中华绒螯蟹重组金属硫蛋白的原核表达与分离纯化  

Expression and Purification of Recombinant Metallothionein of Eriocheir sinensis in E. coli

作　　者：曹晓敏[1] 李冰[1] 陈祯[1] 涂洪斌[1] 付欣[1] 

机构地区：[1]广州医学院实验医学中心,广州510182

出　　处：《生物医学工程学杂志》2007年第2期409-412,419,共5页Journal of Biomedical Engineering

基　　金：广州市科技局重点项目资助(2003Z2-E0193)

摘　　要：金属硫蛋白(MT)由于其独特的结构和性质,而成为潜在的医用蛋白资源。本研究将重组中华绒螯蟹金属硫蛋白基因cDNA克隆入原核表达载体pET-GST,转化大肠杆菌BL21。重组蛋白以可溶和包涵体两种形式表达。根据金属硫蛋白的特性,优化了表达条件。Western blot证实了表达产物的正确性,用锌柱亲合层析得到高纯度的重组金属硫蛋白。为以后金属硫蛋白的生物学功能研究奠定了良好的基础。Metallothioneins（MT） are potential candidates for medicine development and application. For the purpose of expressing recombinant MT in E. coli, a crab MT cDNA cloned into pGEM-T was subcloned into pETGST and then transformed into Escherichia Coli BL21. The fusion protein was proved to be expressed in both soluble and insoluble form by SDS-PAGE and western blot. Since metallothionein chelate metal ions,which may effects the physiological process of E. coli, caused the production of recombinant protein was lower than expected. Optimization of the ions content in the culture medium improved expression. The protein was purified by Zn^2＋ affinity chromatography, and rinsed off with high imidazole （1.5M） which was the result of MT chelating instead of His-tag. This fusion protein laid a foundation of further study on the structural and functional biology of metallothionein.

关 键 词：金属硫蛋白 原核表达 重组融合蛋白 亲合层析 

分 类 号：Q78[生物学—分子生物学]