融合大肠杆菌素DNA结合域的Taq DNA聚合酶的性质表征  

Characterization of a Taq DNA polymerase fused with a DNA binding domain of Escherichia coli colicin

作　　者：王亚平[1] 平啸寅 赵艺 刘阳 吴林 马立新[1] WANG Yaping;PING Xiaoyin;ZHAO Yi;LIU Yang;WU Lin;MA Lixin(State Key Laboratory of Biocatalysis and Enzyme Engineering,School of Life Sciences,Hubei University,Wuhan 430062,Hubei,China)

机构地区：[1]湖北大学生命科学学院省部共建生物催化与酶工程国家重点实验室,湖北武汉430062

出　　处：《生物工程学报》2024年第3期812-820,共9页Chinese Journal of Biotechnology

基　　金：武汉东湖新技术开发区“揭榜挂帅”项目(2022KJB001);省级科技创新专项基金(科技人才服务企业项目:2023DJC123)。

摘　　要：Taq DNA聚合酶发现自水生热栖菌(Thermus aquaticus),是一种同时具有逆转录酶活性以及DNA聚合酶活性的工具酶。Colicin E(简称CE)蛋白质是一类以维生素受体BtuB为跨膜受体的大肠杆菌素,其中CE2、CE7、CE8和CE9是非特异性的DNase型大肠杆菌素。Taq DNA聚合酶由5′→3′核酸外切酶结构域、3′→5′核酸外切酶结构域以及聚合酶结构域组成。缺失5′→3′核酸外切酶结构域的Taq DNA聚合酶(ΔTaq)具有更高的产量,但是其进行性很低,无法扩增长片段。为了提高ΔTaq的进行性,本研究融合dCE和ΔTaq,发现dCE-ΔTaq的进行性相比于Taq DNA聚合酶和dCE-Taq显著提升,并且其逆转录酶活性也比ΔTaq更高。dCE8-ΔTaq的提升最明显,不仅能够1 min内扩增8 kb的DNA片段,并且产量高于其他突变体。综上所述,本研究通过将ΔTaq DNA聚合酶和dCE进行融合,提升了Taq DNA聚合酶的PCR效率和逆转录活性,为改造Taq DNA聚合酶提供了新的方法,有望开发出性质更好的Taq DNA聚合酶。Taq DNA polymerase,which was discovered from a thermophilic aquatic bacterium(Thermus aquaticus),is an enzyme that possesses both reverse transcriptase activity and DNA polymerase activity.Colicin E(CE)protein belongs to a class of Escherichia coli toxins that utilize the vitamin receptor BtuB as a transmembrane receptor.Among these toxins,CE2,CE7,CE8,and CE9 are classified as non-specific DNase-type colicins.Taq DNA polymerase consists of a 5′→3′exonuclease domain,a 3′→5′exonuclease domain,and a polymerase domain.Taq DNA polymerase lacking the 5′→3′exonuclease domain(ΔTaq)exhibits higher yield but lower processivity,making it unable to amplify long fragments.In this study,we aimed to enhance the processivity ofΔTaq.To this end,we fused dCE withΔTaq and observed a significant improvement in the processivity of the resulting dCE-ΔTaq compared to Taq DNA polymerase and dCE-Taq.Furthermore,its reverse transcriptase activity was also higher than that ofΔTaq.The most notable improvement was observed in dCE8-ΔTaq,which not only successfully amplified 8 kb DNA fragments within 1 minute,but also yielded higher results compared to other mutants.In summary,this study successfully enhanced the PCR efficiency and reverse transcription activity of Taq DNA polymerase by fusingΔTaq DNA polymerase with dCE.This approach provides a novel approach for modifying Taq DNA polymerase and holds potential for the development of improved variants of Taq DNA polymerase.

关 键 词：Taq DNA聚合酶 CE PCR效率 逆转录活性 

分 类 号：Q78[生物学—分子生物学]