Mazor Yariv, Van Blarcom Thomas, Iverson Brent L, Georgiou George
University of Texas, Austin, TX, USA.
Methods Mol Biol. 2009;525:217-39, xiv. doi: 10.1007/978-1-59745-554-1_11.
We have developed a technology for the facile isolation of full-length IgG antibodies with desired specificity from combinatorial libraries expressed in Escherichia coli. Full-length heavy and light chains are expressed from a bicistronic operon and are secreted into the periplasm where they assemble 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 disruption, clones expressing IgGs that specifically recognize fluorescently labeled antigen are selected by flow cytometry. This technique was used for the isolation of several IgGs with nanomolar affinities toward the protective antigen of Bacillus anthracis from immune libraries. High-throughput isolation of E. coli-derived full-length IgG can greatly expedite the discovery and production of antibodies for therapeutic and diagnostic applications.
我们开发了一种技术，可从在大肠杆菌中表达的组合文库中轻松分离出具有所需特异性的全长IgG抗体。全长重链和轻链从双顺反子操纵子表达，并分泌到周质中，在那里它们组装成无糖基化的IgG，这些IgG对抗原结合具有完全功能。内膜连接的Fc结合蛋白的表达用于捕获IgG分子并将它们锚定到细胞上。在外膜破坏后，通过流式细胞术选择表达特异性识别荧光标记抗原的IgG的克隆。该技术用于从免疫文库中分离出几种对炭疽芽孢杆菌保护性抗原有纳摩尔亲和力的IgG。高通量分离大肠杆菌来源的全长IgG可极大地加快用于治疗和诊断应用的抗体的发现和生产。
