Inducible expression of calreticulin-N58 in Pichia pastoris by high density cell culture.
Mol Biol Rep
; 38(8): 5003-8, 2011 Nov.
Article
en En
| MEDLINE
| ID: mdl-21181274
ABSTRACT
Calreticulin-N58 (CRT-N58), an active fragment of calreticulin with anti-angiogenesis activity, was expressed in P. pastoris by high density cell culture. Calreticulin-N58 DNA was synthesized by PCR and cloned to plasmid pPIC9 K resulting in the plasmid pPIC9 K-crt-N58 which was then transformed into P. pastoris GS115. The fermentation was carried out in a 50 l bioreactor with 20 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 was grown to A(600) = 135, methanol-PTM4 trace salts was added to induce the expression of calreticulin-N58. During the fermentation, dissolved oxygen level was maintained at 20-30%, pH was controlled at 5 by adding 7 M NH(4)OH. After 52 h of induction, the yield of secreted calreticulin-N58 was 70 mg/l and biomass growth was 293 as measured by absorption of 600 nm. The secreted calreticulin-N58 was purified to a purity of 100% by the use of SP-Sepharose FF ion-exchange chromatography (Pharmacia Biotech. NJ, USA) and desalted with ultrafiltration device (Millipore, Bedford, MA, USA). The recombinant calreticulin-N58 induced endothelial cell apoptosis and inhibited the angiogenesis on the CAM.
Texto completo:
1
Colección:
01-internacional
Banco de datos:
MEDLINE
Asunto principal:
Fragmentos de Péptidos
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Pichia
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Expresión Génica
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Técnicas de Cultivo de Célula
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Calreticulina
Límite:
Animals
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Humans
Idioma:
En
Revista:
Mol Biol Rep
Año:
2011
Tipo del documento:
Article
País de afiliación:
China