提高重组表达体系相对活性促进外源蛋白表达:人干扰素毕赤酵母表达体系研究  

Promote the Expression of Foreign Protein by Improving Relative Cell Viability:Expression of the Fusion Human Interferon in Pichia pastoris

作　　者：窦文芳[1] 张旦旦[1] 许泓瑜[1] 金坚[1,2] 许正宏[1,2] 史劲松[1] 陆震鸣[1] 

机构地区：[1]江南大学医药学院生物制药系,江苏无锡214122 [2]江南大学教育部工业生物技术重点实验室,江苏无锡214122

出　　处：《食品与发酵工业》2011年第11期11-15,共5页Food and Fermentation Industries

基　　金：长江学者和创新团队发展计划(No IRT0532)资助;国家重大新药创制专项(N02009ZX09102-221);国家自然基金(31000016);江苏省自然科学基金(企业博士创新项目BK2009518);江苏省科技支撑计划(BE2009629)

摘　　要：以毕赤酵母KM71/IH的hIFNβ-HAS表达体系为研究对象,较为系统地研究了不同因素引起的细胞相对活性变化与毕赤酵母外源蛋白表达的相关性。结果表明:(1)细胞相对活性变化与外源蛋白的表达成正相关;(2)处在对数生长中后期的细胞相对活性最高,更适合用于外源蛋白的表达;(3)诱导阶段的环境因素直接影响诱导期细胞相对活性,合适的甲醇浓度、诱导pH及温度是维持细胞高活性,促进外源蛋白高表达的关键。对于hIFNβ-HAS表达体系最佳诱导条件为:以培养21～24h的细胞为诱导种子,初始甲醇浓度为2%(V/V),pH6.0,25℃,细胞活性维持在98%以上,hIFNβ-HSA表达量高达45.6mg/L,较优化前提高了62.3%。To systematically study the relationship between foreign protein expression and relative cell viability caused by different modules,we investigated the recombinant human hIFNβ-HSA fusion protein in Pichia pastoris KM71/IH.The results showed that;（1） There were positive correlations between the expression of foreign protein and relative cell viability;（2） Cells on midanaphase of logarithmic growth were much more suitable for foreign protein expression,because of high cell viability;（3 ） Environmental factors of induction period directly influenced the relative cell viability.The methanol concentration,induction pH and temperature were optimized to promote expression of proteins.The optimal induction conditions to express hIFNβ-HAS is;21～24 h yeast cells,2%（V/V） methanol,pH 6.0,25℃.The relative cell viability could remain more than 98%under those conditions,and the yield of hIFNβ-HSA reached 45.6 mg/L,which was 62.3%higher than that of the control.

关 键 词：毕赤酵母 细胞相对活性 外源蛋白表达 人β干扰素 

分 类 号：Q78[生物学—分子生物学]