School of Biology and Biological Engineering, South China University of Technology, Guangzhou, 510006, China.
Guangdong Provincial Key Laboratory of Fermentation and Enzyme Engineering, South China University of Technology, Guangzhou, 510006, China.
Appl Microbiol Biotechnol. 2024 Aug 21;108(1):445. doi: 10.1007/s00253-024-13276-2.
Chimeric DNA polymerase with notable performance has been generated for wide applications including DNA amplification and molecular diagnostics. This rational design method aims to improve specific enzymatic characteristics or introduce novel functions by fusing amino acid sequences from different proteins with a single DNA polymerase to create a chimeric DNA polymerase. Several strategies prove to be efficient, including swapping homologous domains between polymerases to combine benefits from different species, incorporating additional domains for exonuclease activity or enhanced binding ability to DNA, and integrating functional protein along with specific protein structural pattern to improve thermal stability and tolerance to inhibitors, as many cases in the past decade shown. The conventional protocol to develop a chimeric DNA polymerase with desired traits involves a Design-Build-Test-Learn (DBTL) cycle. This procedure initiates with the selection of a parent polymerase, followed by the identification of relevant domains and devising a strategy for fusion. After recombinant expression and purification of chimeric polymerase, its performance is evaluated. The outcomes of these evaluations are analyzed for further enhancing and optimizing the functionality of the polymerase. This review, centered on microorganisms, briefly outlines typical instances of chimeric DNA polymerases categorized, and presents a general methodology for their creation. KEY POINTS: • Chimeric DNA polymerase is generated by rational design method. • Strategies include domain exchange and addition of proteins, domains, and motifs. • Chimeric DNA polymerase exhibits improved enzymatic properties or novel functions.
已经产生了具有显著性能的嵌合 DNA 聚合酶，可广泛应用于 DNA 扩增和分子诊断等领域。这种合理的设计方法旨在通过融合来自不同蛋白质的氨基酸序列来改善特定的酶学特性或引入新的功能，从而创建嵌合 DNA 聚合酶。几种策略被证明是有效的，包括在聚合酶之间交换同源结构域，以结合来自不同物种的优势；引入额外的结构域以增加外切核酸酶活性或增强与 DNA 的结合能力；以及整合具有特定蛋白结构模式的功能性蛋白，以提高热稳定性和对抑制剂的耐受性，过去十年中的许多案例都证明了这一点。开发具有所需特性的嵌合 DNA 聚合酶的传统方案涉及设计-构建-测试-学习（DBTL）循环。该过程首先选择亲本聚合酶，然后确定相关结构域并设计融合策略。在重组表达和纯化嵌合聚合酶后，评估其性能。分析这些评估的结果，以进一步增强和优化聚合酶的功能。本综述以微生物为中心，简要概述了分类的典型嵌合 DNA 聚合酶实例，并提出了创建它们的一般方法。关键点：
嵌合 DNA 聚合酶是通过合理的设计方法产生的。
策略包括结构域交换以及添加蛋白质、结构域和基序。
嵌合 DNA 聚合酶表现出改善的酶学特性或新的功能。
