Charge Engineering Improves the Performance of Bst DNA Polymerase Fusions.
Charge Engineering Improves the Performance of Bst DNA Polymerase Fusions.
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DOI：
10.1021/acssynbio.1c00559
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发表时间：
2022-04-15
影响因子：
4.7
通讯作者：
Ellington, Andrew D.
中科院分区：
文献类型：
--
作者：
Paik, Inyup;Bhadra, Sanchita;Ellington, Andrew D.
蛋白质的电荷状态可以极大地影响其稳定性以及与底物的相互作用，并且添加多个电荷（增压）已被证明是工程蛋白质稳定性和功能的成功方法。在嗜热脂肪芽孢杆菌 DNA 聚合酶中添加快速折叠融合结构域改善了其在等温扩增测定中的功能，并且该结构域的进一步电荷工程提高了蛋白质稳定性和诊断性能。当与稳定蛋白质核心的突变相结合时，电荷工程融合结构域能够在高达 74°C 的温度或存在高浓度尿素的情况下进行环介导等温扩增 (LAMP)，检测时间低于 10 分钟。向融合结构域添加正电荷和负电荷会导致聚合酶的相对逆转录酶和 DNA 聚合酶活性发生变化。总体而言，其带电表面可随意修饰的模块化融合结构域的开发应该被证明可用于其他聚合酶的工程设计，并且通常可能被证明对蛋白质稳定有用。
The charge states of proteins can greatly influence their stabilities and interactions with substrates, and the addition of multiple charges (supercharging) has been shown to be a successful approach for engineering protein stability and function. The addition of a fast-folding fusion domain to the Bacillus stearothermophilus DNA polymerase improved its functionality in isothermal amplification assays, and further charge engineering of this domain has increased both protein stability and diagnostics performance. When combined with mutations that stabilize the core of the protein, the charge-engineered fusion domain leads to the ability to carry out loop-mediated isothermal amplification (LAMP) at temperatures up to 74° C or in the presence of high concentrations of urea, with detection times under 10 min. Adding both positive and negative charges to the fusion domain led to changes in the relative reverse transcriptase and DNA polymerase activities of the polymerase. Overall, the development of a modular fusion domain whose charged surface can be modified at will should prove to be of use in the engineering of other polymerases and, in general, may prove useful for protein stabilization.
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影响因子：
3.1
作者：
Howson ELA;Kidd SP;Armson B;Goring A;Sawyer J;Cassar C;Cross D;Lewis T;Hockey J;Rivers S;Cawthraw S;Banyard A;Anderson P;Rahou S;Andreou M;Morant N;Clark D;Walsh C;Laxman S;Houghton R;Slater-Jefferies J;Costello P;Brown I;Cortes N;Godfrey KM;Fowler VL
通讯作者：
Fowler VL
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14.9
作者：
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Thornton, JM
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作者：
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14.9
作者：
Oscorbin, Igor P.;Belousova, Ekaterina A.;Filipenko, Maksim L.
通讯作者：
Filipenko, Maksim L.
影响因子：
7.4
作者：
Cai, Sheng;Jung, Cheulhee;Ellington, Andrew D.
通讯作者：
Ellington, Andrew D.