Nanobodies are single-domain antibodies derived from the variable regions of Camelidae atypical immunoglobulins. They show promise as high-affinity reagents for research, diagnostics and therapeutics owing to their high specificity, small size (∼15 kDa) and straightforward bacterial expression. However, identification of repertoires with sufficiently high affinity has proven time consuming and difficult, hampering nanobody implementation. Our approach generates large repertoires of readily expressible recombinant nanobodies with high affinities and specificities against a given antigen. We demonstrate the efficacy of this approach through the production of large repertoires of nanobodies against two antigens, GFP and mCherry, with Kd values into the subnanomolar range. After mapping diverse epitopes on GFP, we were also able to design ultrahigh-affinity dimeric nanobodies with Kd values as low as ∼30 pM. The approach presented here is well suited for the routine production of high-affinity capture reagents for various biomedical applications.
Subscribe to Journal
Get full journal access for 1 year
only $9.92 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Tax calculation will be finalised during checkout.
Rent or Buy article
Get time limited or full article access on ReadCube.
All prices are NET prices.
Cristea, I.M., Williams, R., Chait, B.T. & Rout, M.P. Fluorescent proteins as proteomic probes. Mol. Cell. Proteomics 4, 1933–1941 (2005).
Rigaut, G. et al. A generic protein purification method for protein complex characterization and proteome exploration. Nat. Biotechnol. 17, 1030–1032 (1999).
Ho, Y. et al. Systematic identification of protein complexes in Saccharomyces cerevisiae by mass spectrometry. Nature 415, 180–183 (2002).
Domanski, M. et al. Improved methodology for the affinity isolation of human protein complexes expressed at near endogenous levels. Biotechniques doi:10.2144/000113864 (May 2012)
Gingras, A.C., Aebersold, R. & Raught, B. Advances in protein complex analysis using mass spectrometry. J. Physiol. (Lond.) 563, 11–21 (2005).
Cortez-Retamozo, V. et al. Efficient cancer therapy with a nanobody-based conjugate. Cancer Res. 64, 2853–2857 (2004).
Hamers-Casterman, C. et al. Naturally occurring antibodies devoid of light chains. Nature 363, 446–448 (1993).
Muyldermans, S. Nanobodies: natural single-domain antibodies. Annu. Rev. Biochem. 82, 775–797 (2013).
Harmsen, M.M. & De Haard, H.J. Properties, production, and applications of camelid single-domain antibody fragments. Appl. Microbiol. Biotechnol. 77, 13–22 (2007).
Romer, T., Leonhardt, H. & Rothbauer, U. Engineering antibodies and proteins for molecular in vivo imaging. Curr. Opin. Biotechnol. 22, 882–887 (2011).
Dumoulin, M. et al. Single-domain antibody fragments with high conformational stability. Protein Sci. 11, 500–515 (2002).
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).
Arbabi-Ghahroudi, M., Tanha, J. & MacKenzie, R. Prokaryotic expression of antibodies. Cancer Metastasis Rev. 24, 501–519 (2005).
Rothbauer, U. et al. Targeting and tracing antigens in live cells with fluorescent nanobodies. Nat. Methods 3, 887–889 (2006).
Muyldermans, S. et al. Camelid immunoglobulins and nanobody technology. Vet. Immunol. Immunopathol. 128, 178–183 (2009).
Bird, R.E. et al. Single-chain antigen-binding proteins. Science 242, 423–426 (1988).
Skerra, A. & Pluckthun, A. Assembly of a functional immunoglobulin Fv fragment in Escherichia coli. Science 240, 1038–1041 (1988).
Wörn, A. & Pluckthun, A. Stability engineering of antibody single-chain Fv fragments. J. Mol. Biol. 305, 989–1010 (2001).
Scheid, J.F. et al. Sequence and structural convergence of broad and potent HIV antibodies that mimic CD4 binding. Science 333, 1633–1637 (2011).
Shagin, D.A. et al. GFP-like proteins as ubiquitous metazoan superfamily: evolution of functional features and structural complexity. Mol. Biol. Evol. 21, 841–850 (2004).
Dörner, T. & Radbruch, A. Antibodies and B cell memory in viral immunity. Immunity 27, 384–392 (2007).
Benner, R., Hijmans, W. & Haaijman, J.J. The bone marrow: the major source of serum immunoglobulins, but still a neglected site of antibody formation. Clin. Exp. Immunol. 46, 1–8 (1981).
Becker, R.S. & Knight, K.L. Somatic diversification of immunoglobulin heavy chain VDJ genes: evidence for somatic gene conversion in rabbits. Cell 63, 987–997 (1990).
Knight, K.L. Restricted VH gene usage and generation of antibody diversity in rabbit. Annu. Rev. Immunol. 10, 593–616 (1992).
Conrath, K.E. et al. Beta-lactamase inhibitors derived from single-domain antibody fragments elicited in the camelidae. Antimicrob. Agents Chemother. 45, 2807–2812 (2001).
Alvarez-Rueda, N. et al. Generation of llama single-domain antibodies against methotrexate, a prototypical hapten. Mol. Immunol. 44, 1680–1690 (2007).
Brohawn, S.G., Partridge, J.R., Whittle, J.R. & Schwartz, T.U. The nuclear pore complex has entered the atomic age. Structure 17, 1156–1168 (2009).
Fernandez-Martinez, J. et al. Structure-function mapping of a heptameric module in the nuclear pore complex. J. Cell Biol. 196, 419–434 (2012).
Kirchhofer, A. et al. Modulation of protein properties in living cells using nanobodies. Nat. Struct. Mol. Biol. 17, 133–138 (2010).
Ghaemmaghami, S. et al. Global analysis of protein expression in yeast. Nature 425, 737–741 (2003).
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).
Dolman, N.J., Kilgore, J.A. & Davidson, M.W. A review of reagents for fluorescence microscopy of cellular compartments and structures, part I: BacMam labeling and reagents for vesicular structures. Curr. Protoc. Cytom. 65, 12.30 (2013).
DeGrasse, J.A. et al. Evidence for a shared nuclear pore complex architecture that is conserved from the last common eukaryotic ancestor. Mol. Cell. Proteomics 8, 2119–2130 (2009).
Matz, M.V. et al. Fluorescent proteins from nonbioluminescent Anthozoa species. Nat. Biotechnol. 17, 969–973 (1999).
Shu, X., Shaner, N.C., Yarbrough, C.A., Tsien, R.Y. & Remington, S.J. Novel chromophores and buried charges control color in mFruits. Biochemistry 45, 9639–9647 (2006).
Xia, N.S. et al. Bioluminescence of Aequorea macrodactyla, a common jellyfish species in the East China Sea. Mar. Biotechnol. (NY) 4, 155–162 (2002).
Goldflam, M., Tarrago, T., Gairi, M. & Giralt, E. NMR studies of protein-ligand interactions. Methods Mol. Biol. 831, 233–259 (2012).
Georgescu, J. et al. Backbone HN, N, Cα and Cβ assignment of the GFPuv mutant. J. Biomol. NMR 25, 161–162 (2003).
Khan, F., Stott, K. & Jackson, S. 1H, 15N and 13C backbone assignment of the green fluorescent protein (GFP). J. Biomol. NMR 26, 281–282 (2003).
Zuiderweg, E.R. Mapping protein-protein interactions in solution by NMR spectroscopy. Biochemistry 41, 1–7 (2002).
Battistutta, R., Negro, A. & Zanotti, G. Crystal structure and refolding properties of the mutant F99S/M153T/V163A of the green fluorescent protein. Proteins 41, 429–437 (2000).
Neri, D., Momo, M., Prospero, T. & Winter, G. High-affinity antigen binding by chelating recombinant antibodies (CRAbs). J. Mol. Biol. 246, 367–373 (1995).
Silverman, J. et al. Multivalent avimer proteins evolved by exon shuffling of a family of human receptor domains. Nat. Biotechnol. 23, 1556–1561 (2005).
Vanlandschoot, P. et al. Nanobodies®: new ammunition to battle viruses. Antiviral Res. 92, 389–407 (2011).
Huang, L., Muyldermans, S. & Saerens, D. Nanobodies®: proficient tools in diagnostics. Expert Rev. Mol. Diagn. 10, 777–785 (2010).
Revets, H., De Baetselier, P. & Muyldermans, S. Nanobodies as novel agents for cancer therapy. Expert Opin. Biol. Ther. 5, 111–124 (2005).
Vincke, C. et al. General strategy to humanize a camelid single-domain antibody and identification of a universal humanized nanobody scaffold. J. Biol. Chem. 284, 3273–3284 (2009).
Els Conrath, K., Lauwereys, M., Wyns, L. & Muyldermans, S. Camel single-domain antibodies as modular building units in bispecific and bivalent antibody constructs. J. Biol. Chem. 276, 7346–7350 (2001).
Jähnichen, S. 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).
Roovers, R.C. et al. A biparatopic anti-EGFR nanobody efficiently inhibits solid tumour growth. Int. J. Cancer 129, 2013–2024 (2011).
Ulrichts, H. et al. Antithrombotic drug candidate ALX-0081 shows superior preclinical efficacy and safety compared with currently marketed antiplatelet drugs. Blood 118, 757–765 (2011).
Ormö, M. et al. Crystal structure of the Aequorea victoria green fluorescent protein. Science 273, 1392–1395 (1996).
Pletneva, N.V. et al. Yellow fluorescent protein phiYFPv (Phialidium): structure and structure-based mutagenesis. Acta Crystallogr. D Biol. Crystallogr. 69, 1005–1012 (2013).
Wall, M.A., Socolich, M. & Ranganathan, R. The structural basis for red fluorescence in the tetrameric GFP homolog DsRed. Nat. Struct. Biol. 7, 1133–1138 (2000).
Kelley, L.A. & Sternberg, M.J. Protein structure prediction on the Web: a case study using the Phyre server. Nat. Protoc. 4, 363–371 (2009).
Rappsilber, J., Ishihama, Y. & Mann, M. Stop and go extraction tips for matrix-assisted laser desorption/ionization, nanoelectrospray, and LC/MS sample pretreatment in proteomics. Anal. Chem. 75, 663–670 (2003).
Alber, F. et al. Determining the architectures of macromolecular assemblies. Nature 450, 683–694 (2007).
Rout, M.P. et al. The yeast nuclear pore complex: composition, architecture, and transport mechanism. J. Cell Biol. 148, 635–651 (2000).
Dereeper, A., Audic, S., Claverie, J.M. & Blanc, G. BLAST-EXPLORER helps you building datasets for phylogenetic analysis. BMC Evol. Biol. 10, 8 (2010).
Dereeper, A. et al. Phylogeny.fr: robust phylogenetic analysis for the non-specialist. Nucleic Acids Res. 36, W465–W469 (2008).
Zacharias, D.A., Violin, J.D., Newton, A.C. & Tsien, R.Y. Partitioning of lipid-modified monomeric GFPs into membrane microdomains of live cells. Science 296, 913–916 (2002).
Krissinel, E. & Henrick, K. Inference of macromolecular assemblies from crystalline state. J. Mol. Biol. 372, 774–797 (2007).
We acknowledge support from US National Institutes of Health grants U54 GM103511 and P41 GM109824 (M.P.R. and B.T.C.), P41 GM103314 (B.T.C.), and AI072529-08 and AI037526-20A1 (M.C.N.) and support from the Howard Hughes Medical Institute (M.C.N.). M.O. was supported by the Natural Sciences and Engineering Research Council of Canada, Canadian Institutes of Health Research and Fonds de recherches du Québec - Santé. We thank A. North for assistance with immunofluorescence microscopy; A. Viale and C. Zhao for support with high-throughput sequencing; A. Luz for assistance with SPR; S. Reed-Paske and the other members of Capralogics, Inc., for advice and animal husbandry; and members of the Rout and Chait laboratories, past and present, for helpful discussions and technical assistance, particularly A. Ferguson, K. Wei, H. Jiang and S. Obado.
B.T.C. and M.P.R. are inventors on a US patent application encompassing the method described in this manuscript.
About this article
Cite this article
Fridy, P., Li, Y., Keegan, S. et al. A robust pipeline for rapid production of versatile nanobody repertoires. Nat Methods 11, 1253–1260 (2014). https://doi.org/10.1038/nmeth.3170
Nature Methods (2021)
Arabidopsis ACINUS is O-glycosylated and regulates transcription and alternative splicing of regulators of reproductive transitions
Nature Communications (2021)
Nature Chemical Biology (2021)
Nature Chemical Biology (2021)
Current Genetics (2021)