人源EC-SOD在毕赤酵母中的构建与表达

Construction of extracellular superoxide dismutase and expression in Pichia pastoris

利用基因工程技术构建表达载体并电转化至毕赤酵母中,成功实现人源EC-SOD在毕赤酵母X33中的表达.重组蛋白经盐酸羟胺法测得25 mL摇瓶发酵液上清酶活为508 U·mg^(-1),5 L发酵液上清酶活为909U·mg^(-1).发酵液上清用硫酸铵沉降后,使用DEAE弱阴离子交换树脂初步分离纯化出较纯的EC-SOD,测得比活为1 700 U·mg^(-1),并进行氯化硝基四氮唑蓝(NBT)活性定性染色.结果表明,毕赤酵母成功胞外表达具有一定活性的SOD蛋白.

A highly expression strain was constructed by genetic engineering technology.Furthermore,EC-SOD derived from Pichia showed a high activity.EC-SOD was exported into the 25 mL culture medium with an antioxidative activity of 508 U·mg-1,5 L culture medium with an antioxidativeactivity of 909 U·mg-1.The supernatant was concentrated and purified by DEAE rsin.And the purified EC-SOD show the antioxidative activity of 1 700 U·mg-1.Finally the recombined protein show the activity of enzyme has been identified through NBT analysis.