E-clonal antibodies: selection of full-length IgG antibodies using bacterial periplasmic display
E-clonal antibodies: selection of full-length IgG antibodies using bacterial periplasmic display
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DOI：
10.1038/nprot.2008.176
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发表时间：
2008-01-01
期刊：
影响因子：
14.8
通讯作者：
Georgiou, George
中科院分区：
文献类型：
--
作者：
Mazor, Yariv;Van Blarcom, Thomas;Georgiou, George
在这里，我们描述了一个协议的选择全长IgG抗体的剧目显示在大肠杆菌。在本文所述的方法中，全长重链和轻链在周质中组装成完全具有抗原结合功能的无糖基化IgG。内膜栓系的Fc结合蛋白的表达用于捕获IgG分子并将其锚于细胞。外膜透化后，通过多轮荧光激活细胞分选选择荧光标记的配体结合文库克隆。一组完整的IgG克隆的选择通常可以在3-4周内获得，这是与最流行的抗体展示技术相当的时间尺度。分离的抗体在细菌中良好表达，并且表现出纳摩尔范围内的每个结合位点的亲和力。
Here we describe a protocol for the selection of full-length IgG antibodies from repertoires displayed on Escherichia coli. In the method described here, full-length heavy and light chains are assembled in the periplasm into aglycosylated IgGs that are fully functional for antigen binding. Expression of an inner membrane-tethered Fc-binding protein is used to capture the IgG molecules and anchor them to the cell. Following outer-membrane permeabilization, fluorescently labeled ligand-binding library clones are selected by multiple rounds of fluorescence-activated cell sorting. Selection of a comprehensive set of IgG clones can typically be obtained within 3-4 weeks, a timescale that is comparable with most prevalent antibody display technologies. The isolated antibodies are well expressed in bacteria and exhibit affinities per binding site in the nanomolar range.