NDRG2蛋白的毕赤酵母可溶表达与纯化 |
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引用本文: | 张文红,刘新平,王琰,韩月恒,药立波. NDRG2蛋白的毕赤酵母可溶表达与纯化[J]. 医学争鸣, 2003, 24(12): 1061-1065 |
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作者姓名: | 张文红 刘新平 王琰 韩月恒 药立波 |
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作者单位: | 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 |
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基金项目: | 国家自然科学基金资助项目 (30 0 70 773) |
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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G418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G418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。
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关 键 词: | NDRG2蛋白 毕赤酵母 基因表达 亲合纯化 |
文章编号: | 1000-2790(2003)12-1061-05 |
修稿时间: | 2002-08-13 |
Soluble expression of NDRG2 protein in pichia yeast and purification of the expression product |
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Abstract: | 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. |
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Keywords: | NDRG2 protein pichia gene expression affinity purification |
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