Pardon Els, Laeremans Toon, Triest Sarah, Rasmussen Søren G F, Wohlkönig Alexandre, Ruf Armin, Muyldermans Serge, Hol Wim G J, Kobilka Brian K, Steyaert Jan
1] Structural Biology Brussels, Vrije Universiteit Brussel (VUB), Brussels, Belgium. [2] Structural Biology Research Center, Vlaams Instituut voor Biotechnologie (VIB), Brussels, Belgium.
Department of Neuroscience and Pharmacology, The Panum Institute, University of Copenhagen, Copenhagen, Denmark.
Nat Protoc. 2014 Mar;9(3):674-93. doi: 10.1038/nprot.2014.039. Epub 2014 Feb 27.
There is growing interest in using antibodies as auxiliary tools to crystallize proteins. Here we describe a general protocol for the generation of Nanobodies to be used as crystallization chaperones for the structural investigation of diverse conformational states of flexible (membrane) proteins and complexes thereof. Our technology has a competitive advantage over other recombinant crystallization chaperones in that we fully exploit the natural humoral response against native antigens. Accordingly, we provide detailed protocols for the immunization with native proteins and for the selection by phage display of in vivo-matured Nanobodies that bind conformational epitopes of functional proteins. Three representative examples illustrate that the outlined procedures are robust, making it possible to solve by Nanobody-assisted X-ray crystallography in a time span of 6-12 months.
将抗体用作蛋白质结晶辅助工具的兴趣与日俱增。在此，我们描述了一种通用方案，用于生成纳米抗体，作为结晶伴侣，用于研究柔性（膜）蛋白及其复合物的各种构象状态的结构。我们的技术相对于其他重组结晶伴侣具有竞争优势，因为我们充分利用了针对天然抗原的天然体液免疫反应。因此，我们提供了用天然蛋白进行免疫以及通过噬菌体展示选择结合功能蛋白构象表位的体内成熟纳米抗体的详细方案。三个代表性例子表明，所概述的程序是可靠的，使得在6至12个月的时间内通过纳米抗体辅助X射线晶体学解决问题成为可能。
