Enhancing helicase-dependent amplification by fusing the helicase with the DNA polymerase

被引：38

作者：

Motre, Aurelie ^{[1
]}

Li, Ying ^{[1
]}

Kong, Huimin ^{[1
]}

机构：

[1] BioHelix, Beverly, MA 01915 USA

来源：

GENE | 2008年 / 420卷 / 01期

关键词：

protein engineering; helicase; DNA polymerase; protein fusion;

D O I：

10.1016/j.gene.2008.04.017

中图分类号：

Q3 [遗传学];

学科分类号：

071007 ; 090102 ;

摘要：

In this study, we have engineered a new bifunctional protein named "helimerase", by physically linking Thermoanaerobacter tengcongensis UvrD helicase (TteUvrD) and Bacillus stearothermophilus DNA polymerase 1 Large Fragment (Bstpol) using a coiled-coil. TteUvrD is fused with one part of the coiled-coil, WinZip-A2 (WZA2), through a linker L1 and possesses a Maltose Binding Protein (MBP) tag at the N terminal end. Bstpol is fused with the other part of the coiled-coil, WinZip-B1 (WZB1), through the same linker and possesses a His tag at the N terminal end. Therefore, the two fusion proteins MBP-WZA2-L1-TteUvrD and His-WZB1-Li-Bstpot composed the helimerase. We showed that this complex could be formed in vivo as well as in vitro and possessed specific activities of both TteUvrD and Bstpol. Moreover, we demonstrated that significantly longer fragments could be amplified by the helimerase in Helicase-Dependent Amplification (HDA) reactions instead of using TteUvrD and Bstpol proteins that do not form a complex. (C) 2008 Elsevier B.V. All rights reserved.

引用

页码：17 / 22

页数：6

共 15 条

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