Biotinylation of Membrane Proteins for Binder Selections
Biotinylation of Membrane Proteins for Binder Selections
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DOI：
10.1007/978-1-0716-0373-4_11
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发表时间：
2020-01-01
期刊：
影响因子：
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通讯作者：
Geertsma, Eric R.
中科院分区：
文献类型：
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作者：
Kuhn, Benedikt T.;Zimmermann, Iwan;Geertsma, Eric R.
蛋白质的选择性固定是选择结合蛋白（例如抗体）的重要步骤。固定化策略决定了目标蛋白如何呈现给结合剂，从而直接影响实验结果。由于膜蛋白固有的稳定性缺乏和暴露的亲水表面有限，这给膜蛋白带来了特殊的挑战。在这里，我们详细介绍了基于强生物素-亲和素相互作用的膜蛋白选择性固定的方法，并特别关注其在纳米抗体和sybodies选择中的应用。我们讨论了产生等摩尔生物素与靶蛋白比例的挑战和好处。
The selective immobilization of proteins represents an essential step in the selection of binding proteins such as antibodies. The immobilization strategy determines how the target protein is presented to the binders and thereby directly affects the experimental outcome. This poses specific challenges for membrane proteins due to their inherent lack of stability and limited exposed hydrophilic surfaces. Here we detail methodologies for the selective immobilization of membrane proteins based on the strong biotin-avidin interaction and with a specific focus on its application for the selection of nanobodies and sybodies. We discuss the challenges in generating and benefits of obtaining an equimolar biotin to target-protein ratio.