A general protocol for the generation of Nanobodies for structural biology.

TitleA general protocol for the generation of Nanobodies for structural biology.
Publication TypeJournal Article
Year of Publication2014
AuthorsPardon, E., Laeremans T., Triest S., Rasmussen S. G. F., Wohlkonig A., Ruf A., Muyldermans S., Hol W. G. J., Kobilka B. K., and Steyaert J.
JournalNat Protoc
Volume9
Issue3
Pagination674-93
Date Published2014 Mar
ISSN1750-2799
KeywordsAnimals, Biotechnology, Camels, Cell Surface Display Techniques, Cloning, Molecular, Crystallization, DNA Primers, Models, Molecular, Protein Conformation, Single-Domain Antibodies
Abstract

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.

DOI10.1038/nprot.2014.039
Alternate JournalNat Protoc
PubMed ID24577359
PubMed Central IDPMC4297639
Grant ListR01GM083118 / GM / NIGMS NIH HHS / United States
R56 AI034501 / AI / NIAID NIH HHS / United States
R01 GM077418 / GM / NIGMS NIH HHS / United States
GM077418 / GM / NIGMS NIH HHS / United States
AI34501 / AI / NIAID NIH HHS / United States
R01 GM083118 / GM / NIGMS NIH HHS / United States
R01NS028471 / NS / NINDS NIH HHS / United States
R01 AI034501 / AI / NIAID NIH HHS / United States
R01 NS028471 / NS / NINDS NIH HHS / United States
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