin by AOXI promoter in P. pastoris using high-density cell culture

• Published in: Molecular biology reports 2009-11, Vol.36 (8), p.2265-2270
• Main Authors: Zhang, Ai-Lian, Zhang, Tian-Yuan, Luo, Jin-Xian, Fu, Ce-Yi, Qu, Zhi, Yi, Guo-Hui, Su, Dong-Xiao, Tu, Fa-Zhi, Pan, Ying-Wen
• Format: Article
• Language: English
• Subjects: Alcohol Oxidoreductases - genetics; Angiogenesis Inhibitors - genetics; Angiogenesis Inhibitors - metabolism; Angiogenesis Inhibitors - pharmacology; Angiostatins - biosynthesis; Angiostatins - genetics; Angiostatins - pharmacology; Animal Anatomy; Animal Biochemistry; Animals; Biological products; Biomedical and Life Sciences; Bioreactors; Blotting, Western; Carcinoma, Lewis Lung - drug therapy; Carcinoma, Lewis Lung - pathology; Cell Count; Cell culture; Cell Growth Processes - drug effects; Chick Embryo; Chorioallantoic Membrane - blood supply; Fermentation; Genetic recombination; Histology; Humans; Life Sciences; Male; Melanoma, Experimental - drug therapy; Melanoma, Experimental - pathology; Mice; Mice, Inbred C57BL; Molecular biology; Morphology; Neovascularization, Physiologic - drug effects; Pichia - enzymology; Pichia - genetics; Pichia - metabolism; Promoter Regions, Genetic; Recombinant Proteins - biosynthesis; Recombinant Proteins - genetics; Recombinant Proteins - pharmacology; Rodents
• ISSN: 0301-4851; 1573-4978
• DOI: 10.1007/s11033-008-9443-9

A high-density cell culture method was successfully established in P. pastoris with the alcohol oxidase I (AOXI) promoter in order to produce large quantities of recombinant human angiostatin (AS) which has been reported to have antiangiogenic activity. A preliminary study on fermentation conditions in shaking flasks indicated that adequacy of biomass is beneficial to obtain more products. The fermentation was carried out in a 10 l bioreactor with 5 l modified growth medium recommended by Invitrogen at 30°C. The cells were first grown in glycerol-PTM4 trace salts for 24 h. When the cell density reached A₆₀₀ = 125, methanol-PTM4 trace salts was added to induce the expression of AS. During the fermentation, dissolved oxygen level was maintained at 20-30%, pH was controlled at 5 by the addition of 7 M NH₄OH and the biomass was maintained at about A₆₀₀ = 200. After 60 h of induction, the secreted AS was 153 mg/l. The recombinant AS inhibited the angiogenesis on CAM and suppressed the growth of B16 melanoma in C57BL/6J mice (P < 0.01).