Department of Structural and Molecular Biology, University College London, Gower Street, London, WC1E 6BT, UK.
Department of Biological Sciences, University of London, Malet Street, Birkbeck, WC1E 7HX, UK.
Sci Rep. 2021 Apr 28;11(1):9134. doi: 10.1038/s41598-021-88708-4.
Insertions and deletions (indels) are known to affect function, biophysical properties and substrate specificity of enzymes, and they play a central role in evolution. Despite such clear significance, this class of mutation remains an underexploited tool in protein engineering with few available platforms capable of systematically generating and analysing libraries of varying sequence composition and length. We present a novel DNA assembly platform (InDel assembly), based on cycles of endonuclease restriction digestion and ligation of standardised dsDNA building blocks, that can generate libraries exploring both composition and sequence length variation. In addition, we developed a framework to analyse the output of selection from InDel-generated libraries, combining next generation sequencing and alignment-free strategies for sequence analysis. We demonstrate the approach by engineering the well-characterized TEM-1 β-lactamase Ω-loop, involved in substrate specificity, identifying multiple novel extended spectrum β-lactamases with loops of modified length and composition-areas of the sequence space not previously explored. Together, the InDel assembly and analysis platforms provide an efficient route to engineer protein loops or linkers where sequence length and composition are both essential functional parameters.
插入和缺失（indels）已知会影响酶的功能、生物物理特性和底物特异性，并且在进化中起着核心作用。尽管这种突变具有如此明显的意义，但在蛋白质工程中，这种类型的突变仍然是一种未充分利用的工具，因为很少有可用的平台能够系统地生成和分析具有不同序列组成和长度的文库。我们提出了一种新的 DNA 组装平台（InDel 组装），该平台基于内切酶限制性消化和标准化 dsDNA 构建块的连接的循环，能够生成探索组成和序列长度变化的文库。此外，我们开发了一种分析 InDel 生成文库中选择结果的框架，将下一代测序和无序列比对策略相结合进行序列分析。我们通过对参与底物特异性的 TEM-1 β-内酰胺酶 Ω 环进行工程改造来证明该方法，鉴定出多个具有修饰长度和组成的新型扩展谱 β-内酰胺酶的环，这些环是以前未探索过的序列空间区域。总之，InDel 组装和分析平台为工程化蛋白质环或连接子提供了一种有效的途径，其中序列长度和组成都是必需的功能参数。
