Production and Application of Nanobodies for Membrane Protein Structural Biology
Production and Application of Nanobodies for Membrane Protein Structural Biology
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DOI：
10.1007/978-1-0716-0373-4_12
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发表时间：
2020-01-01
期刊：
影响因子：
--
通讯作者：
Schenck, Stephan
中科院分区：
文献类型：
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作者：
Brunner, Janine Denise;Schenck, Stephan
纳米抗体是来源于骆驼科动物单链抗体的小型重组结合剂，已成为与膜蛋白相关的多种学科中广泛使用的工具。它们在许多其他应用中用作结晶中的分子伴侣和蛋白质活性的阻断剂或改性剂。与经典抗体相比，它们作为单一多肽链的简单结构能够实现简单的克隆、文库生成和重组表达。抗原结合位点的小直径和尖的楔形形状是与膜蛋白的中空和裂缝结合的基础，并使纳米抗体通常具有构象特异性，使其成为优选类型的分子伴侣。在这里，我们描述了一个简单的协议，重组生产的纳米抗体在大肠杆菌。大肠杆菌中进行纯化。我们通过描述一种在不干扰其原始特征的情况下在其C-末端扩大纳米抗体以产生“巨抗体”的程序来进一步扩展当前的使用方法。这些放大的纳米抗体扩展了作为晶体学中的伴侣的应用，并且可以用于增加单颗粒电子冷冻显微镜中的小目标的质量，这是一个纳米抗体由于其小尺寸而迄今为止仅具有有限效果的领域。
Nanobodies, small recombinant binders derived from camelid single chain antibodies, have become widely used tools in a diversity of disciplines related to membrane proteins. They are applied as chaperones in crystallization and blockers or modifiers of protein activity among numerous other applications. Their simple architecture as a single polypeptide chain, in contrast to classical antibodies, enables straightforward cloning, library generation, and recombinant expression. The small diameter and the pointed wedge-like shape of the antigen-binding site underlies binding to hollows and crevices of membrane proteins and renders nanobodies often conformation specific making them a preferred type of chaperone. Here we describe a simple protocol for the recombinant production of nanobodies in E. coli and their purification. We expand the current repertoire of usage further by describing a procedure for enlarging nanobodies on their C-terminal end to generate "macrobodies," without interfering with their original characteristics. These enlarged nanobodies extend the application as a chaperone in crystallography and can serve to increase the mass for small targets in single particle electron cryo-microscopy, a field where nanobodies had so far only limited effect because of their small size.