KTH - Royal Institute of Technology, Department of Protein Science, 106 91 Stockholm, Sweden.
KTH - Royal Institute of Technology, Science for Life Laboratory, Solna 171 65, Sweden.
Nucleic Acids Res. 2019 Apr 8;47(6):e34. doi: 10.1093/nar/gkz050.
Mutagenesis libraries are essential for combinatorial protein engineering. Despite improvements in gene synthesis and directed mutagenesis, current methodologies still have limitations regarding the synthesis of complete antibody single-chain variable fragment (scFv) genes and simultaneous diversification of all six CDRs. Here, we describe the generation of mutagenesis libraries for antibody affinity maturation using a cell-free solid-phase technique for annealing of single-strand mutagenic oligonucleotides. The procedure consists of PCR-based incorporation of uracil into a wild-type template, bead-based capture, elution of single-strand DNA, and in vitro uracil excision enzyme based degradation of the template DNA. Our approach enabled rapid (8 hours) mutagenesis and automated cloning of 50 position-specific alanine mutants for mapping of a scFv antibody paratope. We further exemplify our method by generating affinity maturation libraries with diversity introduced in critical, nonessential, or all CDR positions randomly. Assessment with Illumina deep sequencing showed less than 1% wild-type in two libraries and the ability to diversify all CDR positions simultaneously. Selections of the libraries with bacterial display and deep sequencing evaluation of the selection output showed that diversity introduced in non-essential positions allowed for a more effective enrichment of improved binders compared to the other two diversification strategies.
突变文库对于组合蛋白工程至关重要。尽管基因合成和定向诱变技术有所改进，但目前的方法在合成完整的抗体单链可变片段（scFv）基因和同时多样化所有六个 CDR 方面仍存在局限性。在这里，我们描述了一种使用无细胞固相技术退火单链诱变寡核苷酸来生成抗体亲和力成熟突变文库的方法。该程序包括基于 PCR 将尿嘧啶掺入野生型模板、基于珠子的捕获、单链 DNA 的洗脱以及基于体外尿嘧啶切除酶的模板 DNA 降解。我们的方法能够快速（8 小时）进行突变，并自动克隆用于 scFv 抗体结合位映射的 50 个位置特异性丙氨酸突变体。我们通过在关键的、非必需的或所有 CDR 位置随机引入多样性来进一步举例说明我们的方法。使用 Illumina 深度测序评估显示，两个文库中不到 1%的野生型，并且能够同时多样化所有 CDR 位置。文库的细菌展示选择和对选择输出的深度测序评估表明，与其他两种多样化策略相比，在非必需位置引入的多样性允许更有效地富集改善的结合物。
