Genetically engineered horseradish peroxidase for facilitated purification from baculovirus cultures by cation-exchange chromatography
Genetically engineered horseradish peroxidase for facilitated purification from baculovirus cultures by cation-exchange chromatography
复制标题
DOI：
10.1016/j.jbiotec.2005.05.015
复制
发表时间：
2005-09-10
影响因子：
4.1
通讯作者：
Miranda, MV
中科院分区：
文献类型：
--
作者：
Levin, G;Mendive, F;Miranda, MV
利用聚合酶链式反应技术，在6xHis-HRP C的基础上加入6xArg融合尾巴，构建了辣根过氧化物酶同工酶C(HRP C)基因工程载体，并在侵染野油菜夜蛾核型多角体病毒的Sf9昆虫细胞系中表达了6xHis-6xArg-HRP C基因，经等电聚焦鉴定，重组过氧化物酶等电点为9.5，经阳离子交换层析或固定化金属离子亲和层析，于感染后第6天直接从培养基中纯化。前者的得率为98.5%，纯化倍数为130，而后者的得率仅为68%，纯化倍数为140。结果表明，Poly-Arg标签比PolyHis标签更适合于杆状病毒表达系统中过氧化物酶的纯化。(C)2005 Elsevier B.V.保留所有权利。
An engineered horseradish peroxidase isozyme C (HRP C) gene was constructed by the addition of a 6xArg fusion tail to 6xHis-HRP C by the PCR strategy.The 6xHis-6xArg-HRP C cDNA, was expressed in the Sf9 insect cell line from Spodoptera frugiperda infected with Autographa californica nuclear polyhedrosis virus.The recombinant peroxidase isoelectric point was 9.5 as judged by isoelectric focusing and was purified directly from the culture medium at day-6 post-infection by cation-exchange chromatography or immobilised metal ion-affinity chromatography. While the former technique gave a yield of 98.5% with a purification factor of 130, the latter gave only a 68% yield with a purification factor of 140.Results obtained provide evidence that the poly-Arg tag is more effective than the poly-His tag for peroxidase purification from a baculovirus expression system. (C) 2005 Elsevier B.V. All rights reserved.