大肠杆菌表达的Taq DNA聚合酶的纯化.pdfVIP

HEREDITAS (Beijing) 2012 3 , 34(3): 371―378 ISSN 0253-9772 技术与方法 DOI: 10.3724/SP.J.1005.2012.00371 大肠杆菌表达的Taq DNA 聚合酶的纯化 1 2 1 1 1 1 1 刘天磊 , 薛守斌 , 王芳 , 朱琳颖 , 梁微微 , 曲圣轩 , 蔡文博 1. , 212013; 2. , 730000 Taq DNA 聚合酶是分子生物学研究中最常用的热稳定DNA 聚合酶之一, 与其他热稳定DNA 聚合酶具有 相似的特征, 其纯化策略不但有潜在的应用前景, 也对同类聚合酶的分离具有指导意义。已报道的适宜大量制 备Taq酶的方案所需成本较高, 而文章介绍了一种利用国产阳离子交换树脂廉价制备Taq酶的方案。在本方案中, 采用热变性、(NH )SO 沉淀与 724 离子交换层析分离大肠杆菌表达的Taq酶, 约 18 g Na型树脂干粉一次可回收 4 4 比活约 8 131.98 U/mg 、总酶活 2.2×105 U 、近 27.07 mg Taq 酶。纯化的产率可达 48.92%, 纯化倍数约 59.35 。 所制酶SDS电泳只检测到 94 kDa单一蛋白条带, 未检测到DNA核酸酶污染, 与商品酶的PCR扩增能力无 区别。此纯化方法成本低, 适合实验室一般性的制备和生产应用。 Taq DNA 聚合酶; 724 树脂; 阳离子交换层析 Purification of Taq DNA polymerase expressed in Escherichia coli 1 2 1 1 1 LIU Tian-Lei , XUE Shou-Bin , WANG Fang , ZHU Lin-Ying , LIANG Wei-Wei , QU 1 1 Sheng-Xuan , CAI Wen-Bo 1. School of Food and Bioengineering, Jiangsu University , Zhenjiang 212013, China; 2. The Second Clinical Medical College, Lanzhou University , Lanzhou 730000, China Abstract: Taq DNA polymerase is one of the most commonly thermostable DNA polymerases in molecular biological researches, which shares its basic characters with others of the family, thereby its purifying strategy could be used not only in itself production but also in the extraction of the others as a reference. At present, the protocols reported for large scale preparation of Taq DNA are high cost, so a cheaper method was described here. In this protoc