Protocol | Published:

A general protocol for the generation of Nanobodies for structural biology

Nature Protocols volume 9, pages 674693 (2014) | Download Citation

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.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Accessions

NCBI Reference Sequence

References

  1. 1.

    et al. Structure of a nanobody-stabilized active state of the β2 adrenoceptor. Nature 469, 175–180 (2011).

  2. 2.

    et al. Crystal structure of the β2 adrenergic receptor-Gs protein complex. Nature 477, 549–555 (2011).

  3. 3.

    et al. Nanobody mediated crystallization of an archeal mechanosensitive channel. PLoS ONE 8, e77984 (2013).

  4. 4.

    et al. SbsB structure and lattice reconstruction unveil Ca2+ triggered S-layer assembly. Nature 487, 119–122 (2012).

  5. 5.

    , , , & Crystal structure of a heterodimer of editosome interaction proteins in complex with two copies of a cross-reacting nanobody. Nucleic Acids Res. 40, 1828–1840 (2012).

  6. 6.

    et al. Structural and functional studies on the interaction of GspC and GspD in the type II secretion system. PLoS Pathog. 7, e1002228 (2011).

  7. 7.

    et al. Crystal structure of the intrinsically flexible addiction antidote MazE. J. Biol. Chem. 278, 28252–28257 (2003).

  8. 8.

    et al. Combining in situ proteolysis and microseed matrix screening to promote crystallization of PrPc-nanobody complexes. Protein Eng. Des. Sel. 24, 737–741 (2011).

  9. 9.

    et al. Atomic structure of a nanobody-trapped domain-swapped dimer of an amyloidogenic β2-microglobulin variant. Proc. Natl. Acad. Sci. USA 108, 1314–1319 (2011).

  10. 10.

    et al. Nanobodies raised against monomeric α-synuclein distinguish between fibrils at different maturation stages. J. Mol. Biol. 425, 2397–2411 (2013).

  11. 11.

    , & Recognition of antigens by single-domain antibody fragments: the superfluous luxury of paired domains. Trends Biochem. Sci. 26, 230–235 (2001).

  12. 12.

    Nanobodies: natural single-domain antibodies. Annu. Rev. Biochem. 82, 775–797 (2013).

  13. 13.

    et al. Potent enzyme inhibitors derived from dromedary heavy-chain antibodies. EMBO J. 17, 3512–3520 (1998).

  14. 14.

    et al. Molecular basis for the preferential cleft recognition by dromedary heavy-chain antibodies. Proc. Natl. Acad. Sci. USA 103, 4586–4591 (2006).

  15. 15.

    et al. A general protocol for the crystallization of membrane proteins for X-ray structural investigation. Nat. Protoc. 4, 619–637 (2009).

  16. 16.

    et al. Single domain antibodies from llama effectively and specifically block T cell ecto-ADP-ribosyltransferase ART2.2 in vivo. FASEB J. 21, 3490–3498 (2007).

  17. 17.

    et al. Genetic immunization for producing immunoglobulins against cell-associated antigens such as P2X7, CXCR7 or CXCR4. WO patent 2,010,070,145 (2010).

  18. 18.

    et al. Efficient inhibition of EGFR signaling and of tumour growth by antagonistic anti-EFGR Nanobodies. Cancer Immunol. Immunother. 56, 303–317 (2007).

  19. 19.

    et al. Crystal structure of a camel single-domain VH antibody fragment in complex with lysozyme. Nat. Struct. Biol. 3, 803–811 (1996).

  20. 20.

    et al. A single-domain antibody fragment in complex with RNase A: non-canonical loop structures and nanomolar affinity using two CDR loops. Structure 7, 361–370 (1999).

  21. 21.

    et al. Three camelid VHH domains in complex with porcine pancreatic α-amylase. Inhibition and versatility of binding topology. J. Biol. Chem. 277, 23645–23650 (2002).

  22. 22.

    et al. A camelid antibody fragment inhibits the formation of amyloid fibrils by human lysozyme. Nature 424, 783–788 (2003).

  23. 23.

    , , & Crystal structure of the N-terminal domain of the secretin GspD from ETEC determined with the assistance of a nanobody. Structure 17, 255–265 (2009).

  24. 24.

    et al. The structure of the C-terminal domain of the largest editosome interaction protein and its role in promoting RNA binding by RNA-editing ligase L2. Nucleic Acids Res. 40, 6966–6977 (2012).

  25. 25.

    et al. Structure of an early native-like intermediate of β2-microglobulin amyloidogenesis. Protein Sci. 22, 1349–1357 (2013).

  26. 26.

    et al. Structures of P-glycoprotein reveal its conformational flexibility and an epitope on the nucleotide-binding domain. Proc. Natl. Acad. Sci. USA 110, 13386–13391 (2013).

  27. 27.

    et al. Mapping the conformational space accessible to BACE2 using surface mutants and cocrystals with Fab fragments, Fynomers and Xaperones. Acta Crystallogr. D Biol. Crystallogr. 69, 1124–1137 (2013).

  28. 28.

    et al. Toward chaperone-assisted crystallography: protein engineering enhancement of crystal packing and X-ray phasing capabilities of a camelid single-domain antibody (VHH) scaffold. Protein Sci. 17, 1175–1187 (2008).

  29. 29.

    et al. Lactococcal bacteriophage p2 receptor-binding protein structure suggests a common ancestor gene with bacterial and mammalian viruses. Nat. Struct. Mol. Biol. 13, 85–89 (2006).

  30. 30.

    et al. Structures of a key interaction protein from the Trypanosoma brucei editosome in complex with single domain antibodies. J. Struct. Biol. 174, 124–136 (2011).

  31. 31.

    Engineering of recombinant crystallization chaperones. Curr. Opin. Struct. Biol. 19, 449–457 (2009).

  32. 32.

    & New concepts and aids to facilitate crystallization. Curr. Opin. Struct. Biol. 23, 409–416 (2013).

  33. 33.

    & Nanobody stabilization of G protein-coupled receptor conformational states. Curr. Opin. Struct. Biol. 21, 567–572 (2011).

  34. 34.

    et al. Structural flexibility of the Gαs α-helical domain in the β2-adrenoceptor Gs complex. Proc. Natl. Acad. Sci. USA 108, 16086–16091 (2011).

  35. 35.

    et al. Structure of a bacterial type IV secretion core complex at subnanometre resolution. EMBO J. 32, 1195–1204 (2013).

  36. 36.

    et al. Measuring cooperative Rev protein-protein interactions on Rev responsive RNA by fluorescence resonance energy transfer. RNA Biol. 8, 316–324 (2011).

  37. 37.

    et al. Targeting and tracing antigens in live cells with fluorescent nanobodies. Nat. Methods 3, 887–889 (2006).

  38. 38.

    et al. Conformational biosensors reveal GPCR signalling from endosomes. Nature 495, 534–538 (2013).

  39. 39.

    , , , & An intrabody based on a llama single-domain antibody targeting the N-terminal α-helical multimerization domain of HIV-1 rev prevents viral production. J. Biol. Chem. 285, 21768–21780 (2010).

  40. 40.

    Antibody-enabled small-molecule drug discovery. Nat. Rev. Drug Discov. 11, 519–525 (2012).

  41. 41.

    Structural Genomics Consortium. et al. Protein production and purification. Nat. Methods 5, 135–146 (2008).

  42. 42.

    , , , & Biophysical characterization of recombinant proteins: a key to higher structural genomics success. J. Struct. Biol. 172, 107–119 (2010).

  43. 43.

    et al. CXCR4 nanobodies (VHH-based single variable domains) potently inhibit chemotaxis and HIV-1 replication and mobilize stem cells. Proc. Natl. Acad. Sci. USA 107, 20565–20570 (2010).

  44. 44.

    et al. The development of activating and inhibiting camelid VHH domains against human protein kinase Cɛ. Eur. J. Pharm. Sci. 42, 332–339 (2011).

  45. 45.

    & Trapping moving targets with small molecules. Science 324, 213–215 (2009).

  46. 46.

    , & A case of convergence: why did a simple alternative to canonical antibodies arise in sharks and camels? PLoS Biol. 9, e1001120 (2011).

  47. 47.

    , , & Antibody repertoire development in camelids. Dev. Comp. Immunol. 30, 187–198 (2006).

  48. 48.

    et al. Beta-lactamase inhibitors derived from single-domain antibody fragments elicited in the camelidae. Antimicrob. Agents Chemother. 45, 2807–2812 (2001).

  49. 49.

    et al. Induction of immune responses and molecular cloning of the heavy chain antibody repertoire of Lama glama. J. Immunol. Methods 240, 185–195 (2000).

  50. 50.

    , , & Alpaca (Lama pacos) as a convenient source of recombinant camelid heavy chain antibodies (VHHs). J. Immunol. Methods 324, 13–25 (2007).

  51. 51.

    et al. A single-step procedure of recombinant library construction for the selection of efficiently produced llama VH binders directed against cancer markers. J. Immunol. Methods 350, 54–62 (2009).

  52. 52.

    Selecting and screening recombinant antibody libraries. Nat. Biotechnol. 23, 1105–1116 (2005).

  53. 53.

    & Affinity maturation of single-domain antibodies by yeast surface display. in Single-Domain Antibodies 431–443 (Springer, 2012).

  54. 54.

    , , & Isolation of antigen-binding camelid heavy chain antibody fragments (nanobodies) from an immune library displayed on the surface of Pichia pastoris. J. Biotechnol. 145, 93–98 (2010).

  55. 55.

    et al. Surface display of a single-domain antibody library on Gram-positive bacteria. Cell Mol. Life Sci. 70, 1081–1093 (2013).

  56. 56.

    , , & Selection and application of recombinant antibodies as sensors of Rab protein conformation. Methods Enzymol. 403, 135–153 (2005).

  57. 57.

    & Selection by phage display of single-domain antibodies specific to antigens in their native conformation. in Single-Domain Antibodies 81–104 (Springer, 2012).

  58. 58.

    Single-domain camel antibodies: current status. J. Biotechnol. 74, 277–302 (2001).

  59. 59.

    & DNA mismatch-repair in Escherichia coli counteracting the hydrolytic deamination of 5-methyl-cytosine residues. EMBO J. 6, 1809–1815 (1987).

  60. 60.

    , , , & Selection and identification of single-domain antibody fragments from camel heavy-chain antibodies. FEBS Lett. 414, 521–526 (1997).

  61. 61.

    et al. Structure and properties of a complex of α-synuclein and a single-domain camelid antibody. J. Mol. Biol. 402, 326–343 (2010).

  62. 62.

    et al. An improved protocol for rapid freezing of protein samples for long-term storage. Acta Crystallogr. D Biol. Crystallogr. 60, 203–204 (2004).

  63. 63.

    et al. Naturally occurring antibodies devoid of light chains. Nature 363, 446–448 (1993).

  64. 64.

    , , , & Application of monoclonal antibodies in functional and comparative investigations of heavy-chain immunoglobulins in new world camelids. Clin. Diagn. Lab. Immunol. 12, 380–386 (2005).

  65. 65.

    & The single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction: twenty-something years on. Nat. Protoc. 1, 581–585 (2006).

  66. 66.

    et al. Bace2 is a beta cell–enriched protease that regulates pancreatic beta cell function and mass. Cell Metab. 14, 365–377 (2011).

  67. 67.

    Amino and carboxyl terminal modifications to facilitate the production and purification of a G protein–coupled receptor. Anal. Biochem. 231, 269–271 (1995).

  68. 68.

    et al. Maltose-neopentyl glycol (MNG) amphiphiles for solubilization, stabilization and crystallization of membrane proteins. Nat. Methods 7, 1003–1008 (2010).

  69. 69.

    et al. A monomeric G protein–coupled receptor isolated in a high-density lipoprotein particle efficiently activates its G protein. Proc. Natl. Acad. Sci. USA 104, 7682–7687 (2007).

  70. 70.

    , , , & Nanobody-aided structure determination of the EpsI:EpsJ pseudopilin heterodimer from Vibrio vulnificus. J. Struct. Biol. 166, 8–15 (2009).

  71. 71.

    , & Panning phage libraries with lipoprotein particles expressing the target antigen. WO patent 2,011,083,141 (2011).

  72. 72.

    et al. Reactivity and applications of new amine reactive cross-linkers for mass spectrometric detection of protein-protein complexes. Anal. Chem. 82, 172–179 (2010).

Download references

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.

Author information

Affiliations

  1. Structural Biology Brussels, Vrije Universiteit Brussel (VUB), Brussels, Belgium.

    • Els Pardon
    • , Toon Laeremans
    • , Sarah Triest
    • , Alexandre Wohlkönig
    •  & Jan Steyaert
  2. Structural Biology Research Center, Vlaams Instituut voor Biotechnologie (VIB), Brussels, Belgium.

    • Els Pardon
    • , Toon Laeremans
    • , Sarah Triest
    • , Alexandre Wohlkönig
    • , Serge Muyldermans
    •  & Jan Steyaert
  3. Department of Neuroscience and Pharmacology, The Panum Institute, University of Copenhagen, Copenhagen, Denmark.

    • Søren G F Rasmussen
  4. Pharma Research and Early Development (pRED), Small Molecule Research, Discovery Technologies, F. Hoffmann-La Roche, Basel, Switzerland.

    • Armin Ruf
  5. Cellular and Molecular Immunology, VUB, Brussels, Belgium.

    • Serge Muyldermans
  6. Department of Biochemistry, Biomolecular Structure Center, School of Medicine, University of Washington, Seattle, Washington, USA.

    • Wim G J Hol
  7. Department of Molecular and Cellular Physiology, School of Medicine, Stanford University, Stanford, California, USA.

    • Brian K Kobilka

Authors

  1. Search for Els Pardon in:

  2. Search for Toon Laeremans in:

  3. Search for Sarah Triest in:

  4. Search for Søren G F Rasmussen in:

  5. Search for Alexandre Wohlkönig in:

  6. Search for Armin Ruf in:

  7. Search for Serge Muyldermans in:

  8. Search for Wim G J Hol in:

  9. Search for Brian K Kobilka in:

  10. Search for Jan Steyaert in:

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.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Jan Steyaert.

Integrated supplementary information

Supplementary information

PDF files

  1. 1.

    Supplementary Figure 1

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

  2. 2.

    Supplementary Figure 2

    Map and sequence information for phage display vector pMES4.

  3. 3.

    Supplementary Figure 3

    Map and sequence information for phage display vector pMESy4.

About this article

Publication history

Published

DOI

https://doi.org/10.1038/nprot.2014.039

Further reading

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.