Constitutive expression of human angiostatin in Pichia pastoris by high-density cell culture.
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 NH4OH. 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.
Zhang
A L
AL
Institute of Tropical Bioscience and Biotechnology/National Key Biotechnology Laboratory for Tropical Crops, CATAS, Haikou Hainan, 571101, China. zhangailian6@yahoo.com.cn
Zhang
T Y
TY
Luo
J X
JX
Chen
S C
SC
Guan
W J
WJ
Fu
C Y
CY
Peng
S Q
SQ
Li
H L
HL
D020533:Angiogenesis Inhibitors / Q000096:biosynthesis* / Q000235:genetics / Q000494:pharmacology / Q000627:therapeutic use; D043168:Angiostatins / Q000096:biosynthesis* / Q000235:genetics / Q000494:pharmacology / Q000627:therapeutic use; D000818:Animals; D019149:Bioreactors; D001709:Biotechnology / Q000379:methods; D045744:Cell Line, Tumor; D002642:Chick Embryo; D003470:Culture Media; D015966:Gene Expression Regulation, Fungal; D005818:Genetic Engineering / Q000379:methods; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008297:Male; D008546:Melanoma, Experimental / Q000188:drug therapy; D051379:Mice; D008810:Mice, Inbred C57BL; D009172:Mycology / Q000379:methods; D010843:Pichia / Q000235:genetics / Q000254:growth & development* / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000096:biosynthesis; D016896:Treatment Outcome
D020533:Angiogenesis Inhibitors; D003470:Culture Media; D011994:Recombinant Proteins; D043168:Angiostatins; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases