Department of Molecular Genetics, Charles Best Institute, University of Toronto, 112 College Street, Room 70, Toronto, ON, M5G 1L6, Canada.
Mol Biotechnol. 2019 Jun;61(6):410-420. doi: 10.1007/s12033-019-00171-9.
Current developments in meta-data analysis and predictive computational models offer alternative routes for the identification of antibodies. In silico-based technologies and NGS data analysis from Ab phage-display selections offer expanded selections of Ab candidates. Accordingly, the identified de novo Abs with predicted selectivity for a target antigen must undergo rapid gene synthesis for downstream Ab characterizations. Here we describe a high-throughput strategy for the generation of synthetic Ab clones for expression as Fab proteins in Escherichia coli. Our approach utilizes simultaneous single-stranded site-directed mutagenesis of diversified Ab regions of a phagemid template with engineered complementary determining regions that contain multiple stop codon and restriction enzyme sites. Subsequently, we perform rapid screening of Ab DNA clones for correct gene assemblies by high-throughput Ab-phage protein expression screens. Identified sequences are corroborated by Sanger DNA sequencing analysis. In summary, our work describes a rapid and cost-effective platform for the high-throughput synthesis of synthetic Ab genes as Fab proteins for implementation into downstream protein validation pipelines.
当前，元数据分析和预测计算模型的发展为抗体的鉴定提供了替代途径。基于计算机的技术和 Ab 噬菌体展示选择的 NGS 数据分析提供了扩展的 Ab 候选物选择。因此，必须对具有针对靶抗原的预测选择性的新鉴定的 de novo Abs 进行快速基因合成，以进行下游 Ab 特性分析。在这里，我们描述了一种用于生成用于在大肠杆菌中表达为 Fab 蛋白的合成 Ab 克隆的高通量策略。我们的方法利用同时对噬菌体模板的多样化 Ab 区域进行单链定点诱变，该噬菌体模板包含带有多个终止密码子和限制酶位点的工程互补决定区。随后，我们通过高通量 Ab 噬菌体蛋白表达筛选快速筛选正确基因组装的 Ab DNA 克隆。通过 Sanger DNA 测序分析验证鉴定的序列。总之，我们的工作描述了一种快速且具有成本效益的平台，用于高通量合成作为 Fab 蛋白的合成 Ab 基因，以实施到下游蛋白质验证管道中。
