NDRG2蛋白的毕赤酵母可溶表达与纯化  

Soluble expression of NDRG2 protein in pichia yeast and purification of the expression product

作　　者：张文红[1] 刘新平[1] 王琰[2] 韩月恒[1] 药立波[1] 

机构地区：[1]第四军医大学基础部生物化学与分子生物学教研室,陕西西安710033 [2]第四军医大学科研部基因诊断技术应用研究所,陕西西安710033

出　　处：《第四军医大学学报》2003年第12期1061-1065,共5页Journal of the Fourth Military Medical University

基　　金：国家自然科学基金资助项目 (30 0 70 773)

摘　　要：目的 :酵母表达并纯化可溶性NDRG2蛋白 .方法 :从 pRSET A ndrg2重组质粒中扩增出 6his ndrg2融合基因 ,克隆入酵母表达载体 pPIC3.5K ;酵母重组表达载体pPIC3.5K 6his ndrg2电转化入毕赤酵母GS115 ,并进行营养缺陷筛选和G4 18抗性筛选 ;对高拷贝整合的重组酵母表达菌株诱导表达 ,可溶性表达产物经Ni NTA亲合层析纯化 .结果 :构建得到酵母融合重组表达载体 pPIC3.5K 6his ndrg2 ;经G4 18浓度梯度筛选得到串联整合 16个重组表达载体以上的重组酵母表达菌株 ;诱导后表达得到 6his NDRG2融合蛋白 ,并成功纯化得到可溶性表达产物 .结论 :表达并纯化得到可溶性NDRG2蛋白 ,为进一步研究NDRG2的结构与功能打下了必要的基础 .AIM: To express the NDRG2 protein in soluble form in yeast. METHODS: 6his ndrg 2 fusion gene was amplified from pRSET A ndrg 2 recombinant plasmid, and cloned into pPIC3.5K yeast expression vector. pPIC3.5K 6his ndrg 2 recombiant vector was transformed into pichia GS115 by electroporation. Positive recombinant yeast strains were screened through nutrition defect and G418 resistance. Expression of the integrated multicopy recombinant yeast strains were induced. The soluble expression products were purified by Ni NTA affinity chromatography. RESULTS: The yeast recombinant expression vector pPIC3.5K 6his ndrg 2 was constructed. Recombiant yeast strains which integrated over 16 recombinant vectors were obtained. The NDRG2 protein was expressed in soluble form in pichia . After expression and purification by metal chelate affinity chromatography, 6his NDRG2 fusion protein were obtained. CONCLUSION: The soluble NDRG2 protein will be useful for further research on its functions.

关 键 词：NDRG2蛋白 毕赤酵母 基因表达 亲合纯化 

分 类 号：R349.83[医药卫生—基础医学]