Laboratory of Biomolecular Research, Division of Biology and Chemistry, Paul Scherrer Institute, Villigen PSI, Switzerland.
Methods Mol Biol. 2020;2127:167-184. doi: 10.1007/978-1-0716-0373-4_12.
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.
纳米抗体是从小型骆驼科单链抗体衍生而来的小分子重组结合蛋白，已广泛应用于与膜蛋白相关的多个领域。它们可作为结晶过程中的分子伴侣，也可用于阻断或修饰蛋白质的活性，应用广泛。与传统抗体不同，纳米抗体结构简单，只有一条多肽链，因此可以直接进行克隆、文库构建和重组表达。纳米抗体的抗原结合部位呈小直径、楔形，这使它们能够与膜蛋白的凹陷和缝隙结合，且通常具有构象特异性，使其成为首选的分子伴侣类型。本研究描述了在大肠杆菌中重组生产纳米抗体及其纯化的简单方案。我们进一步扩展了纳米抗体的使用范围，描述了在其 C 末端扩大纳米抗体的方法，从而生成“巨抗体”，而不会干扰其原始特性。这些扩大的纳米抗体可作为结晶学中的分子伴侣，也可用于增加单颗粒电子冷冻显微镜中小目标的质量，而在此之前，由于纳米抗体的尺寸较小，它们在该领域的应用受到限制。
