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Nat Protoc. 2014 Mar;9(3):674-93. doi: 10.1038/nprot.2014.039. Epub 2014 Feb 27.

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

Author information

1
1] Structural Biology Brussels, Vrije Universiteit Brussel (VUB), Brussels, Belgium. [2] Structural Biology Research Center, Vlaams Instituut voor Biotechnologie (VIB), Brussels, Belgium.
2
Department of Neuroscience and Pharmacology, The Panum Institute, University of Copenhagen, Copenhagen, Denmark.
3
Pharma Research and Early Development (pRED), Small Molecule Research, Discovery Technologies, F. Hoffmann-La Roche, Basel, Switzerland.
4
1] Structural Biology Research Center, Vlaams Instituut voor Biotechnologie (VIB), Brussels, Belgium. [2] Cellular and Molecular Immunology, VUB, Brussels, Belgium.
5
Department of Biochemistry, Biomolecular Structure Center, School of Medicine, University of Washington, Seattle, Washington, USA.
6
Department of Molecular and Cellular Physiology, School of Medicine, Stanford University, Stanford, California, USA.

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.

PMID:
24577359
PMCID:
PMC4297639
DOI:
10.1038/nprot.2014.039
[Indexed for MEDLINE]
Free PMC Article

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