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首页> 外文期刊>Combinatorial Chemistry _High Throughput Screening >The Diversity Challenge in Directed Protein Evolution
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The Diversity Challenge in Directed Protein Evolution

机译：定向蛋白质进化中的多样性挑战

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摘要

Over the past decade, we have witnessed a bloom in the field of evolutive protein engineering which is fueled by advances in molecular biology techniques and high-throughput screening technology. Directed protein evolution is a powerful algorithm using iterative cycles of random mutagenesis and screening for tailoring protein properties to our needs in industrial applications and for elucidating proteins' structure function relationships.nnThis review summarizes, categorizes and discusses advantages and disadvantages of random mutagenesis methods used for generating genetic diversity. These random mutagenesis methods have been classified into four main categories depending on the method employed for nucleotide substitutions: enzyme based methods (Category I), synthetic chemistry based methods (Category II), whole cell methods (Category III) and combined methods (Category I-II, I-III and II-III). The basic principle of each method is discussed and varied mutagenic conditions are summarized in Tables and compared (benchmarked) to each other in terms of: mutational bias, controllable mutation frequency, ability to generate consecutive nucleotide substitutions and subset diversity, dependency on gene length, technical simplicity/robustness and costeffectiveness. The latter comparison shows how highly-biased and limited current diversity creating methods are. Based on these limitations, strategies for generating diverse mutant libraries are proposed and discussed (RaMuS-Flowchart; KISS principle).nnWe hope that this review provides, especially for researchers just entering the field of directed evolution, a guide for developing successful directed evolution strategies by selecting complementary methods for generating diverse mutant libraries.
机译：在过去的十年中，我们见证了进化性蛋白质工程领域的蓬勃发展，这得益于分子生物学技术和高通量筛选技术的发展。定向蛋白质进化是一种使用随机诱变和筛选的迭代循环的强大算法，可以根据我们在工业应用中的需求量身定制蛋白质特性，并阐明蛋白质的结构功能关系。产生遗传多样性。根据用于核苷酸取代的方法，这些随机诱变方法已分为四个主要类别：基于酶的方法（类别I），基于合成化学的方法（类别II），全细胞方法（类别III）和组合方法（类别I） -II，I-III和II-III）。讨论了每种方法的基本原理，并在表中总结了各种诱变条件，并就以下方面进行了比较（标为基准）：突变偏倚，可控制的突变频率，产生连续核苷酸取代和子集多样性的能力，对基因长度的依赖性，技术的简单性/鲁棒性和成本效益。后面的比较显示了高度偏向和有限的电流分集创建方法的程度。基于这些限制，提出并讨论了生成各种突变体文库的策略（RaMuS-Flowchart； KISS原理）。nn我们希望这篇评论特别为刚刚进入定向进化领域的研究人员提供指导，以制定成功的定向进化策略通过选择用于生成各种突变体文库的互补方法。

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