Selection and production of camelid VHHs/nanobodies
This PBSIM service offers the expertise for selection of camelid VHHs/nanobodies followed by their production and characterization (SPR, BLI, micro-cal).
The use of single-domains from camelid immunoglobulins, termed VHHs or nanobodies (Fig. 1), has seen an increasing growth in recent years. The usefulness of nanobodies in structural biology is now firmly established, as they provide access to new epitopes in concave and hinge regions – and stabilize them.
Remarkable results have been achieved by using nanobodies with flexible multi-domain proteins, large complexes and, last but not least, membrane proteins such as GPCRs or other receptors or channels. The structural biology group at the AFMB lab has been among the pioneers in the work on VHHs and nanobodies.
This tecnological facility offers immunization of llamas (Lama glama glama) or camels (Camelus dromedarius) in view to obtain target specific libraries, panning of these libraries by phage display from which several vHHs/nanobodies are cloned (service 1), expressed (service 2) and characterized (service 3) (Fig. 2). We also propose our “naive” library obtained by gathering the lymphocytes of non-immunized animals – in our case, 3 camels and 4 llamas. Randomizing one of the three CDRs (generally CDR3) from a selected VHH/nanobody followed by a new panning cycle (service 4) leads to sub-nanomolar affinities.
Accessibility
Academia and industries
Cost
To be defined from the service requested.
Comment faire une demande ?
- Contact : Anaïs Gaubert
Specific equipement
- FPLC Äkta Purifier and Xpress (GE Heathcare)
- Microcalorimetry: MicroCal iTC200 (GE Heathcare)
- Microscale thermophoresis: MST Monolith NT.115 (NanoTemper Technologies GmbH)
- Bio-layer interferometry: Octet RED96 system and Blitz system (Pall ForteBio LLC)
- Size exclusion chromatography / multi-angle light scattering / refractometry: HPLC Alliance (Waters) / Minidawn Treos (Wyatt) / Optilab rEX (Wyatt)
Team contacts
Publications
- Eric Durand, Van Son Nguyen, Abdelrahim Zoued, Laureen Logger, Gérard Péhau-Arnaudet, Marie-Stéphanie Aschtgen, Silvia Spinelli, Aline Desmyter, Benjamin Bardiaux, Annick Dujeancourt, Alain Roussel, Christian Cambillau, Eric Cascales and Rémi Fronzes. Biogenesis and structure of the bacterial Type VI secretion membrane core complex. Nature, 300 :555-560 [doi: 10.1038/nature14667] (2015).
- Nguyen, V.S., Logger, L., Spinelli, S., Desmyter, A., Le, T.T.H., Kellenberger, C., Douzi, B., Durand, E., Roussel, A., Cascales, E. and Cambillau, C. Inhibition of Type VI secretion by an anti-TssM llama nanobody, PloS-ONE, 10(3): e0122187. [doi:10.1371/journal.pone.0122187] (2015).
- Nguyen, V.S., Spinelli, S., Desmyter, A., Le, T.T.H., Kellenberger, C., Cascales, E., Cambillau, C. and Roussel, A., Production, crystallization and X-ray diffraction analysis of a complex between a fragment of the TssM T6SS protein and a Camelid nanobody, Acta Crystallogr. F, F71, 266-271 (2015)
- Desmyter, A., Spinelli, S., Roussel, A. and Cambillau, C., Camelid nanobodies : killing two birds with one stone, Curr. Opin. Struct. Biol., 32C:1–8 [doi: 10.1016/j.sbi.2015.01.001] (2015)
- Hassaine G, Deluz C, Grasso L, Wyss R, Tol MB, Hovius R, Graff A, Stahlberg H, Tomizaki T, Desmyter A, et al.: X-ray structure of the mouse serotonin 5-HT3 receptor. Nature 2014, 512:276-281.
- Aline Desmyter, Carine Farenc, Jennifer Mahony, Silvia Spinelli, Cecilia Bebeacua, Stéphanie Blangy, David Veesler, Douwe van Sinderen and Christian Cambillau, Viral infection modulation and neutralization by camelid nanobodies, Proc. Natl. Acad. Sci. USA. 110 : E1371-9 (2013).
- Giuliano Sciara, Cecilia Bebeacua, Patrick Bron, Denise Tremblay, Miguel Ortiz-Lombardia, Julie Lichière, Marin van Heel, Valérie Campanacci, Sylvain Moineau and Christian Cambillau. Structures of Lactococcal Phage p2 Baseplate and Mechanism of Action. Proc. Natl. Acad. Sci. USA, 107:6852-7 (2010).
- Katja Conrath, Alice S. Pereira, Carlos Martins, Cristina G. Timóteo, Pedro Tavares, Silvia Spinelli, Ulrich Werner, Christophe Flaudrops, Christian Cambillau, Serge Muyldermans, Isabel Moura, Jose Moura, Mariella Tegoni and Aline Desmyter, Camelid Nanobodies Raised against an Integral Membrane Enzyme, Nitric Oxide Reductase Protein Science, 18:619-628 (2009).
- Roos G, Brosens E, Wahni K, Desmyter A, Spinelli S, Wyns L, et al. Combining site-specific mutagenesis and seeding as a strategy to crystallize ’difficult’ proteins: the case of Staphylococcus aureus thioredoxin. Acta Crystallogr Sect F Struct Biol Cryst Commun. 2006;62:1255-8.
- Silvia Spinelli, Aline Desmyter, C.Theo Verrips, Hans J. W. de Haard, Sylvain Moineau and Christian Cambillau, Structure of lactococcal bacteriophage p2 receptor binding protein suggests gene transfer with bacterial and mammalian viruses, Nature Struct Molec Biol, 13, 85-89 (2006)
- Denise M. Tremblay, Mariella Tegoni, Silvia Spinelli, Valérie Campanacci, Stéphanie Blangy, Céline Huyghe, Aline Desmyter, Steve Labrie, Sylvain Moineau and Christian Cambillau. Receptor Binding Protein of Lactococcus lactis phages: Identification and Characterization of the Saccharide Receptor Binding Site, J. Bact. 188, 2400-2410 (2006).
- Edward Dolk, Cees van Vliet, Janice M.J. Perez , Gert Vriend, Christian Cambillau, Leon G.J. Frenken and Theo Verrips, induced refolding of a temperature denatured llama heavy chain antibody fragment by its antigen, Proteins. 59, 555-564 (2005).
- Silvia Spinelli, Aline Desmyter, Leon Frenken, Theo Verrips, Mariella Tegoni and Christian Cambillau, Domain Swapping in a Llama VHH Domain Leads to Amyloid-like Structures in the Crystal Packing, FEBS letters 564, 35-40 (2004).
- Jean-Guillaume Renisio, Janice Pérez, Michael Czisch, Marc Guenneugues, Olivier Bornet, Leon Frenken, Christian Cambillau and Hervé Darbon. Solution structure and relaxation studies of an antigen-free Heavy Chain Variable Domain (VHH) from Llama, Proteins. 47, 546-555 (2002).
- Stefan Ewert, Christian Cambillau, Katja Conrath and Andreas Plückthun, Biophysical Properties of Camelid VHH Domains Compared to Those of Human VH3 Domains, Biochemistry 41, 3228-3236 (2002).
- Desmyter, A., Spinelli, S., Lauwereys, M., Wyns,L., Muyldermans, S. and Cambillau,C. 2002. Camelids VHH Domains in Complex with Porcine Amylase: Inhibition and Involvement of Framework in Interactions. J.Biol.Chem. 277, 23645-50 .
- Gilles Ferrat, Jean-Guillaume Renisio, Xavier Morelli , Jerry Slootstra, Christian Cambillau and Hervé Darbon, A Peptide Mimic of an Antigenic Loop of a-hCG: Solution Structure and Interaction with a Ll ama VHH Domain. Biochem. J. 366 415-421 (2002).
- Janice, M.J. Pérez, Jean-Guillaume Renisio, Jeanine J. Prompers, Chris J. van Platerink, Christian Cambillau, Hervé Darbon, and Leon G.J. Frenken, Intrinsic Stability of a Llama Antibody domain as Determined by Circular Dichroism, Nuclear Magnetic Resonance and Electrospray Ionisation Mass spectrometry, Biochemistry 40, 74-83 (2001).
- Serge Muyldermans, Christian Cambillau and Lode Wyns, Recognition of antigens by single domain antibody fragments: the superfluous luxury of paired domains. TIBS 26, 230-235 (2001).
- Silvia Spinelli, Mariella Tegoni , Leon Frenken, Cees van Vliet and Christian Cambillau, Lateral Recognition of a Dye Hapten by a Llama VHH Domain , J.Mol.Biol. 311:123-9 (2001).
- Silvia Spinelli, G.J. Leon Frenken , Pim Hermans, Theo Verrips, Kieron Brown, Mariella Tegoni and Christian Cambillau. Camelids heavy chain variable domains provide efficient combining sites to haptens. Biochemistry, 39, 1217-1222 (2000).
- Silvia Spinelli , Leon Frenken, Dominique Bourgeois, Lian de Ron, Will Bos, Theo Verrips, Christelle Anguille, Christian Cambillau and Mariella Tegoni The Crystal Structure of a Llama Heavy Chain Variable Domain. Nature Struct. Biol., 3, 752-756 (1996).
