Yeast display immunoprecipitation is a combinatorial library screening platform for the discovery and engineering of antibodies against membrane proteins using detergent-solubilized membrane fractions or cell lysates as antigen sources. Here, we present the extension of this method for the screening of antibodies that bind to membrane protein complexes, enabling discovery of antibodies that target antigens involved in a functional protein-protein interaction of interest. For this proof-of-concept study, we focused on the receptor-mediated endocytosis machinery at the blood-brain barrier, and adaptin 2 (AP-2) was chosen as the functional interaction hub. The goal of this study was to identify antibodies that bound to blood-brain barrier (BBB) membrane protein complexes containing AP-2. Screening of a nonimmune yeast display antibody library was carried out using detergent-solubilized BBB plasma membranes as an antigen pool, and antibodies that could interact with protein complexes containing AP-2 were identified. Downstream characterization of isolated antibodies confirmed targeting of proteins known to play important roles in membrane trafficking. This functional yeast display immunoprecipitation screen may be applied to other systems where antibodies against other functional classes of protein complexes are sought.

中文翻译：

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酵母展示免疫沉淀筛选，用于靶向发现针对膜蛋白复合物的抗体。

酵母展示免疫沉淀是一种组合文库筛选平台，用于使用去污剂溶解的膜组分或细胞裂解物作为抗原源来发现和工程化针对膜蛋白的抗体。在这里，我们提出了这种方法的扩展，用于筛选与膜蛋白复合物结合的抗体，从而能够发现针对参与感兴趣的功能性蛋白质-蛋白质相互作用的抗原的抗体。在这项概念验证研究中，我们重点关注血脑屏障处受体介导的内吞机制，并选择适应素 2 (AP-2) 作为功能相互作用中心。本研究的目的是鉴定与含有 AP-2 的血脑屏障 (BBB) 膜蛋白复合物结合的抗体。使用去污剂溶解的 BBB 质膜作为抗原库对非免疫酵母展示抗体库进行筛选，并鉴定出可以与含有 AP-2 的蛋白质复合物相互作用的抗体。分离抗体的下游表征证实了已知在膜运输中发挥重要作用的蛋白质的靶向性。这种功能性酵母展示免疫沉淀筛选可应用于寻求针对其他功能类别的蛋白质复合物的抗体的其他系统。