重组毕赤酵母表达人血清白蛋白-人白介素2融合蛋白过程的代谢特性

分析了重组甲醇营养型毕赤酵母在5L多参数自控发酵罐上生产人血清白蛋白-人白介素2融合蛋白(HSA-IL-2)的发酵过程。在使用多种在线传感器和离线检测参数的基础上,应用相关性分析的方法将细胞生长的关键酶(乙醇氧化酶、葡萄糖-6-磷酸脱氢酶和异柠檬酸脱氢酶)酶活和代谢特性相关联。实验结果显示,甲醇诱导后葡萄糖-6-磷酸脱氢酶和异柠檬酸脱氢酶的酶活分别下降了46.1%和66.8%,说明胞内代谢流发生了迁移:三羧酸循环(TCA)和磷酸戊糖途径(HMP)中代谢流通量下降,甲醇完全氧化代谢成为主要代谢流。此外,在不同控制策略下,乙醇氧化酶的酶活和重组蛋白HSA-IL-2表达量也呈现出一定的差异。高溶氧低甲醇控制方式下HSA-IL2的表达量达到27mg/L,比低溶氧高甲醇条件下的表达量高33.7%。

Metabolic Characteristics of Recombinant Pichia pastoris Producing a Fusion Protein-HSA-IL2

The characteristics of recombinant Pichia pastoris fermentation process that produces HSA-IL2 in a 5 L fermentor were explored.Based on the on-line and off-line measurements,as well as the correlation analysis,the activities of three key enzymes(i.e.alcohol oxidase,glucose-6-phosphate dehydrogenase and isocitrate dehydrogenase)were characterized with oxygen uptake rate and carbon dioxide evolution rate.The results showed that the activities of glucose-6-phosphate dehydrogenase and isocitrate dehydrogenase sharply decreased in the methanol induction phase,indicating that metabolic flux shift occurred during fermentation process.Furthermore,the changes of alcohol oxidase activity and HSA-IL2 expression exhibited different patterns with different control strategies.With the control strategy of higher dissolved oxygen(DO)and lower methanol concentration,the final HSA-IL2 expression amount reached 27 mg/L,which was 33.7% higher than that achieved with the control strategy of lower DO and higher methanol concentration.