A Novel Nanobody Scaffold Optimized for Bacterial Expression and Suitable for the Construction of Ribosome Display Libraries

  • Davide FerrariEmail author
  • Valentina Garrapa
  • Massimo Locatelli
  • Angelo Bolchi
Original paper


Single-domain antigen-binding fragments of camelid antibodies, known as VHHs or nanobodies, are widely used affinity reagents. However, their production involving animal immunization is time- and resource-intensive. Starting from a sequence dataset of llama VHHs, we designed a novel scaffold, based on conserved framework sequences, suitable for bacterial nanobody expression and synthetic library construction. The consensus scaffold was validated by grafting the CDRs from two known nanobodies. While maintaining their binding properties, the two chimeric nanobodies showed higher levels of expression and solubility in E. coli when compared to the corresponding wild types. A proof-of-concept synthetic combinatorial library, suitable for ribosome display (RD) selection, was obtained by encoding three randomized complementarity determining regions within the consensus framework. The library, made of linear DNA fragments, has an estimated complexity of > 1012 that is three orders of magnitude higher than common phage display libraries. The bacterial expression of several library clones showed a high production of soluble recombinant proteins. The high complexity of the library, confirmed by sequencing of a subset of clones, as well as a preliminary RD selection of a maltose binding protein binder, indicated this approach as a starting point in the construction of synthetic combinatorial libraries to be used as animal-free tools for the low-cost selection of target-specific nanobodies.


Nanobody VHH Scaffold Library Ribosome display 



V.G. is the recipient of a PhD student fellowship from the Fondazione Cariparma. This work was supported in part by a grant from Regione Emilia-Romagna, Italy (Programma di Ricerca Regione-Università 2010-2012; PRUa1RI-2012-006). Support from the Interuniversity Consortium for Biotechnologies (CIB) and European Molecular Biology Organization (EMBO) is also gratefully acknowledged.


This work has benefited from the equipment and framework of the COMP-HUB Initiative, funded by the ‘Departments of Excellence’ program of the Italian Ministry for Education, University, and Research (MIUR, 2018-2022).

Compliance with Ethical Standards

Conflict of interest

The authors declare no financial or commercial conflict of interest.

Supplementary material

12033_2019_224_MOESM1_ESM.docx (1.3 mb)
Supplementary material 1 (DOCX 1315 kb)


  1. 1.
    Hamers-Casterman, C., Atarhouch, T., Muyldermans, S., Robinson, G., Hammers, C., Songa, E. B., et al. (1993). Naturally occurring antibodies devoid of light chains. Nature,363(6428), 446–448. Scholar
  2. 2.
    Oliveira, S., Heukers, R., Sornkom, J., Kok, R. J., & Van Bergen En Henegouwen, P. M. P. (2013). Targeting tumors with nanobodies for cancer imaging and therapy. Journal of Controlled Release. Scholar
  3. 3.
    Smolarek, D., Bertrand, O., & Czerwinski, M. (2012). Variable fragments of heavy chain antibodies (VHHs): A new magic bullet molecule of medicine? Postepy Higieny i Medycyny Doswiadczalnej,66, 348–358. Scholar
  4. 4.
    Muyldermans, S., Baral, T. N., Retamozzo, V. C., De Baetselier, P., De Genst, E., Kinne, J., et al. (2009). Camelid immunoglobulins and nanobody technology. Veterinary Immunology and Immunopathology. Scholar
  5. 5.
    Harmsen, M. M., & De Haard, H. J. (2007). Properties, production, and applications of camelid single-domain antibody fragments. Applied Microbiology and Biotechnology. Scholar
  6. 6.
    Lauwereys, M., Ghahroudi, M. A., Desmyter, A., Genst, E. De, Wyns, L., & Muyldermans, S. (1998). Potent enzyme inhibitors derived from dromedary heavy-chain antibodies. Early Intervention in Psychiatry,17(13), 3512–3520.Google Scholar
  7. 7.
    De Genst, E., Silence, K., Decanniere, K., Conrath, K., Loris, R., Kinne, J., et al. (2006). Molecular basis for the preferential cleft recognition by dromedary heavy-chain antibodies. Proceedings of the National Academy of Sciences,103(12), 4586–4591. Scholar
  8. 8.
    Chakravarty, R., Goel, S., & Cai, W. (2014). Nanobody: The “magic bullet” for molecular imaging? Theranostics. Scholar
  9. 9.
    Hassanzadeh-Ghassabeh, G., Devoogdt, N., De Pauw, P., Vincke, C., & Muyldermans, S. (2013). Nanobodies and their potential applications. Nanomedicine. Scholar
  10. 10.
    De Meyer, T., Muyldermans, S., & Depicker, A. (2014). Nanobody-based products as research and diagnostic tools. Trends in Biotechnology. Scholar
  11. 11.
    Desmyter, A., Spinelli, S., Roussel, A., & Cambillau, C. (2015). Camelid nanobodies: Killing two birds with one stone. Current Opinion in Structural Biology,32, 1–8. Scholar
  12. 12.
    Olichon, A., & De Marco, A. (2012). Preparation of a naïve library of camelid single domain antibodies. Methods in Molecular Biology. Scholar
  13. 13.
    Pardon, E., Laeremans, T., Triest, S., Rasmussen, S. G. F., Wohlkönig, A., Ruf, A., et al. (2014). A general protocol for the generation of Nanobodies for structural biology. Nature Protocols. Scholar
  14. 14.
    Verheesen, P., Roussis, A., de Haard, H. J., Groot, A. J., Stam, J. C., den Dunnen, J. T., et al. (2006). Reliable and controllable antibody fragment selections from Camelid non-immune libraries for target validation. Biochimica et Biophysica Acta - Proteins and Proteomics. Scholar
  15. 15.
    Monegal, A., Ami, D., Martinelli, C., Huang, H., Aliprandi, M., Capasso, P., et al. (2009). Immunological applications of single-domain llama recombinant antibodies isolated from a naïve library. Protein Engineering, Design & Selection. Scholar
  16. 16.
    Kehoe, J. W., & Kay, B. K. (2005). Filamentous phage display in the new millennium. Chemical Reviews. Scholar
  17. 17.
    Moutel, S., Bery, N., Bernard, V., Keller, L., Lemesre, E., De Marco, A., et al. (2016). NaLi-H1: A universal synthetic library of humanized nanobodies providing highly functional antibodies and intrabodies. eLife,5, 1–31. Scholar
  18. 18.
    McMahon, C., Baier, A. S., Pascolutti, R., Wegrecki, M., Zheng, S., Ong, J. X., et al. (2018). Yeast surface display platform for rapid discovery of conformationally selective nanobodies. Nature Structural & Molecular Biology,25(3), 289–296. Scholar
  19. 19.
    Koide, A., Tereshko, V., Uysal, S., Margalef, K., Kossiakoff, A. A., & Koide, S. (2007). Exploring the capacity of minimalist protein interfaces: Interface energetics and affinity maturation to picomolar KD of a single-domain antibody with a flat paratope. Journal of Molecular Biology. Scholar
  20. 20.
    Plückthun, A. (2012). Ribosome display: A perspective. Methods in Molecular Biology. Scholar
  21. 21.
    Yau, K. Y. F., Groves, M. A. T., Li, S., Sheedy, C., Lee, H., Tanha, J., et al. (2003). Selection of hapten-specific single-domain antibodies from a non-immunized llama ribosome display library. Journal of Immunological Methods,281(1–2), 161–175. Scholar
  22. 22.
    Li, R., Kang, G., Hu, M., & Huang, H. (2019). Ribosome display: A potent display technology used for selecting and evolving specific binders with desired properties. Molecular Biotechnology,61(1), 60–71. Scholar
  23. 23.
    Schaffitzel, C., Hanes, J., Jermutus, L., & Plückthun, A. (1999). Ribosome display: An in vitro method for selection and evolution of antibodies from libraries. Journal of Immunological Methods. Scholar
  24. 24.
    Sambrook, J., & W Russell, D. (2001). Molecular cloning: A laboratory manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press.Google Scholar
  25. 25.
    Gorlani, A., Adams, H., Hermans, P., & Verrips, T. (2011). Antibody engineering reveals the important role of J segments in the production efficiency of llama single-domain antibodies in Saccharomyces cerevisiae. Protein Engineering, Design & Selection. Scholar
  26. 26.
    Crameri, A., Whitehorn, E. A., Tate, E., & Stemmer, W. P. C. (1996). Improved green fluorescent protein by molecular evolution using DNA shuffling. Nature Biotechnology. Scholar
  27. 27.
    Amstutz, P., Binz, H. K., Parizek, P., Stumpp, M. T., Kohl, A., Grütter, M. G., et al. (2005). Intracellular kinase inhibitors selected from combinatorial libraries of designed ankyrin repeat proteins. Journal of Biological Chemistry. Scholar
  28. 28.
    Hanes, J., & Plückthun, A. (1997). In vitro selection and evolution of functional proteins by using ribosome display. Proceedings of the National academy of Sciences of the United States of America. Scholar
  29. 29.
    Huber, T., Steiner, D., Röthlisberger, D., & Plückthun, A. (2007). 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. Journal of Structural Biology. Scholar
  30. 30.
    Zahnd, C., Amstutz, P., & Plückthun, A. (2007). Ribosome display: Selecting and evolving proteins in vitro that specifically bind to a target. Nature Methods. Scholar
  31. 31.
    Milovnik, P., Ferrari, D., Sarkar, C. A., & Plückthun, A. (2009). Selection and characterization of DARPins specific for the neurotensin receptor 1. Protein Engineering, Design & Selection,22(6), 357–366. Scholar
  32. 32.
    Sehr, P., Zumbach, K., & Pawlita, M. (2001). A generic capture ELISA for recombinant proteins fused to glutathione S-transferase: Validation for HPV serology. Journal of Immunological Methods,253(1–2), 153–162. Scholar
  33. 33.
    Muyldermans, S. (2013). Nanobodies: Natural single-domain antibodies. Annual Review of Biochemistry. Scholar
  34. 34.
    Kirchhofer, A., Helma, J., Schmidthals, K., Frauer, C., Cui, S., Karcher, A., et al. (2010). Modulation of protein properties in living cells using nanobodies. Nature Structural & Molecular Biology. Scholar
  35. 35.
    Vincke, C., Loris, R., Saerens, D., Martinez-Rodriguez, S., Muyldermans, S., & Conrath, K. (2009). General strategy to humanize a camelid single-domain antibody and identification of a universal humanized nanobody scaffold. Journal of Biological Chemistry,284(5), 3273–3284. Scholar
  36. 36.
    Liu, J. L., Goldman, E. R., Zabetakis, D., Walper, S. A., Turner, K. B., Shriver-Lake, L. C., et al. (2015). Enhanced production of a single domain antibody with an engineered stabilizing extra disulfide bond. Microbial Cell Factories. Scholar
  37. 37.
    Harmsen, M. M., Ruuls, R. C., Nijman, I. J., Niewold, T. A., Frenken, L., & Geus, D. (2001). Llama heavy chain V-regions consist of at least four distinct subfamilies revealing novel sequence features. Molecular Immunology,37(2000), 579–590.Google Scholar
  38. 38.
    Saerens, D., Pellis, M., Loris, R., Pardon, E., Dumoulin, M., Matagne, A., et al. (2005). Identification of a universal VHH framework to graft non-canonical antigen-binding loops of camel single-domain antibodies. Journal of Molecular Biology,352(3), 597–607. Scholar
  39. 39.
    Mitchell, L. S., & Colwell, L. J. (2018). Analysis of nanobody paratopes reveals greater diversity than classical antibodies. Protein Engineering, Design & Selection. Scholar
  40. 40.
    Yan, J., Li, G., Hu, Y., Ou, W., & Wan, Y. (2014). Construction of a synthetic phage-displayed Nanobody library with CDR3 regions randomized by trinucleotide cassettes for diagnostic applications. Journal of Translational Medicine. Scholar
  41. 41.
    Yang, H. Y., Kang, K. J., Chung, J. E., & Shim, H. (2009). Construction of a large synthetic human scFv library with six diversified CDRs and high functional diversity. Molecules and Cells. Scholar
  42. 42.
    Virnekas, B., Ge, L., Plukthun, A., Schneider, K. C., Wellnhofer, G., & Moroney, S. E. (1994). Trinucleotide phosphoramidites: Ideal reagents for the synthesis of mixed oligonucleotides for random mutagenesis. Nucleic Acids Research. Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Department of Chemistry, Life Sciences and Environmental SustainabilityUniversity of ParmaParmaItaly
  2. 2.San Raffaele HospitalMilanItaly
  3. 3.Biopharmanet-Tec LaboratoryUniversity of ParmaParmaItaly

Personalised recommendations