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  

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

作　　者：王舒[1] 郑志永[1] 詹晓北[1] 史仲平[1] 高敏杰[1] 刘立明[1] 金坚[1] 李华钟[1] 

机构地区：[1]江南大学工业生物技术教育部重点实验室

出　　处：《食品与发酵工业》2008年第8期11-17,共7页Food and Fermentation Industries

基　　金：国家自然科学基金(60604028);国家重点基础研究发展规划项目(973)(2007CB714304);江苏省自然科学基金(BK2006532)

摘　　要：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%。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.

关 键 词：重组甲醇营养型毕赤酵母 在线测量 酶活分析 相关性分析 

分 类 号：Q78[生物学—分子生物学]