Inducible expression of calreticulin-N58 in Pichia pastoris by high density cell culture

文献类型： 外文期刊

第一作者： Su, D. X.

作者： Su, D. X.;Zhang, A. L.;Liu, Z. W.;Luo, J. X.;Zhang, T. Y.;Luo, J. X.;Zhang, T. Y.;Yi, G. H.;Rao, L. Y.;Zhou, Z. J.

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关键词： DNA;plasmid;calreticulin;ammonium hydroxide: 1336-21-6;calreticulin-N58: CRT-N58;expression;purification;endothelial cell apoptosis inducer;angiogenesi;modified growth medium;glycerol-PTM4 trace salt;methanol-PTM4 trace sal;biomass growth;dissolved oxygen level;endothelial cell apoptosis;controlled pH

期刊名称：MOLECULAR BIOLOGY REPORTS （ 影响因子：2.316； 五年影响因子：2.357 ）

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收录情况： SCI

摘要： 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 30A degrees 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 NH4OH. 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.

分类号： Q7

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