重组萝卜磷脂氢谷胱甘肽过氧化物酶在毕赤酵母优化表达初步纯化与鉴定  

Optimized Expression and Identification of the Radish Phospholipid Hydroperoxide Glutathione Peroxidase in Pichia pastoris

作　　者：李洋[1] 李晖[2] 祝建波[1] 刘进元[2] 

机构地区：[1]石河子大学农业生物技术重点实验室,石河子832000 [2]清华大学生命科学学院分子生物学实验室,北京100084

出　　处：《中国生物工程杂志》2010年第4期54-59,共6页China Biotechnology

基　　金：国家"863"计划(2007AA100604);国家自然科学基金(30170080;39770078);国家重点基础研究发展规划(2006CB101706)资助项目

摘　　要：将萝卜磷脂氢谷胱甘肽过氧化物酶(RsPHGPx)基因插入到分泌表达载体pPIC9K中,转化巴斯德毕赤酵母GS115细胞,筛选具有G418抗性的单拷贝转化子。经过优化表达条件,RsPHGPx在1%甲醇、pH6.0、28℃条件下诱导60h后得到最大表达量,产率约为102mg/L。通过硫酸铵分级沉淀、脱盐柱脱盐、凝胶过滤等纯化步骤,得到了90%以上纯度的RsPHGPx.活性分析显示纯化获得的RsPHGPx具有依赖于GSH的还原活性,比活性为4.2μmol/min.mg,为获得大量RsPHGPx而用于应用开发研究奠定了基础。The expression of RsPHGPx gene in Pichia pastoris was investigated.The RsPHGPx gene inserted into secretory vector pPIC9K was transformed into Pichia pastoris strain GS115.The singlecopy recombinant strains were screened by G418.In addition,the induction conditions were optimized to get the highest expression of the target protein （the optimum：1%methonal,pH6.0,28℃）.Finally,it was shown that the recombinant RsPHGPx could be secreted into the culture supernatant to a level of 102mg/L after 60 hours of induction.The fractional ammonium sulfate precipitation,desalination,and gel chromatography were used to purify protein and more than 90% purity of RsPHGPx was obtained.The bioactivity of RsPHGPx was high-dependent redox-active of GSH and reached its max secreted volume at 60h,the specific activity is 4.2μmol/min·mg.The research has laid the foundation for gaining and exploiting a large amount of RsPHGPx.

关 键 词：分泌表达 蛋白纯化 质谱鉴定 活性检测 

分 类 号：Q786[生物学—分子生物学]