Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA, USA.
Department of Neuroscience, University of Copenhagen, Copenhagen, Denmark.
Nat Struct Mol Biol. 2018 Mar;25(3):289-296. doi: 10.1038/s41594-018-0028-6. Epub 2018 Feb 12.
Camelid single-domain antibody fragments ('nanobodies') provide the remarkable specificity of antibodies within a single 15-kDa immunoglobulin V domain. This unique feature has enabled applications ranging from use as biochemical tools to therapeutic agents. Nanobodies have emerged as especially useful tools in protein structural biology, facilitating studies of conformationally dynamic proteins such as G-protein-coupled receptors (GPCRs). Nearly all nanobodies available to date have been obtained by animal immunization, a bottleneck restricting many applications of this technology. To solve this problem, we report a fully in vitro platform for nanobody discovery based on yeast surface display. We provide a blueprint for identifying nanobodies, demonstrate the utility of the library by crystallizing a nanobody with its antigen, and most importantly, we utilize the platform to discover conformationally selective nanobodies to two distinct human GPCRs. To facilitate broad deployment of this platform, the library and associated protocols are freely available for nonprofit research.
骆驼科单域抗体片段（“纳米抗体”）在单个 15kDa 免疫球蛋白 V 结构域内提供了抗体的显著特异性。这一独特的特性使得纳米抗体在从生化工具到治疗剂等各个领域得到了应用。纳米抗体在蛋白质结构生物学中已成为特别有用的工具，促进了对构象动态蛋白（如 G 蛋白偶联受体（GPCR））的研究。迄今为止，几乎所有可用的纳米抗体都是通过动物免疫获得的，这一瓶颈限制了该技术的许多应用。为了解决这个问题，我们报告了一种基于酵母表面展示的完全体外纳米抗体发现平台。我们提供了一种鉴定纳米抗体的蓝图，通过对与抗原结合的纳米抗体进行结晶证明了文库的实用性，最重要的是，我们利用该平台发现了两种不同的人 GPCR 的构象选择性纳米抗体。为了促进该平台的广泛应用，该文库和相关协议可供非营利性研究免费使用。
