Research and Development, CSL Limited, 30 Flemington Road, Parkville, Victoria 3010, Australia.
Nucleic Acids Res. 2014 Feb;42(4):e26. doi: 10.1093/nar/gkt1142. Epub 2013 Nov 18.
We describe a novel cloning method, referred to as insert-tagged (InTag) positive selection, for the rapid one-step reformatting of phage-displayed antibody fragments to full-length immunoglobulin Gs (IgGs). InTag positive selection enables recombinant clones of interest to be directly selected without cloning background, bypassing the laborious process of plating out cultures and colony screening and enabling the cloning procedure to be automated and performed in a high-throughput format. This removes a significant bottleneck in the functional screening of phage-derived antibody candidates and enables a large number of clones to be directly reformatted into IgG without the intermediate step of Escherichia coli expression and testing of soluble antibody fragments. The use of InTag positive selection with the Dyax Fab-on-phage antibody library is demonstrated, and optimized methods for the small-scale transient expression of IgGs at high levels are described. InTag positive selection cloning has the potential for wide application in high-throughput DNA cloning involving multiple inserts, markedly improving the speed and quality of selections from protein libraries.
我们描述了一种新颖的克隆方法，称为插入标记（InTag）正向选择，用于快速一步将噬菌体展示的抗体片段重构成全长免疫球蛋白 G（IgG）。InTag 正向选择可直接选择感兴趣的重组克隆，而无需克隆背景，避免了繁琐的培养物平板和菌落筛选过程，并使克隆过程自动化，并以高通量格式进行。这消除了噬菌体衍生抗体候选物功能筛选中的一个主要瓶颈，并能够直接将大量克隆重构成 IgG，而无需大肠杆菌表达和可溶性抗体片段测试的中间步骤。使用 Dyax Fab-on-phage 抗体文库展示了 InTag 正向选择，还描述了用于高水平瞬时表达 IgG 的小规模优化方法。InTag 正向选择克隆具有广泛应用于涉及多个插入物的高通量 DNA 克隆的潜力，显著提高了从蛋白质文库中进行选择的速度和质量。
