Pfu DNA聚合酶在大肠杆菌Rosetta(DE3)中的表达及快速纯化  

The expression and fast purification of Pfu DNA polymerase in E.coli Rosetta( DE3) cells

作　　者：何华华 胡晓韵 王婷[1] 喻婵 HE Huahua;HU Xiaoyun;WANG Ting;YU Chan(Bio-Resource Green Transformation Collaborative Innovation Center,College of Life Sciences,Hubei University,Wuhan 430062,China)

机构地区：[1]湖北大学生命科学学院生物资源绿色转化协同创新中心,湖北武汉430062

出　　处：《湖北大学学报（自然科学版）》2019年第3期223-227,共5页Journal of Hubei University：Natural Science

基　　金：油料脂质化学与营养湖北省重点实验室开放基金资助

摘　　要：来源于Pyrococcus furiosus COM1菌的Pfu DNA聚合酶具有保真性高、延伸速度快、热稳定性强等特点,能够提高PCR反应过程中的扩增效率,减少错误碱基掺入率.以大肠杆菌Rosetta(DE3)为重组细胞,利用一种快速表达纯化方法,让Pfu DNA聚合酶的表达纯化过程变得更加简便,并维持其高活性.结果表明,Pfu DNA聚合酶在大肠杆菌Rosetta(DE3)中能够进行大量表达,同时60℃高温水浴能有效去除杂蛋白,简化纯化步骤及提高Pfu DNA聚合酶纯度.经过一系列纯化步骤处理后,Pfu DNA聚合酶仍然能够保持较好的酶活,且在100μL PCR反应体系中,加入4μL的1 mg/mL Pfu DNA聚合酶液效果最佳.The Pfu DNA polymerase derived from Pyrococcus furiosus COM1 has the characteristics of high fidelity,fast extension,and strong thermal stability. It can increase the amplification efficiency during the PCR reaction and reduce the rate of incorrect base incorporation. Using Escherichia coli Rosetta( DE3) as the recombinant cells,a rapid protein purification method was developed to facilitate the purification of Pfu DNA polymerase while maintaining its high polymerase activity. The results showed Pfu DNA polymerase could be expressed in a large amount in E.coli Rosetta( DE3). In addition,the high-temperature water bath at 60 ℃can effectively remove the miscellaneous proteins,simplify the purification process and increase the purity of Pfu DNA polymerase. After a series of purification steps,Pfu DNA polymerase can still maintain a good enzyme activity,and in 100 μL PCR reaction system,4 μL of 1 mg/m L Pfu DNA polymerase solution gave the best outcomes.

关 键 词：PFU DNA聚合酶 Rosetta(DE3) 亲和层析 PCR检测 

分 类 号：Q786[生物学—分子生物学]