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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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Figure 1: Workflow for the generation of conformational Nanobodies for structural biology.
Figure 2: Native gel analysis of Nanobodies interacting with the oligonucleotide-binding fold of the A1 protein of the editosome of the sleeping sickness parasite Trypanosoma brucei24.
Figure 3: Nb80 is a structural mimic of GαS and stabilizes the active-state conformation of β2AR.
Figure 4: Structure of the β2AR-Gs complex solved by Nanobody-enabled X-ray crystallography.

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Acknowledgements

We thank members and associates of the Steyaert, Muyldermans, Hol and Kobilka laboratories, past and present, for their assorted contributions over the years to this work. In particular, we acknowledge the contributions of N. Buys and Y.J. Park. The Steyaert laboratory was supported by the Fonds Wetenschappelijk Onderzoek-Vlaanderen through research grants G011110N and G049512N, Innoviris Brussels through the Impulse Life Science program BRGEOZ132, the Belgian Federal Science Policy Office through IAP7-40 and by the SBO program IWT120026 from the Flemish Agency for Innovation by Science and Technology. B.K.K. received support from US National Institutes of Health (NIH) grants R01NS028471 and R01GM083118 and from the Mathers Foundation. The research in the laboratory of W.G.J.H. was supported by the National Institute of Allergy and Infectious Diseases (NIAID) and the National Institute of General Medical Sciences (NIGMS) of the NIH under award numbers AI34501 and GM077418. S.T. received a doctoral fellowship from the Fonds Wetenschappelijk Onderzoek-Vlaanderen. S.G.F.R is supported by the Lundbeck Foundation.

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Authors

Contributions

J.S. developed the concept of Nanobody-assisted crystallography in collaboration with W.G.J.H. and B.K.K.; E.P., T.L., S.T., A.W. and J.S. worked out the protocol. E.P., T.L., S.M., W.G.J.H., B.K.K. and J.S. contributed to the Introduction. E.P., T.L., S.G.F.R., A.R., B.K.K. and J.S. performed the experiments described in the Anticipated Results and all authors participated in discussions on technical and conceptual aspects of the protocol and the editing of the manuscript.

Corresponding author

Correspondence to Jan Steyaert.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Strategies to amplify the Nanobody repertoire by PCR from PBL cDNA.

For each primer, we specify the locus of hybridization on the cDNAs encoding the heavy chains of the conventional antibodies (isotype IgG1), on the cDNAs encoding the heavy chain only antibodies of isotype IgG3 or on the cDNAs encoding the heavy chain only antibodies of isotype IgG2. Arrows representing the primers are not drawn to scale. H: hinge; CH2: constant domain 2; CH3: constant domain 3; Fr1 (to 4): framework 1 (to 4); CDR1 (to 3): complementarity determining region 1 (to 3); VH: variable domain of the heavy chain of a conventional antibody (IgG1); VHH: variable domain of a heavy chain only antibody (IgG2 or IgG3). Immunoglobulin domains with highly conserved DNA sequences amongst camelid species (leader, CH2, CH3) are pattern filled. (A) Primers CALL001 and CAL002 to amplify the variable domains of all camelid immunoglobulin heavy chains from PBL cDNA (Step 21) are depicted in black. The primers VHH-Back and VHH-For to amplify the Nanobody repertoire via nested PCR (Step 24) are depicted with open arrows. (B) Van der linden et al. developed dedicated primers to separately amplify the IgG2 and IgG3 isotypes from llama glama49 (C) Maass et al. developed primers to separately amplify the IgG2 and IgG3 isotypes from llama pacos 50(D) Kastelic et al. developed a set of primers that amplify all VHs and VHHs from llama51.

Supplementary Figure 2 Map and sequence information for phage display vector pMES4.

A. Map of phage display vector pMES4 (4501bp, Genbank GQ907248). Nanobodies can be cloned as PstI-Eco91I fragments (Step 26) in between the pelB sequence (pelB) coding for the secretion signal peptide of PelB and a 6xHis-tag (His-tag) followed by the hemagglutinin tag (HA-tag) and gene III of filamentous phage fd (fd geneIII). Other annotations are the lac promotor/operator (Plac/operator), the gene conferring ampicillin resistance (AmpR), the bacterial origin of replication (rep) and the f1 origin of replication (f1ori). The annealing sites for the forward sequencing primer (MP57) and the backward primer (GIII) are also indicated on the map (used in Steps 30 and 68). B. Nucleotide sequence and amino acid sequence of the Nanobody cloning site. An amber stopcodon (TAG) is located downstream of the His-tag.

Supplementary Figure 3 Map and sequence information for phage display vector pMESy4.

A. Map of phage display vector pMESy4 (4513bp; Genbank KF415192). Nanobodies can be cloned as PstI-Eco91I fragments (Step 26) in between the pelB sequence (pelB) coding for the secretion signal peptide of PelB and a 6xHis-tag (His-tag), followed by the CaptureSelectTM C-tag, the hemagglutinin tag (HA-tag) and gene III of filamentous phage fd (fd geneIII). Other annotations are the lac promotor/operator (Plac/operator), the gene conferring ampicillin resistance (AmpR), the bacterial origin of replication (rep) and the f1 origin of replication (f1ori). The annealing sites for the forward sequencing primer (MP57) and the backward primer (GIII) are also indicated on the map (used in Steps 30 and 68). B. Nucleotide sequence and amino acid sequence of the Nanobody cloning site. An amber stopcodon (TAG) is located downstream of the CaptureSelectTM C-tag.

Supplementary information

Supplementary Figure 1

Strategies to amplify the Nanobody repertoire by PCR from PBL cDNA. (PDF 408 kb)

Supplementary Figure 2

Map and sequence information for phage display vector pMES4. (PDF 565 kb)

Supplementary Figure 3

Map and sequence information for phage display vector pMESy4. (PDF 579 kb)

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Pardon, E., Laeremans, T., Triest, S. et al. A general protocol for the generation of Nanobodies for structural biology. Nat Protoc 9, 674–693 (2014). https://doi.org/10.1038/nprot.2014.039

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