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

摘要

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.%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%。