Laboratory of Combinatorial Genetics and Synthetic Biology, School of Biomedical Sciences, The University of Hong Kong, Hong Kong SAR, China.
Department of Electrical and Electronic Engineering, The University of Hong Kong, Hong Kong SAR, China.
Methods Mol Biol. 2021;2199:3-12. doi: 10.1007/978-1-0716-0892-0_1.
Exploring how combinatorial mutations can be combined to optimize protein functions is important to guide protein engineering. Given the vast combinatorial space of changing multiple amino acids, identifying the top-performing variants from a large number of mutants might not be possible without a high-throughput gene assembly and screening strategy. Here we describe the CombiSEAL platform, a strategy that allows for modularization of any protein sequence into multiple segments for mutagenesis and barcoding, and seamless single-pot ligations of different segments to generate a library of combination mutants linked with concatenated barcodes at one end. By reading the barcodes using next-generation sequencing, activities of each protein variant during the protein selection process can be easily tracked in a high-throughput manner. CombiSEAL not only allows the identification of better protein variants but also enables the systematic analyses to distinguish the beneficial, deleterious, and neutral effects of combining different mutations on protein functions.
探索组合突变如何组合以优化蛋白质功能对于指导蛋白质工程很重要。考虑到改变多个氨基酸的组合空间非常庞大，如果没有高通量的基因组装和筛选策略，从大量突变体中识别出表现最佳的变体可能是不可能的。在这里，我们描述了 CombiSEAL 平台，这是一种将任何蛋白质序列模块化成多个片段进行突变和条形码标记的策略，并且可以无缝地将不同片段连接在一起进行单管连接，从而生成一端连接串联条形码的组合突变体文库。通过使用下一代测序读取条形码，可以以高通量的方式轻松跟踪蛋白质选择过程中每个蛋白质变体的活性。CombiSEAL 不仅可以识别更好的蛋白质变体，还可以进行系统分析，以区分不同突变组合对蛋白质功能的有益、有害和中性影响。
