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›› 2007, Vol. 7 ›› Issue (6): 1181-1186.

• 生化工程专栏 • 上一篇    下一篇

提高重组毕赤酵母表达hIFNb-HSA的碳源控制策略

张宏斌,窦文芳,许泓瑜,金坚,李华钟,许正宏   

  1. 江南大学生物工程学院生物制药系
  • 出版日期:2007-12-20 发布日期:2007-12-20

A Carbon-controlling Strategy to Enhancement of the Expression for Human IFNb-HSA Fusion Protein with Pichia pastoris

ZHANG Hong-bin,DOU Wen-fang,XU Hong-yu,JIN Jian,LI Hua-zhong,XU Zheng-hong   

  1. Department of Biopharmaceutics, School of Biotechnology, Southern Yangtze University
  • Online:2007-12-20 Published:2007-12-20

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摘要： 研究了重组毕赤酵母KM71/IH菌体生长阶段饥饿培养对其细胞相对活性及人b干扰素-血清白蛋白融合蛋白(hIFNb-HSA)表达的影响. 结果表明，在以甘油为唯一碳源的细胞培养后期进行饥饿培养以耗尽培养基中的甘油会导致重组细胞相对活性的降低，进而减少hIFNb-HSA的表达量. 在此基础上，提出了菌体培养后期，即过渡阶段流加甘露醇碳源代替饥饿培养的策略，不仅可以消除培养液中甘油残留对甲醇诱导表达hIFNb-HSA的不利影响，而且能够保持重组细胞培养物较高的相对活性(>97%)，从而可以提高甲醇诱导表达hIFNb-HSA的水平，表达量可达37.3 mg/L，最大生产强度为0.78 mg/(L×h)，较对照分别提高了92.3%和44.4%.

关键词: 巴斯德毕赤酵母, 细胞相对活性, 非抑制表达碳源, 人b干扰素-血清白融合蛋白

Abstract: The relationship between the starvation culture and relative cell viability in the growth phase and their effects on the expression of recombinant human IFNb-HSA fusion protein in Pichia pastoris KM71/IH were observed in 5 L fermentor experiments. The results showed that the starvation culture in which glycerol as the solo carbon source was exhausted resulted in the decrease of relative cell viability and expression of recombinant hIFNb-HSA fusion protein. The strategy of mannitol addition in the transition stage not only eliminated the inhibition effect on methanol induction by residual glycerol, but also kept higher relative cell viability (>97%) and then improved the yield of human IFNb-HSA fusion protein. The maximum yield and maximum hIFNb-HSA productivity reached 37.3 mg/L and 0.78 mg/(L×h) respectively, which were 92.3% and 44.4% higher than those in the control.

Key words: Pichia pastoris, relative cell viability, non-repressing carbon source, human IFNb-HSA fusion protein