A general protocol for the generation of Nanobodies for structural biology

By E. Pardon, T. Laeremans, Sarah Triest, S. Rasmussen, Alexandre Wohlkönig, A. Ruf, S. Muyldermans, W. Hol, Brian K. Kobilka, J. Steyaert. 2014

750 citations recorded by the source corpus.

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.

Subfields: Molecular Biology, Radiology, Nuclear Medicine and Imaging

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Citations in the corpus

Structural activation of pro-inflammatory human cytokine IL-23 by cognate IL-23 receptor enables recruitment of the shared receptor IL-12Rβ1 (2017, methodology): “Single domain VHH camelid antibodies were expressed as described by (Pardon et al., 2014) in E. coli WK6.”
Nanobody cocktails potently neutralize SARS-CoV-2 D614G N501Y variant and protect mice (2021, neutral): “Nanobodies are ∼12 to 15 kDa in size, highly stable across a wide range of pH and temperature, display strong binding affinities to target proteins, and can be expressed with high yields in bacterial, yeast, and mammalian expression systems (27, 28).”
Noninvasive imaging of immune responses (2015, neutral): “Two VHHs, VHH7 (anti-mouse class II MHC) and VHHDC13 (anti-mouse CD11b) were generated following standard procedures (SI Materials and Methods) (10).”
Structure of the Pf12 and Pf41 heterodimeric complex of Plasmodium falciparum 6-cysteine proteins (2022, methodology): “Isolation of nanobodies against Pf12-Pf41 Biopanning for anti-Pf12-Pf41 nanobodies using phage display was performed as previously described with the following modifications (Pardon et al. 2014).”