Mazor Yariv, Van Blarcom Thomas, Iverson Brent L, Georgiou George
Institute for Cellular and Molecular Biology, University of Texas at Austin, Austin, Texas 78712, USA.
Nat Protoc. 2008;3(11):1766-77. doi: 10.1038/nprot.2008.176.
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.
在此，我们描述了一种从展示于大肠杆菌上的抗体库中筛选全长IgG抗体的方案。在此所述方法中，重链和轻链全长在周质中组装成无糖基化的IgG，其对抗原结合具有完全功能。利用内膜系留Fc结合蛋白的表达来捕获IgG分子并将其锚定到细胞上。在外膜通透化后，通过多轮荧光激活细胞分选来选择荧光标记的配体结合文库克隆。通常可在3-4周内获得一整套全面的IgG克隆，这一时间尺度与大多数流行的抗体展示技术相当。分离出的抗体在细菌中表达良好，每个结合位点的亲和力在纳摩尔范围内。
