Impact of temperature reduction and expression of yeast pyruvate carboxylase on hGM-CSF-producing CHO cells
Impact of temperature reduction and expression of yeast pyruvate carboxylase on hGM-CSF-producing CHO cells
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DOI：
10.1016/j.jbiotec.2003.10.035
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发表时间：
2004-04-08
影响因子：
4.1
通讯作者：
Kratje, R
中科院分区：
文献类型：
--
作者：
Fogolín, MB;Wagner, R;Kratje, R
最近，我们证明了在BHK-21细胞的细胞质中表达的重组酵母丙酮酸羧化酶被证明部分地重建糖酵解和TCA之间的缺失环节，增加葡萄糖进入TCA的通量，并实现更高的重组促红细胞生成素的产量。在本研究中，使用产生重组人粒细胞巨噬细胞集落刺激因子的CHO细胞系来评价PYC 2表达和降低培养温度的影响。温度从37 ℃降低到33 ℃，表明CHO-K1-hGM-CSF细胞的生长速率降低，稳定期延长，细胞特异性rhGM-CSF产生速率增加2.1倍。与表达rhGM-CSF但不表达PYC 2的对照相比，表达PYC 2的细胞克隆显示出降低的细胞生长和较低的最大细胞浓度。然而，在表达PYC 2的细胞中仅产生65%的乳酸，并且产物产率比对照高200%。与先前报道的BHK细胞相比，CHO细胞获得的结果表明PYC 2表达主要减少乳酸盐形成并增加待生产的重组蛋白的产率。最后，研究了在两种温度条件下表达PYC 2的CHO-K1-hGM-CSF细胞的生长和生产率。在降低的温度条件下，平均细胞特异性rhGM-CSF产量增加了3.2倍。结果显示PYC 2的表达和降低的培养温度对CHO-K1-hGM-CSF细胞的细胞比生产力具有累加效应。(C)2004 Elsevier B. V.保留所有权利。
Recently, we demonstrated that a recombinant yeast pyruvate carboxylase expressed in the cytoplasm of BHK-21 cells was shown to partially reconstitute the missing link between glycolysis and TCA, increasing the flux of glucose into the TCA and achieving higher yields of recombinant erythropoietin. In the present study, a CHO cell line producing recombinant human granulocyte macrophage colony stimulating factor was used to evaluate the impact of PYC2 expression and reduced culture temperature. Temperature reduction from 37 to 33 degreesC revealed a reduced growth rate, a prolonged stationary phase and a 2.1-fold increase of the cell specific rhGM-CSF production rate for CHO-K1-hGM-CSF cells. The PYC2-expressing cell clones showed a decreased cell growth and a lower maximum cell concentration compared to the control expressing rhGM-CSF but no PYC2. However, only 65% lactate were produced in PYC2-expressing cells and the product yield was 200% higher compared to the control. The results obtained for CHO cells compared to BHK cells reported previously, indicated that the PYC2 expression dominantly reduced the lactate formation and increased the yield of the recombinant protein to be produced. Finally, the growth and productivity of PYC2-expressing CHO-K1-hGM-CSF cells under both temperature conditions were investigated. The average cell specific rhGM-CSF production increased by 3.2-fold under reduced temperature conditions. The results revealed that the expression of PYC2 and a reduced culture temperature have an additive effect on the cell specific productivity of CHO-K1-hGM-CSF cells. (C) 2004 Elsevier B.V. All rights reserved.