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A Fusion of Taq DNA Polymerase with the CL7 Protein from Escherichia coli Remarkably Improves DNA Amplification

文献类型： 外文期刊

作者： Li, Zhongchen 1 ; Wang, Yaping 1 ; Wang, Xiangyi 1 ; Niu, Shuhui 1 ; Su, Zhenlong 1 ; Wang, Fei 1 ; Ni, Jing 1 ; Gong, Yan 1 ; Rao, Ben 1 ;

作者机构： 1.Hubei Univ, Hubei Key Lab Ind Biotechnol, Engn Hubei Collaborat Innovat Ctr Green Transforma, State Key Lab Biocatalysis & Enzyme,Biol Fac, Wuhan 430062, Peoples R China

2.Hubei Acad Agr Sci, Hubei Biopesticide Engn Res Ctr, Biopesticide Branch Hubei Innovat Ctr Agr Sci & Te, Wuhan 430064, Peoples R China

关键词： Taq DNA polymerase; CL7; fusion DNA polymerase; PCR amplification

期刊名称：MOLECULES （ 影响因子：4.6； 五年影响因子：4.9 ）

ISSN：

年卷期： 2024 年 29 卷 5 期

页码：

收录情况： SCI

摘要： DNA polymerases are important enzymes that synthesize DNA molecules and therefore are critical to various scientific fields as essential components of in vitro DNA synthesis reactions, including PCR. Modern diagnostics, molecular biology, and genetic engineering require DNA polymerases with improved performance. This study aimed to obtain and characterize a new CL7-Taq fusion DNA polymerase, in which the DNA coding sequence of Taq DNA polymerase was fused with that of CL7, a variant of CE7 (Colicin E7 DNase) from Escherichia coli. The resulting novel recombinant open reading frame was cloned and expressed in E. coli. The recombinant CL7-Taq protein exhibited excellent thermostability, extension rate, sensitivity, and resistance to PCR inhibitors. Our results showed that the sensitivity of CL7-Taq DNA polymerase was 100-fold higher than that of wild-type Taq, which required a template concentration of at least 1.8 x 105 nM. Moreover, the extension rate of CL7-Taq was 4 kb/min, which remarkably exceeded the rate of Taq DNA polymerase (2 kb/min). Furthermore, the CL7 fusion protein showed increased resistance to inhibitors of DNA amplification, including lactoferrin, heparin, and blood. Single-cope human genomic targets were readily available from whole blood, and pretreatment to purify the template DNA was not required. Thus, this is a novel enzyme that improved the properties of Taq DNA polymerase, and thus may have wide application in molecular biology and diagnostics.

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