Popa Serban C, Inamoto Ichiro, Thuronyi B W, Shin Jumi A
Department of Chemistry, University of Toronto, 3359 Mississauga Road, Mississauga, ON L5L 1C6, Canada.
Department of Chemistry, Williams College, 47 Lab Campus Drive, Williamstown, Massachusetts 01267, United States.
ACS Omega. 2020 Oct 16;5(42):26957-26966. doi: 10.1021/acsomega.0c03508. eCollection 2020 Oct 27.
The uptake of directed evolution methods is increasing, as these powerful systems can be utilized to develop new biomolecules with altered/novel activities, for example, proteins with new catalytic functions or substrate specificities and nucleic acids that recognize an intended target. Especially useful are systems that incorporate evolution, where the protein under selective pressure undergoes continuous mutagenesis with little-to-no input from the researcher once the system is started. However, continuous evolution methods can be challenging to implement and a daunting investment of time and resources. Our intent is to provide basic information and helpful suggestions that we have gained from our experience with bacterial phage-assisted continuous evolution (PACE) toward the evolution of proteins that bind to a specific DNA target. We discuss factors to consider before adopting PACE for a given evolution scheme with focus on the PACE bacterial one-hybrid selection system and what optimization of a PACE selection circuit may look like using the evolution of the DNA-binding protein ME47 as a case study. We outline different types of selection circuits and techniques that may be added onto a basic PACE setup. With this information, researchers will be better equipped to determine whether PACE is a valid strategy to adopt for their research program and how to set up a valid selection circuit.
定向进化方法的应用正在增加，因为这些强大的系统可用于开发具有改变的/新活性的新生物分子，例如，具有新催化功能或底物特异性的蛋白质以及识别特定靶标的核酸。特别有用的是纳入进化的系统，在这种系统中，一旦系统启动，处于选择压力下的蛋白质会经历连续诱变，几乎无需研究人员输入。然而，连续进化方法实施起来可能具有挑战性，且需要投入大量的时间和资源。我们的目的是提供我们从细菌噬菌体辅助连续进化（PACE）中获得的基本信息和有用建议，该方法用于进化与特定DNA靶标结合的蛋白质。我们讨论在采用PACE进行特定进化方案之前需要考虑的因素，重点是PACE细菌单杂交选择系统，以及以DNA结合蛋白ME47的进化为例，PACE选择电路的优化可能是什么样的。我们概述了可以添加到基本PACE设置中的不同类型的选择电路和技术。有了这些信息，研究人员将更有能力确定PACE是否是其研究计划中可采用的有效策略，以及如何建立有效的选择电路。
