Generation of Conformation-Specific Antibody Fragments for Crystallization of the Multidrug Resistance Transporter MdfA
Generation of Conformation-Specific Antibody Fragments for Crystallization of the Multidrug Resistance Transporter MdfA
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DOI：
10.1007/978-1-4939-7454-2_7
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发表时间：
2018-01-01
期刊：
影响因子：
--
通讯作者：
Tanabe, Mikio
中科院分区：
文献类型：
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作者：
Jaenecke, Frank;Nakada-Nakura, Yoshiko;Tanabe, Mikio
膜蛋白晶体学的一个主要障碍是产生足以确定结构的衍射晶体。这通常不仅归因于获得毫克量的功能活性蛋白质的困难，还归因于其多种构象的内在灵活性。据报道，膜蛋白与抗体片段的共结晶是通过限制内在灵活性来提高膜蛋白晶体衍射质量的有效方法。分离识别天然膜蛋白单一构象的合适抗体片段并不是一项简单的任务。然而，通过系统的筛选方法，可以减少获得合适抗体片段的时间，从而减少获得衍射晶体的机会。在本章中，我们描述了生成 Fab 片段的协议，该片段识别来自大肠杆菌的主要促进子超家族 (MFS) 型 MDR 转运蛋白 MdfA 的天然构象。我们证实，使用 Fab 片段可有效稳定 MdfA 并改善其结晶特性。
A major hurdle in membrane protein crystallography is generating crystals diffracting sufficiently for structure determination. This is often attributed not only to the difficulty of obtaining functionally active protein in mg amounts but also to the intrinsic flexibility of its multiple conformations. The cocrystallization of membrane proteins with antibody fragments has been reported as an effective approach to improve the diffraction quality of membrane protein crystals by limiting the intrinsic flexibility. Isolating suitable antibody fragments recognizing a single conformation of a native membrane protein is not a straightforward task. However, by a systematic screening approach, the time to obtain suitable antibody fragments and consequently the chance of obtaining diffracting crystals can be reduced. In this chapter, we describe a protocol for the generation of Fab fragments recognizing the native conformation of a major facilitator superfamily (MFS)-type MDR transporter MdfA from Escherichia coli. We confirmed that the use of Fab fragments was efficient for stabilization of MdfA and improvement of its crystallization properties.