Generation of synthetic nanobodies against delicate proteins

Abstract

Here, we provide a protocol to generate synthetic nanobodies, known as sybodies, against any purified protein or protein complex within a 3-week period. Unlike methods that require animals for antibody generation, sybody selections are carried out entirely in vitro under controlled experimental conditions. This is particularly relevant for the generation of conformation-specific binders against labile membrane proteins or protein complexes and allows selections in the presence of non-covalent ligands. Sybodies are especially suited for cases where binder generation via immune libraries fails due to high sequence conservation, toxicity or insufficient stability of the target protein. The procedure entails a single round of ribosome display using the sybody libraries encoded by mRNA, followed by two rounds of phage display and binder identification by ELISA. The protocol is optimized to avoid undesired reduction in binder diversity and enrichment of non-specific binders to ensure the best possible selection outcome. Using the efficient fragment exchange (FX) cloning method, the sybody sequences are transferred from the phagemid to different expression vectors without the need to amplify them by PCR, which avoids unintentional shuffling of complementary determining regions. Using quantitative PCR (qPCR), the efficiency of each selection round is monitored to provide immediate feedback and guide troubleshooting. Our protocol can be carried out by any trained biochemist or molecular biologist using commercially available reagents and typically gives rise to 10–30 unique sybodies exhibiting binding affinities in the range of 500 pM–500 nM.

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Fig. 1: Sybody libraries.
Fig. 2: Sybody selection flowchart.
Fig. 3: Overview of genetic constructs and primers.
Fig. 4: Exemplary DNA gel of sybody pools.
Fig. 5: SEC analysis of sybodies.
Fig. 6: A conformation-specific sybody against the ABC transporter TM287/288.
Fig. 7: Structure of the KDEL receptor in complex with a sybody.

Data availability

All plasmids have been deposited on Addgene. The sybody libraries can be obtained from the authors via an academic material transfer agreement.

References

  1. 1.

    Ries, J., Kaplan, C., Platonova, E., Eghlidi, H. & Ewers, H. A simple, versatile method for GFP-based super-resolution microscopy via nanobodies. Nat. Methods 9, 582–584 (2012).

  2. 2.

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

  3. 3.

    Manglik, A., Kobilka, B. K. & Steyaert, J. Nanobodies to study G protein-coupled receptor structure and function. Annu. Rev. Pharmacol. Toxicol. 57, 19–37 (2017).

  4. 4.

    Harmansa, S., Alborelli, I., Bieli, D., Caussinus, E. & Affolter, M. A nanobody-based toolset to investigate the role of protein localization and dispersal in Drosophila. Elife 6, e22549 (2017).

  5. 5.

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

  6. 6.

    Kirchhofer, A. et al. Modulation of protein properties in living cells using nanobodies. Nat. Struct. Mol. Biol. 17, 133–138 (2010).

  7. 7.

    Bräuer, P. et al. Structural basis for pH-dependent retrieval of ER proteins from the Golgi by the KDEL receptor. Science 363, 1103–1107 (2019).

  8. 8.

    Kaur, H. et al. Identification of conformation-selective nanobodies against the membrane protein insertase BamA by an integrated structural biology approach. J. Biomol. NMR 73, 375–384 (2019).

  9. 9.

    Nevoltris, D. et al. Conformational nanobodies reveal tethered epidermal growth factor receptor involved in EGFR/ErbB2 predimers. ACS Nano 9, 1388–1399 (2015).

  10. 10.

    Vazquez-Lombardi, R. et al. Challenges and opportunities for non-antibody scaffold drugs. Drug Discov. Today 20, 1271–1283 (2015).

  11. 11.

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

  12. 12.

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

  13. 13.

    Kruse, A. C. et al. Activation and allosteric modulation of a muscarinic acetylcholine receptor. Nature 504, 101–106 (2013).

  14. 14.

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

  15. 15.

    Henry, K. A. & MacKenzie, C. R. Antigen recognition by single-domain antibodies: structural latitudes and constraints. MAbs 10, 815–826 (2018).

  16. 16.

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

  17. 17.

    Pardon, E. et al. A general protocol for the generation of Nanobodies for structural biology. Nat. Protoc. 9, 674–693 (2014).

  18. 18.

    Zimmermann, I. et al. Synthetic single domain antibodies for the conformational trapping of membrane proteins. Elife 7, e34317 (2018).

  19. 19.

    Bradbury, A. R. M., Sidhu, S., Dubel, S. & McCafferty, J. Beyond natural antibodies: the power of in vitro display technologies. Nat. Biotechnol. 29, 245–254 (2011).

  20. 20.

    Hutter, C. A. J. et al. The extracellular gate shapes the energy profile of an ABC exporter. Nat. Commun. 10, 2260 (2019).

  21. 21.

    Arbabi Ghahroudi, M., Desmyter, A., Wyns, L., Hamers, R. & Muyldermans, S. Selection and identification of single domain antibody fragments from camel heavy-chain antibodies. FEBS Lett. 414, 521–526 (1997).

  22. 22.

    Verheesen, P. et al. Reliable and controllable antibody fragment selections from Camelid non-immune libraries for target validation. Biochim. Biophys. Acta 1764, 1307–1319 (2006).

  23. 23.

    Olichon, A. & de Marco, A. Preparation of a naïve library of camelid single domain antibodies. Methods Mol. Biol. 911, 65–78 (2012).

  24. 24.

    Monegal, A. et al. Immunological applications of single-domain llama recombinant antibodies isolated from a naïve library. Protein Eng. Des. Sel. 22, 273–280 (2009).

  25. 25.

    Moutel, S. et al. NaLi-H1: a universal synthetic library of humanized nanobodies providing highly functional antibodies and intrabodies. Elife 5, e16228 (2016).

  26. 26.

    Geertsma, E. R. et al. Structure of a prokaryotic fumarate transporter reveals the architecture of the SLC26 family. Nat. Struct. Mol. Biol. 22, 803–808 (2015).

  27. 27.

    Egloff, P. et al. Engineered peptide barcodes for in-depth analyses of binding protein libraries. Nat. Methods 16, 421–428 (2019).

  28. 28.

    Ehrnstorfer, I. A., Geertsma, E. R., Pardon, E., Steyaert, J. & Dutzler, R. Crystal structure of a SLC11 (NRAMP) transporter reveals the basis for transition-metal ion transport. Nat. Struct. Mol. Biol. 21, 990–996 (2014).

  29. 29.

    Prado, N. D. et al. Inhibition of the myotoxicity induced by Bothrops jararacussu venom and isolated phospholipases A2 by specific camelid single-domain antibody fragments. PLoS One 11, e0151363 (2016).

  30. 30.

    Koch, K. et al. Selection of nanobodies with broad neutralizing potential against primary HIV-1 strains using soluble subtype C gp140 envelope trimers. Sci. Rep. 7, 8390 (2017).

  31. 31.

    Hussack, G., Arbabi-Ghahroudi, M., Mackenzie, C. R. & Tanha, J. Isolation and characterization of Clostridium difficile toxin-specific single-domain antibodies. Methods Mol. Biol. 911, 211–239 (2012).

  32. 32.

    McMahon, C. et al. Yeast surface display platform for rapid discovery of conformationally selective nanobodies. Nat. Struct. Mol. Biol. 25, 289–296 (2018).

  33. 33.

    Yan, J. R., Li, G. H., Hu, Y. H., Ou, W. J. & Wan, Y. K. Construction of a synthetic phage-displayed Nanobody library with CDR3 regions randomized by trinucleotide cassettes for diagnostic applications. J. Transl. Med. 12, 343 (2014).

  34. 34.

    Sircar, A., Sanni, K. A., Shi, J. & Gray, J. J. Analysis and modeling of the variable region of camelid single-domain antibodies. J. Immunol. 186, 6357–6367 (2011).

  35. 35.

    Dumoulin, M. et al. Single-domain antibody fragments with high conformational stability. Protein Sci. 11, 500–515 (2002).

  36. 36.

    Govaert, J. et al. Dual beneficial effect of interloop disulfide bond for single domain antibody fragments. J. Biol. Chem. 287, 1970–1979 (2012).

  37. 37.

    Ring, A. M. et al. Adrenaline-activated structure of beta2-adrenoceptor stabilized by an engineered nanobody. Nature 502, 575–579 (2013).

  38. 38.

    Kuhn, B. T. et al. Biotinylation of membrane proteins for binder selections. Methods Mol. Biol. 2127, 151–165 (2020).

  39. 39.

    Zahnd, C., Amstutz, P. & Plückthun, A. Ribosome display: selecting and evolving proteins in vitro that specifically bind to a target. Nat. Methods 4, 269–279 (2007).

  40. 40.

    Shimizu, Y. et al. Cell-free translation reconstituted with purified components. Nat. Biotechnol. 19, 751–755 (2001).

  41. 41.

    Geertsma, E. R. & Dutzler, R. A versatile and efficient high-throughput cloning tool for structural biology. Biochemistry 50, 3272–3278 (2011).

  42. 42.

    Geertsma, E. R. FX cloning: a simple and robust high-throughput cloning method for protein expression. Methods Mol. Biol. 1116, 153–164 (2014).

  43. 43.

    Sidhu, S. S., Lowman, H. B., Cunningham, B. C. & Wells, J. A. Phage display for selection of novel binding peptides. Methods Enzymol. 328, 333–363 (2000).

  44. 44.

    McCafferty, J., Griffiths, A. D., Winter, G. & Chiswell, D. J. Phage antibodies: filamentous phage displaying antibody variable domains. Nature 348, 552–554 (1990).

  45. 45.

    Zahnd, C., Sarkar, C. A. & Pluckthun, A. Computational analysis of off-rate selection experiments to optimize affinity maturation by directed evolution. Protein Eng. Des. Sel. 23, 175–184 (2010).

  46. 46.

    Seeger, M. A. et al. Design, construction, and characterization of a second-generation DARPin library with reduced hydrophobicity. Protein Sci. 22, 1239–1257 (2013).

  47. 47.

    Huber, T., Steiner, D., Röthlisberger, D. & Plückthun, A. In vitro selection and characterization of DARPins and Fab fragments for the co-crystallization of membrane proteins: the Na+-citrate symporter CitS as an example. J. Struct. Biol. 159, 206–221 (2007).

  48. 48.

    Garza, J. A., Taylor, A. B., Sherwood, L. J., Hart, P. J. & Hayhurst, A. Unveiling a drift resistant cryptotope within Marburgvirus nucleoprotein recognized by llama single-domain antibodies. Front. Immunol. 8, 1234 (2017).

  49. 49.

    Zabetakis, D., Anderson, G. P., Bayya, N. & Goldman, E. R. Contributions of the complementarity determining regions to the thermal stability of a single-domain antibody. PLoS One 8, e77678 (2013).

  50. 50.

    Hussack, G. et al. Neutralization of Clostridium difficile toxin A with single-domain antibodies targeting the cell receptor binding domain. J. Biol. Chem. 286, 8961–8976 (2011).

  51. 51.

    Casadaban, M. J. & Cohen, S. N. Analysis of gene control signals by DNA fusion and cloning in Escherichia coli. J. Mol. Biol. 138, 179–207 (1980).

  52. 52.

    Hohl, M., Briand, C., Grütter, M. G. & Seeger, M. A. Crystal structure of a heterodimeric ABC transporter in its inward-facing conformation. Nat. Struct. Mol. Biol. 19, 395–402 (2012).

  53. 53.

    Hohl, M. et al. Structural basis for allosteric cross-talk between the asymmetric nucleotide binding sites of a heterodimeric ABC exporter. Proc. Natl Acad. Sci. USA 111, 11025–11030 (2014).

  54. 54.

    Timachi, M. H. et al. Exploring conformational equilibria of a heterodimeric ABC transporter. Elife 6, e20236 (2017).

  55. 55.

    Lu, F. et al. Structure and mechanism of the uracil transporter UraA. Nature 472, 243–246 (2011).

  56. 56.

    Yu, X. et al. Dimeric structure of the uracil:proton symporter UraA provides mechanistic insights into the SLC4/23/26 transporters. Cell Res. 27, 1020–1033 (2017).

  57. 57.

    Cull, M. G. & Schatz, P. J. Biotinylation of proteins in vivo and in vitro using small peptide tags. Methods Enzymol. 326, 430–440 (2000).

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Acknowledgements

We wish to thank all members of the Seeger and Geertsma laboratoriess for scientific discussions. We acknowledge Jenifer Cuesta Bernal for critical reading. E.R.G. acknowledges financial support from the German Research Foundation via the Cluster of Excellence Frankfurt (Macromolecular Complexes) and the CRC807 (Transport and Communication across Biological Membranes). Work in the Seeger group was supported by an SNSF Professorship of the Swiss National Science Foundation (PP00P3_144823, to M.A.S.), an SNSF NRP 72 grant (407240_177368, to M.A.S.), an SNSF BRIDGE proof-of-concept grant (20B1-1_175192, to P.E.) and a BioEntrepreneur-Fellowship of the University of Zurich (BIOEF-17-002, to I.Z.). R.J.P.D, E.R.G., and M.A.S. acknowledge a grant from the Commission for Technology and Innovation CTI (16003.1 PFLS-LS). Work in the group of S.N. was supported by a Wellcome award (102890/Z/13/Z).

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Authors

Contributions

M.A.S., E.R.G. and R.J.P.D. conceived the sybody project. E.R.G. and M.A.S. designed the sybody library. I.Z. and P.E. established the sybody selection platform. C.A.J.H. established the ELISA setup. I.Z., C.A.J.H., B.T.K., P.B. and E.R.G. selected sybodies against protein targets. I.Z., S.N., E.R.G. and M.A.S. supervised students and postdocs. I.Z., B.T.K., E.R.G. and M.A.S. wrote the manuscript. P.E., C.A.J.H., S.N. and R.J.P.D. edited the manuscript.

Corresponding authors

Correspondence to Eric R. Geertsma or Markus A. Seeger.

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Competing interests

The authors declare competing financial interests. I.Z., P.E., R.J.P.D. and M.A.S. are co-founders and shareholders of Linkster Therapeutics AG.

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Peer review information Nature Protocols thanks Serge Muyldermans, Jamshid Tanha and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Key references using this protocol

Zimmermann, I. et al. Elife 7, e34317 (2018): https://doi.org/10.7554/eLife.34317

Hutter, C. et al. Nat. Commun. 10, 2260 (2019): https://doi.org/10.1038/s41467-019-09892-6

Bräuer, P. et al. Science 363, 1103–1107 (2019): https://doi.org/10.1126/science.aaw2859

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Zimmermann, I., Egloff, P., Hutter, C.A.J. et al. Generation of synthetic nanobodies against delicate proteins. Nat Protoc 15, 1707–1741 (2020). https://doi.org/10.1038/s41596-020-0304-x

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