人Cu,Zn-SOD基因在毕赤酵母中的表达及稳定性

目的构建人Cu,Zn-SOD基因的真核表达载体,转化毕赤酵母,并检测表达产物的稳定性。方法根据酵母密码子偏好性,合成人Cu,Zn-SOD基因,克隆至真核表达载体pPIC9k中,转化毕赤酵母GS115。甲醇进行诱导表达,SDS-PAGE和Western blot鉴定表达产物。优化表达条件,并检测粗酶液的稳定性。结果经SDS-PAGE和Western blot检测,表达的目的蛋白相对分子质量为18000左右;最佳表达条件为pH6.0,甲醇浓度1.5%,持续培养96h;目的蛋白占分泌蛋白总量的27%,最高表达量为91mg/L;最佳条件下培养液上清的酶活性最高达334U/ml,粗酶对氯仿和乙醇稳定,对H2O2不稳定,对温度有一定的耐受性,pH在6和8时较稳定。结论在毕赤酵母GS115中成功表达了具备酶活性的人Cu,Zn-SOD基因。

Expression of Human Cu, Zn-SOD Gene in Pichia pastoris and Stability of Expressed Product

Objective To construct a eukaryotic expression vector for human Cu,Zn-SOD gene and express in Pichia pastoris.Methods Digest recombinant plasmid pU57-SOD with SnaB Ⅰ and Not Ⅰ and insert the obtained SOD gene into expression vector pPIC9k.Transform the constructed recombinant plasmid pPIC9k/SOD to Pichia pastoris GS115 for expression under induction of methanol.Identify the expressed product by SDS-PAGE and Western blot.Optimize the condition for expression and determine the expressed product for protein content by Lowry method and for activity by modified pyrogallol method.Test the stability of crude SOD to chemical reagent,temperature and pH value.Results The expressed product,with a relative molecular mass of about 18 000,contained 27% of total somatic protein.The maximum expression level of target protein reached 91 mg/L.The optimal pH value,methanol concentration and time for expression were 6.0,1.5% and 96 h respectively.Under the optimal condition,the enzyme activity of expressed protein was 334 U/ml.The crude SOD was stable to chloroform and ethanol,but unstable to hydrogen peroxide.However,it showed a certain resistance to temperature and was stable at pH 6 and 8.Conclusion The human Cu,Zn-SOD with enzyme activity was successfully expressed in Pichia pastoris GS115.