昆明鼠肠激酶轻链的原核表达、复性与纯化  

Expression,Refolding and Purification of the Light Chain of Enterokinase from Kunming Mice in Escherichia coli

作　　者：陈展歌[1] 陈锡贤 杨梦琳[1] 蔡洪敏[1] 李黄金[1] 

机构地区：[1]广东药学院生命科学与生物制药学院,广东广州510006

出　　处：《生物技术》2014年第1期48-51,共4页Biotechnology

基　　金：国家自然科学基金项目("基于EGFR"二聚体界面"靶向策略的单克隆抗体与多肽疫苗研究";编号:81273423)资助

摘　　要：目的:构建昆明小鼠十二指肠肠激酶轻链(mEKL)大肠杆菌高效表达系统,并建立复性与纯化方法。方法:RT-PCR法扩增mEKL全长cDNA,以融合表达载体pET32a克隆于大肠杆菌BL21(DE3)和Origami(DE3),采用阴离子交换层析纯化复性表达产物,经牛肠激酶消化回收mEKL。结果:扩增得到723bp的mEKL全长cDNA,经序列分析发现,昆明鼠来源的mEKLcDNA序列的Ser131密码子中存在1个同义点突变。mEKL在2种宿主菌中的表达产物均为包涵体蛋白,经稀释复性、阴离子交换层析纯化和肠激酶切割可得高纯度mEKL。结论:成功构建昆明鼠源mEKL高效表达工程菌,并建立相应的纯化方法。Objective：To construct recombinant Escherichia bacteria over - expressing the light chain of entcrokinase from Kunming mice （ mEKL ） and develop a purification procedure of the expression product. Method ： The full - length cDNA of mEKL was amplified by RT - PCR, and cloned into E. coli BL21 （DE3） and Origami （DE3） in pET32a for fusion -expressing with thioredoxin（Trx）under the control of promoter T7. The expression product was purified by anion exchange chromatography （AEC）after refolding, from which mEKL was recov- ered by enterokinase digestion. Result： A 723 bp cDNA coding mEKL was from Kunming mice＇ s duodenum,in which a synonymous point mutation was found in the codon of Ser^131. The fusion gene of Trx - mEKL was over - expressed as inclusion proteins in both host cells. The refolded fusion protein was purified by AEC to an over 80% purity,from which mEKL was recovered by enterokinase digestion and can be further purified by Ni - chelating affinity chromatography. Conclusion：An over - expression system for the recombinant mEKL was established,Which laid a good foundation for the over- production of enterokinase.

关 键 词：鼠肠激酶 表达 复性 纯化 

分 类 号：Q786[生物学—分子生物学]