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

Molecular Biology Reports
Ai-Lian ZhangYing-Wen Pan

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 degrees C. The cells were first grown in glycerol-PTM4 trace salts for 24 h. When the cell density reached A(600) = 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 NH(4)OH and the biomass was maintained at about A(600) = 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).

References

Jan 21, 2000·FEMS Microbiology Reviews·J L Cereghino, J M Cregg
Dec 19, 2003·Biochemical and Biophysical Research Communications·Guo-An HeRui-Fang Li
Jun 11, 2005·Protein Expression and Purification·Jean-Marc DelroisseMicheline Vandenbol
Sep 22, 2006·Journal of Industrial Microbiology & Biotechnology·A L ZhangH L Li

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