Inducible expression of human angiostatin by AOXI promoter in P. pastoris using high-density cell culture.
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).
Zhang
Ai-Lian
AL
Institute of Tropical Bioscience and Biotechnology, Chinese Academy of Tropical Agricultural Sciences, Haikou, Hainan 571101, China. zhangailian6@yahoo.com.cn
Zhang
Tian-Yuan
TY
Luo
Jin-Xian
JX
Fu
Ce-Yi
CY
Qu
Zhi
Z
Yi
Guo-Hui
GH
Su
Dong-Xiao
DX
Tu
Fa-Zhi
FZ
Pan
Ying-Wen
YW
D000429:Alcohol Oxidoreductases / Q000235:genetics*; D020533:Angiogenesis Inhibitors / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology; D043168:Angiostatins / Q000096:biosynthesis* / Q000235:genetics / Q000494:pharmacology; D000818:Animals; D019149:Bioreactors; D015153:Blotting, Western; D018827:Carcinoma, Lewis Lung / Q000188:drug therapy / Q000473:pathology; D002452:Cell Count; D048708:Cell Growth Processes / Q000187:drug effects; D002642:Chick Embryo; D049033:Chorioallantoic Membrane / Q000098:blood supply; D005285:Fermentation; D006801:Humans; D008297:Male; D008546:Melanoma, Experimental / Q000188:drug therapy / Q000473:pathology; D051379:Mice; D008810:Mice, Inbred C57BL; D018919:Neovascularization, Physiologic / Q000187:drug effects; D010843:Pichia / Q000201:enzymology / Q000235:genetics* / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000494:pharmacology
D020533:Angiogenesis Inhibitors; D011994:Recombinant Proteins; D043168:Angiostatins; D000429:Alcohol Oxidoreductases