A general protocol for the generation of Nanobodies for structural biology

Els Pardon; Toon Laeremans; Sarah Triest; Søren Gøgsig Faarup Rasmussen; Alexandre Wohlkönig; 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, Søren Gøgsig Faarup Rasmussen, Alexandre Wohlkönig, Armin Ruf, Serge Muyldermans, Wim G J Hol, Brian K Kobilka, Jan Steyaert

Neuropharm and Genetics

285 Citationer (Scopus)

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.

Originalsprog	Engelsk
Tidsskrift	Nature Protocols (Print)
Vol/bind	9
Udgave nummer	3
Sider (fra-til)	674-93
Antal sider	20
ISSN	1754-2189
DOI	https://doi.org/10.1038/nprot.2014.039
Status	Udgivet - mar. 2014

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10.1038/nprot.2014.039

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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.",

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AU - Pardon, Els

AU - Laeremans, Toon

AU - Triest, Sarah

AU - Rasmussen, Søren Gøgsig Faarup

AU - Wohlkönig, Alexandre

AU - Ruf, Armin

AU - Muyldermans, Serge

AU - Hol, Wim G J

AU - Kobilka, Brian K

AU - Steyaert, Jan

PY - 2014/3

Y1 - 2014/3

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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DO - 10.1038/nprot.2014.039

M3 - Journal article

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JO - Nature Protocols

JF - Nature Protocols

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A general protocol for the generation of Nanobodies for structural biology

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