title: A general protocol for the generation of Nanobodies for structural biology.
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.
authors.affiliation: 1] Structural Biology Brussels, Vrije Universiteit Brussel (VUB), Brussels, Belgium. [2] Structural Biology Research Center, Vlaams Instituut voor Biotechnologie (VIB), Brussels, Belgium.
authors.forename: Els
authors.initials: E
authors.lastname: Pardon
authors.affiliation: 1] Structural Biology Brussels, Vrije Universiteit Brussel (VUB), Brussels, Belgium. [2] Structural Biology Research Center, Vlaams Instituut voor Biotechnologie (VIB), Brussels, Belgium.
authors.forename: Toon
authors.initials: T
authors.lastname: Laeremans
authors.affiliation: 1] Structural Biology Brussels, Vrije Universiteit Brussel (VUB), Brussels, Belgium. [2] Structural Biology Research Center, Vlaams Instituut voor Biotechnologie (VIB), Brussels, Belgium.
authors.forename: Sarah
authors.initials: S
authors.lastname: Triest
authors.affiliation: Department of Neuroscience and Pharmacology, The Panum Institute, University of Copenhagen, Copenhagen, Denmark.
authors.forename: Søren G F
authors.initials: SG
authors.lastname: Rasmussen
authors.affiliation: 1] Structural Biology Brussels, Vrije Universiteit Brussel (VUB), Brussels, Belgium. [2] Structural Biology Research Center, Vlaams Instituut voor Biotechnologie (VIB), Brussels, Belgium.
authors.forename: Alexandre
authors.initials: A
authors.lastname: Wohlkönig
authors.affiliation: Pharma Research and Early Development (pRED), Small Molecule Research, Discovery Technologies, F. Hoffmann-La Roche, Basel, Switzerland.
authors.forename: Armin
authors.initials: A
authors.lastname: Ruf
authors.affiliation: 1] Structural Biology Research Center, Vlaams Instituut voor Biotechnologie (VIB), Brussels, Belgium. [2] Cellular and Molecular Immunology, VUB, Brussels, Belgium.
authors.forename: Serge
authors.initials: S
authors.lastname: Muyldermans
authors.affiliation: Department of Biochemistry, Biomolecular Structure Center, School of Medicine, University of Washington, Seattle, Washington, USA.
authors.forename: Wim G J
authors.initials: WG
authors.lastname: Hol
authors.affiliation: Department of Molecular and Cellular Physiology, School of Medicine, Stanford University, Stanford, California, USA.
authors.forename: Brian K
authors.initials: BK
authors.lastname: Kobilka
authors.affiliation: 1] Structural Biology Brussels, Vrije Universiteit Brussel (VUB), Brussels, Belgium. [2] Structural Biology Research Center, Vlaams Instituut voor Biotechnologie (VIB), Brussels, Belgium.
authors.forename: Jan
authors.initials: J
authors.lastname: Steyaert
mesh_terms: D000818:Animals; D001709:Biotechnology / Q000379:methods*; D002162:Camelus; D061505:Cell Surface Display Techniques; D003001:Cloning, Molecular; D003460:Crystallization / Q000379:methods*; D017931:DNA Primers / Q000235:genetics; D008958:Models, Molecular; D011487:Protein Conformation; D061905:Single-Domain Antibodies / Q000096:biosynthesis*
chemical_list: D017931:DNA Primers; D061905:Single-Domain Antibodies