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Inducible expression of human angiostatin by AOXI promoter in P. pastoris using high-density cell culture
- Source :
- Molecular Biology Reports. 36:2265-2270
- Publication Year :
- 2009
- Publisher :
- Springer Science and Business Media LLC, 2009.
-
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).
- Subjects :
- Male
Angiogenesis
Blotting, Western
Melanoma, Experimental
Neovascularization, Physiologic
Angiogenesis Inhibitors
Cell Count
Cell Growth Processes
Chick Embryo
Biology
Chorioallantoic Membrane
Pichia
law.invention
Carcinoma, Lewis Lung
Mice
chemistry.chemical_compound
Bioreactors
law
Genetics
Bioreactor
Animals
Humans
Promoter Regions, Genetic
Angiostatins
Molecular Biology
Growth medium
Angiostatin
General Medicine
Molecular biology
Recombinant Proteins
Alcohol oxidase
Mice, Inbred C57BL
Alcohol Oxidoreductases
Biochemistry
chemistry
Cell culture
Fermentation
Recombinant DNA
Subjects
Details
- ISSN :
- 15734978 and 03014851
- Volume :
- 36
- Database :
- OpenAIRE
- Journal :
- Molecular Biology Reports
- Accession number :
- edsair.doi.dedup.....ef510305eb4be267b92d22181fcc8e24
- Full Text :
- https://doi.org/10.1007/s11033-008-9443-9