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.
出处
《中国生物工程杂志》
CAS
CSCD
北大核心
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
关键词
分泌表达
蛋白纯化
质谱鉴定
活性检测
Secreted expression Protein purification Mass spectrum identification Analysis of enzymatic activity
作者简介
通讯作者，电子信箱：liujy@mail．tsinghua．edu．cn
