Derivatives of Bst-like Gss-polymerase with improved processivity and inhibitor tolerance
Derivatives of Bst-like Gss-polymerase with improved processivity and inhibitor tolerance
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DOI：
10.1093/nar/gkx645
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发表时间：
2017-09-19
影响因子：
14.9
通讯作者：
Filipenko, Maksim L.
中科院分区：
文献类型：
--
作者：
Oscorbin, Igor P.;Belousova, Ekaterina A.;Filipenko, Maksim L.
目前，医学诊断的实际趋势之一是开发用于实际应用的方法，例如即时检测，POCT或研究工具，例如全基因组扩增，WGA。所有技术都基于使用具有链置换活性、高合成持续合成能力、保真度以及最重要的是对污染物的耐受性的特定DNA聚合酶，所述污染物从分析的生物样品中出现或在纯化程序下收集。在此，我们设计了一套基于Geobacillussp.777的DNA聚合酶I的催化结构域与DNA连接酶Pyrococcusabyssi的DNA结合结构域和Sulfolobustokodaii的Sto 7 d蛋白（Sso 7 d的类似物）的融合酶。设计的嵌合DNA聚合酶DBD-Gss、Sto-Gss和Gss-Sto表现出与天然Gss相同水平的热稳定性、热转移酶活性和保真度;然而，持续合成能力增加高达3倍，导致WGA中的DNA产物增加约4倍，这更加令人兴奋。DNA结合蛋白的附着增强了嵌合聚合酶在环介导的等温扩增中对几种最常见的DNA样品污染物-尿素和全血、肝素、乙二胺四乙酸、NaCl、乙醇的抑制剂耐受性。因此，嵌合BST样Gss-聚合酶由于增加的持续合成能力和抑制剂耐受性，将是WGA和POCT的有希望的工具。
At the moment, one of the actual trends in medical diagnostics is a development of methods for practical applications such as point-of-care testing, POCT or research tools, for example, whole genome amplification, WGA. All the techniques are based on using of specific DNA polymerases having strand displacement activity, high synthetic processivity, fidelity and, most significantly, tolerance to contaminants, appearing from analysed biological samples or collected under purification procedures. Here, we have designed a set of fusion enzymes based on catalytic domain of DNA polymerase I from Geobacillus sp. 777 with DNA-binding domain of DNA ligase Py-rococcus abyssi and Sto7d protein from Sulfolobus tokodaii, analogue of Sso7d. Designed chimeric DNA polymerases DBD-Gss, Sto-Gss and Gss-Sto exhibited the same level of thermal stability, thermal transferase activity and fidelity as native Gss; however, the processivity was increased up to 3-fold, leading to about 4-fold of DNA product in WGA which is much more exiting. The attachment of DNAbinding proteins enhanced the inhibitor tolerance of chimeric polymerases in loop-mediated isothermal amplification to several of the most common DNA sample contaminants--urea and whole blood, heparin, ethylenediaminetetraacetic acid, NaCl, ethanol. Therefore, chimeric Bst-like Gss-polymerase will be promising tool for both WGA and POCT due to increased processivity and inhibitor tolerance.