Department of Molecular Biosciences, College of Natural Sciences, The University of Texas at Austin, Austin, Texas 78712, United States.
Center for Systems and Synthetic Biology, The University of Texas at Austin, Austin, Texas 78712, United States.
ACS Synth Biol. 2022 Apr 15;11(4):1488-1496. doi: 10.1021/acssynbio.1c00559. Epub 2022 Mar 23.
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 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.
蛋白质的电荷状态会极大地影响其稳定性和与底物的相互作用，而添加多个电荷（超电荷）已被证明是一种成功的工程蛋白质稳定性和功能的方法。将快速折叠融合结构域添加到 DNA 聚合酶中，提高了其在等温扩增测定中的功能，而对该结构域的进一步电荷工程化则提高了蛋白质的稳定性和诊断性能。当与稳定蛋白质核心的突变结合使用时，电荷工程化的融合结构域可实现高达 74°C 的环介导等温扩增（LAMP）或在高浓度脲存在下进行，检测时间不到 10 分钟。在融合结构域中添加正电荷和负电荷会导致聚合酶的逆转录酶和 DNA 聚合酶相对活性发生变化。总的来说，开发一种模块化的融合结构域，其带电表面可以随意修饰，这对于其他聚合酶的工程化应该是有用的，而且通常可能对蛋白质稳定化有用。
