Proteoliposome-based selection of a recombinant antibody fragment against the human M2 muscarinic acetylcholine receptor.
Proteoliposome-based selection of a recombinant antibody fragment against the human M2 muscarinic acetylcholine receptor.
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基于蛋白脂质体的抗人 M2 毒蕈碱乙酰胆碱受体重组抗体片段的选择。
DOI：
10.1089/mab.2014.0041
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发表时间：
2014
期刊：
影响因子：
--
通讯作者：
Nomura N.
中科院分区：
文献类型：
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作者：
Suharni;Nomura Y;Arakawa T;Hino T;Abe H;Nakada-Nakura Y;Sato Y;Iwanari H;Shiroishi M;Asada H;Shimamura T;Murata T;Kobayashi T;Hamakubo T;Iwata S;Nomura N.
针对人g蛋白偶联受体（gpcr）的抗体的开发取得了有限的成功，这主要归因于它们在洗涤剂溶解状态下的低稳定性。我们在这里描述了一种通常可以应用于选择噬菌体展示文库的方法，该文库是在脂质体中重组的人gpcr。这种方法的一个关键特点是生产生物素化的蛋白脂质体，这些蛋白脂质体可以固定在链霉亲和素偶联的微孔板或顺磁珠的表面，并用作抗体的结合靶标。作为一个例子，我们从免疫噬菌体文库中分离出一个单链Fv片段，该片段特异性地与人M2毒蕈碱乙酰胆碱受体结合，具有纳米摩尔亲和力。所选择的抗体片段可以识别洗涤剂溶解和膜包埋形式的GPCR，这表明它可能是GPCR结构和功能研究的潜在有价值的工具。利用蛋白脂质体作为免疫原和筛选诱饵，将有利于噬菌体展示技术在这类困难的膜蛋白中的应用。
The development of antibodies against human G-protein-coupled receptors (GPCRs) has achieved limited success, which has mainly been attributed to their low stability in a detergent-solubilized state. We herein describe a method that can generally be applied to the selection of phage display libraries with human GPCRs reconstituted in liposomes. A key feature of this approach is the production of biotinylated proteoliposomes that can be immobilized on the surface of streptavidin-coupled microplates or paramagnetic beads and used as a binding target for antibodies. As an example, we isolated a single chain Fv fragment from an immune phage library that specifically binds to the human M2 muscarinic acetylcholine receptor with nanomolar affinity. The selected antibody fragment recognized the GPCR in both detergent-solubilized and membrane-embedded forms, which suggests that it may be a potentially valuable tool for structural and functional studies of the GPCR. The use of proteoliposomes as immunogens and screening bait will facilitate the application of phage display to this difficult class of membrane proteins.