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首页> 外文期刊>Proceedings of the National Academy of Sciences of the United States of America >APEx 2-hybrid, a quantitative protein-protein interaction assay for antibody discovery and engineering
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APEx 2-hybrid, a quantitative protein-protein interaction assay for antibody discovery and engineering

机译：APEx 2-hybrid，定量的蛋白质-蛋白质相互作用测定法，用于抗体发现和工程

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摘要

We have developed a bacterial system for the discovery of interacting proteins that, unlike other two-hybrid technologies, allows for the selection of protein pairs on the basis of affinity or expression. This technology relies on the anchored periplasmic expression (APEx) of one protein (bait) on the periplasmic side of the inner membrane of Escherichia coli and its interacting partner (prey) as a soluble, epitope-tagged, periplasmic protein. Upon removal of the outer membrane by spheroplasting, periplasmic proteins, including any unbound epitope-tagged prey, are released into the extracellular fluid. However, if the epitope-tagged prey can bind to the membrane-anchored bait, it remains associated with the cell and can be detected quantitatively by using fluorescent anti-epitope tag antibodies. Cells expressing prey.bait pairs exhibiting different affinities can be readily distinguished by flow cytometry. The utility of this technology, called APEx two-hybrid, was demonstrated in two demanding antibody engineering applications: First, single-chain variable fragment (scFvs) with increased affinity to the protective antigen of Bacillus anthracis were isolated from cells coexpressing libraries of scFv random mutants, together with endogenously expressed antigen. Second, APEx two-hybrid coupled with multicolor FACS analysis to account for protein expression was used for the selection of mutant Fab antibody fragments exhibiting improved expression in the bacterial periplasm.
机译：我们已经开发了一种用于发现相互作用蛋白的细菌系统，与其他两种杂交技术不同，该系统允许根据亲和力或表达选择蛋白对。这项技术依赖于一种蛋白（诱饵）在大肠杆菌内膜的周质侧及其相互作用的伴侣（猎物）上锚定的周质表达（APEx），它是一种可溶性的，被表位标记的周质蛋白。通过原生质球去除外膜后，周质蛋白（包括任何未结合的表位标记的猎物）被释放到细胞外液中。但是，如果带有表位标签的猎物可以与膜锚定的诱饵结合，则它仍与细胞结合并可以通过使用荧光抗表位标签抗体进行定量检测。表达表现出不同亲和力的诱饵对的细胞可以通过流式细胞仪轻松区分。在两项要求苛刻的抗体工程应用中证明了该技术的实用性，称为APEx两杂交：首先，从共表达scFv随机库的细胞中分离出对炭疽芽孢杆菌保护性抗原具有更高亲和力的单链可变片段（scFvs）。突变体，以及内源表达的抗原。其次，APEx两杂交结合多色FACS分析以说明蛋白质表达，用于筛选在细菌周质中表现出改善表达的突变Fab抗体片段。

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