High-Throughput Protein Engineering by Massively Parallel Combinatorial Mutagenesis.

Yuk Kei Wan, Gigi C G Choi, Alan S L Wong

Methods in molecular biology (Clifton, N.J.) 2021

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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.

Citation

Yuk Kei Wan, Gigi C G Choi, Alan S L Wong. High-Throughput Protein Engineering by Massively Parallel Combinatorial Mutagenesis. Methods in molecular biology (Clifton, N.J.). 2021;2199:3-12