A general protocol for the generation of Nanobodies for structural Biology

Els Pardon; Toon Laeremans; Sarah Triest; Soren G. F. Rasmussen; Armin Ruf; Serge Muyldermans; Wim G. J. Hol; Brian K Kobilka; Jan Steyaert

doi:10.1038/nprot.2014.039

A general protocol for the generation of Nanobodies for structural Biology

Els Pardon, Toon Laeremans, Sarah Triest, Soren G. F. Rasmussen, Armin Ruf, Serge Muyldermans, Wim G. J. Hol, Brian K Kobilka, Jan Steyaert

Department of Bio-engineering Sciences

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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.

Original language	English
Pages (from-to)	674-693
Number of pages	20
Journal	Nature Protocols
Volume	9
DOIs	https://doi.org/10.1038/nprot.2014.039
Publication status	Published - 27 Feb 2014

Keywords

Nanobodies

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nprot.2014.039Final published version, 1.22 MB

http://www.nature.com/nprot/journal/v9/n3/abs/nprot.2014.039.html

SRP13: Strategic Research Programme: Structure and dynamics of macromolecular complexes in health and disease
Steyaert, J., Remaut, H. K., Loris, R., Efremov, R., Steyaert, J., Loris, R. & Remaut, H. K.
1/11/12 → 31/10/24
Project: Fundamental

Cite this

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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.",

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AU - Pardon, Els

AU - Laeremans, Toon

AU - Triest, Sarah

AU - Rasmussen, Soren G. F.

AU - Ruf, Armin

AU - Muyldermans, Serge

AU - Hol, Wim G. J.

AU - Kobilka, Brian K

AU - Steyaert, Jan

PY - 2014/2/27

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N2 - 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.

AB - 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.

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JF - Nature Protocols

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A general protocol for the generation of Nanobodies for structural Biology

Abstract

Keywords

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Projects

SRP13: Strategic Research Programme: Structure and dynamics of macromolecular complexes in health and disease

Cite this