| 参与DNA修复的PIF1解链酶的纯化和ATP酶活性分析 |
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| 引用本文： | 顾永清,王国卫. 参与DNA修复的PIF1解链酶的纯化和ATP酶活性分析[J]. 中国地方病学杂志, 2006, 28(1): 276-279. DOI: 10.3760/cma.j.issn.1000-4955.2009.03.011 |
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| 作者姓名： | 顾永清 王国卫 |
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| 作者单位： | 新疆石河子大学医学院生物化学教研室,832002;新疆石河子大学医学院化学化工学院实验中心,832002; |
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| 基金项目： | 在日外国人女子大学院留学生奖学金资助项目 |
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| 摘 要： | 目的 克隆并表达纯化人类PIF1蛋白,分析其生物化学活性.方法 从HeLa细胞的cDNA文库中克隆得到PIF1解链酶基因,插入融合有组氨酸的表达载体pET24b,构建pET24b-PIF1重组质粒,转化RosettaTM2(DE3)感受态细胞,十二烷基磺酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)观察PIF1蛋白表达;通过快速液相蛋白色谱纯化(FPLC)系统,采用亲和层析和凝胶过滤,纯化人类重组PIF1蛋白.薄层层析(TLC)法检测纯化PIF1蛋白的ATP酶活性.结果 克隆了人类PIF1基因,PIF1蛋白在大肠埃希菌(E.coli)中成功表达.结论 建立了PIF1蛋白纯化的方法,PIF1蛋白具有依赖镁离子和DNA的ATP酶活性.
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| 关 键 词： | DNA螺旋酶类 基因表达 腺苷三磷酸酶类 |
Purification and ATPase activity analysis of human PIF1 helicase participating in DNA repair |
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| GU Yong-qing,WANG Guo-wei. Purification and ATPase activity analysis of human PIF1 helicase participating in DNA repair[J]. Chinese Jouranl of Endemiology, 2006, 28(1): 276-279. DOI: 10.3760/cma.j.issn.1000-4955.2009.03.011 |
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| Authors: | GU Yong-qing WANG Guo-wei |
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| Abstract: | Objective To clone, express and purify human PIF1 protein and analyze its ATPase activity. Methods The PIF1 cDNA was amplified by PCR from HeLa cell cDNA library and inserted to pET24b with histidine tag at its terminus to form pET24b-PIF1 plasmid. The recombinant pET24b-PIF1 plasmid was transformed to RosettaTM 2 (DE3) and the expression of PIF1 protein was monitored by SDS-PAGE analysis. By using fast protein liquid chromatograph (FPLC) system, the PIF1 protein was purified by affinity chromatograph and gel filtration. The ATPase activity of PIF1 was checked by thin layer chromatograph(TLC). Results The PIF1 protein was successfully cloned and expressed in E.coli. Conclusions The purification procedure of PIF1 protein was established using FPLC. The overexpressed and the purified PIF1 helicase has DNA and Mg2+ dependent ATPase activity. |
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| Keywords: | DNA helicasesGene expressionAdenosine triphosphatases |
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