Abstract
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).
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Supported by the Natural Science Foundation of China (30760061).
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Zhang, AL., Zhang, TY., Luo, JX. et al. Inducible expression of human angiostatin by AOXI promoter in P. pastoris using high-density cell culture. Mol Biol Rep 36, 2265–2270 (2009). https://doi.org/10.1007/s11033-008-9443-9
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DOI: https://doi.org/10.1007/s11033-008-9443-9