Efficiently Exploring Functional Space In Loop Engineering With Variations In Length And Composition
Efficiently Exploring Functional Space In Loop Engineering With Variations In Length And Composition
复制标题
有效探索循环工程中长度和组成变化的功能空间
DOI：
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发表时间：
2017
期刊：
影响因子：
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通讯作者：
Vitor B. Pinheiro
中科院分区：
文献类型：
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作者：
Pedro A. G. Tizei;Emma Harris;M. Renders;Vitor B. Pinheiro
插入和缺失 (indels) 可以影响酶的功能、生物物理特性和底物特异性，它们在进化中发挥着核心作用。尽管具有如此明显的相关性，此类突变仍然是蛋白质工程中尚未充分利用的工具，没有能够系统生成和分析不同序列组成和长度的文库的可用平台。在这里，我们提出了一种新型 DNA 组装平台（InDeL 组装），该平台基于核酸内切酶限制和连接循环，与基于 k 聚体的序列分析框架相结合，能够在不同组成和长度之间系统且有效地导航序列空间。我们通过设计充分表征的 TEM-1 β-内酰胺酶 Ω 环来演示该方法，该环涉及底物特异性，在之前未探索过的序列空间区域中识别新型广谱 β-内酰胺酶。 InDel 组装提供了优化蛋白质环或接头的途径，其中序列长度和组成都是重要的功能参数。
Insertions and deletions (indels) can affect function, biophysical properties and substrate specificity of enzymes, and they play a central role in evolution. Despite such clear relevance, this class of mutation remains an underexploited tool in protein engineering with no available platforms capable of systematically generating and analyzing libraries of varying sequence composition and length. Here, we present a novel DNA assembly platform (InDeL assembly), based on cycles of endonuclease restriction and ligation, that coupled to a k-mer based sequence analysis framework, enables systematic and efficient navigation of the sequence space across different compositions and lengths. We demonstrate the approach by engineering the well-characterized TEM-1 β-lactamase Ω-loop, involved in substrate specificity, identifying novel extended spectrum β-lactamases in areas of the sequence space not previously explored. InDel assembly provides a route to optimize protein loops or linkers where sequence length and composition are both essential functional parameters.