A Novel Nanobody Scaffold Optimized for Bacterial Expression and Suitable for the Construction of Ribosome Display Libraries
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.
KeywordsNanobody 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.
- 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.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. https://doi.org/10.1073/pnas.0505379103.CrossRefGoogle Scholar
- 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. https://doi.org/10.1016/j.bbapap.2006.05.011.CrossRefGoogle Scholar
- 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. https://doi.org/10.1038/s41594-018-0028-6.CrossRefGoogle Scholar
- 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. https://doi.org/10.1016/j.jmb.2007.08.027.CrossRefPubMedPubMedCentralGoogle Scholar
- 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. https://doi.org/10.1016/j.jim.2003.07.011.CrossRefPubMedGoogle Scholar
- 24.Sambrook, J., & W Russell, D. (2001). Molecular cloning: A laboratory manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press.Google Scholar
- 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. https://doi.org/10.1093/protein/gzr057.CrossRefGoogle Scholar
- 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. https://doi.org/10.1074/jbc.M501746200.CrossRefPubMedGoogle Scholar
- 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. https://doi.org/10.1016/j.jsb.2007.01.013.CrossRefPubMedGoogle Scholar
- 33.Muyldermans, S. (2013). Nanobodies: Natural single-domain antibodies. Annual Review of Biochemistry. https://doi.org/10.1146/annurev-biochem-063011-092449.CrossRefPubMedGoogle Scholar
- 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. https://doi.org/10.1074/jbc.M806889200.CrossRefPubMedGoogle Scholar
- 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. https://doi.org/10.1186/s12934-015-0340-3.CrossRefPubMedPubMedCentralGoogle Scholar
- 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.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. https://doi.org/10.1016/j.jmb.2005.07.038.CrossRefPubMedGoogle Scholar
- 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. https://doi.org/10.1186/s12967-014-0343-6.CrossRefPubMedPubMedCentralGoogle Scholar
- 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. https://doi.org/10.1093/nar/22.25.5600.CrossRefPubMedPubMedCentralGoogle Scholar