Enhanced performance of Thermococcus kodakarensis KOD1 polymerase in PCR via fusion to Sulfolobus tokodaii Sto7d

IF 1.2 4区生物学 Q4 BIOCHEMICAL RESEARCH METHODS

Protein expression and purification Pub Date : 2025-08-31 DOI:10.1016/j.pep.2025.106809

Leheng Chen , Dawei Fu

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引用次数: 0

Abstract

The DNA polymerase from Thermococcus kodakarensis KOD1 (KOD) is widely utilized in polymerase chain reaction (PCR) due to its high processivity and fidelity. However, like many other B-family DNA polymerases, it faces limitations in extension efficiency, amplicon length, and resistance to PCR inhibitors. In order to further enhance its capability, novel mutants were engineered by fusing a 7 kDa nonspecific double-stranded DNA (dsDNA)-binding protein from Sulfolobus tokodaii (Sto7d) to the C-terminus of KOD via distinct peptide linkers, resulting in a set of KOD-Sto7d polymerase variants. These constructs were expressed, purified, and characterized. Among the variants, KOD-GT4G-Sto7d exhibited the best PCR performance and was selected as the representative variant for subsequent assays. Compared with wild-type KOD (KOD-WT), KOD-Sto7d demonstrated significantly improved extension efficiency that successfully amplified 7 kb targets with only 10 s elongation time, increased salt tolerance up to 120 mM NaCl for 2 kb targets, and an improved capacity to amplify long DNA fragments up to 10 kb within 4 min. In comparison with a commercially available KOD mutant fused to a dsDNA-binding protein (Sso7d from Saccharolobus solfataricus) at its C-terminus (KOD-Sso7d), KOD-Sto7d demonstrated greater salt tolerance and sensitivity. These results suggest that KOD-Sto7d is a robust polymerase suitable for time-saving and high-demanding PCR.

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通过融合tokodaisulfolobus Sto7d提高柯达热球菌KOD1聚合酶的PCR性能

kodakarensis热球菌（Thermococcus KOD1， KOD）的DNA聚合酶因其高精密度和保真度被广泛应用于聚合酶链反应（polymerase chain reaction， PCR）中。然而，像许多其他b家族DNA聚合酶一样，它在延伸效率、扩增子长度和对PCR抑制剂的抗性方面存在局限性。为了进一步增强其能力，我们将tokodaii Sulfolobus （Sto7d）中一个7 kDa的非特异性双链DNA （dsDNA）结合蛋白通过不同的肽连接体融合到KOD的c端，从而构建了一组KOD-Sto7d聚合酶变体。这些结构被表达、纯化和表征。其中，KOD-GT4G-Sto7d表现出最好的PCR性能，并被选为后续检测的代表性变异。与野生型KOD （KOD- wt）相比，KOD- sto7d扩增效率显著提高，在10 s的延伸时间内扩增出7 kb的目标，对2 kb目标的耐盐性提高到120 mM NaCl，在4 min内扩增10 kb长的DNA片段的能力也有所提高。与市售的在其c端融合dsdna结合蛋白（来自Saccharolobus solfataricus的Sso7d）的KOD突变体（KOD-Sso7d）相比，KOD- sto7d表现出更强的耐盐性和敏感性。这些结果表明KOD-Sto7d是一种强大的聚合酶，适合于节省时间和高要求的PCR。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

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来源期刊

Protein expression and purification 生物-生化研究方法

CiteScore

3.70

自引率

6.20%

发文量

120

审稿时长

32 days

期刊介绍： Protein Expression and Purification is an international journal providing a forum for the dissemination of new information on protein expression, extraction, purification, characterization, and/or applications using conventional biochemical and/or modern molecular biological approaches and methods, which are of broad interest to the field. The journal does not typically publish repetitive examples of protein expression and purification involving standard, well-established, methods. However, exceptions might include studies on important and/or difficult to express and/or purify proteins and/or studies that include extensive protein characterization, which provide new, previously unpublished information.