Abstract
Single-domain antibodies libraries of heavy-chain only immunoglobulins from camelids or shark are enriched for high-affinity antigen-specific binders by a short in vivo immunization. Thus, potent binders are readily retrieved from relatively small-sized libraries of 107–108 individual transformants, mostly after phage display and panning on a purified target. However, the remaining drawback of this strategy arises from the need to generate a dedicated library, for nearly every envisaged target. Therefore, all the procedures that shorten and facilitate the construction of an immune library of best possible quality are definitely a step forward. In this chapter, we provide the protocol to generate a high-quality immune VHH library using the Golden Gate Cloning strategy employing an adapted phage display vector where a lethal ccdB gene has to be substituted by the VHH gene. With this procedure, the construction of the library can be shortened to less than a week starting from bleeding the animal. Our libraries exceed 108 individual transformants and close to 100% of the clones harbor a phage display vector having an insert with the length of a VHH gene. These libraries are also more economic to make than previous standard approaches using classical restriction enzymes and ligations. The quality of the Nanobodies that are retrieved from immune libraries obtained by Golden Gate Cloning is identical to those from immune libraries made according to the classical procedure.
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References
Muyldermans S (2013) Nanobodies: natural single-domain antibodies. Annu Rev Biochem 82:775–797
Hamers-Casterman C, Atarhouch T, Muyldermans S, Robinson G, Hamers C, Songa EB, Bendahman N, Hamers R (1993) Naturally-occurring antibodies devoid of light-chains. Nature 363:446–448
Greenberg AS, Avila D, Hughes M, Hughes A, McKinney EC, Flajnik MF (1995) A new antigen receptor gene family that undergoes rearrangement and extensive somatic diversification in sharks. Nature 374:168–173
Dmitriev OY, Lutsenko S, Muyldermans S (2016) Nanobodies as probes for protein dynamics in vitro and in cells. J Biol Chem 291:3767–3775
Engler C, Gruetzner R, Kandzia R, Marillonet S (2009) Golden gate shuffling: a one-pot DNA shuffling method based on type IIs restriction enzymes. PLoS One 4:e5553
Bernard P, Gabant P, Bahassi E, Couturier M (1994) Positive-selectionvectors using the F plasmid ccdB killer gene. Gene 148:71–74
Vincke C, Gutiérrez C, Wernery U, Devoogdt N, Hassanzadeh-Ghassabeh G, Muyldermans S (2012) Chapter 8: Generation of single domain antibody fragments derived from camelids and generation of manifold constructs. In: Chames P (ed) Antibody engineering: methods and protocols, second edition. Methods in molecular biology, vol 917. Humana, Louisville, KY, pp 145–176
Pardon E, Laeremans T, Triest S, Rasmussen SGF, Wohlkönig A, Ruf A, Muyldermans S, Hol WGJ, Kobilka BK, Steyaert J (2014) A general protocol for the generation of Nanobodies for structural biology. Nat Protoc 9:674–693
Nguyen VK, Hamers R, Wyns L, Muyldermans S (2000) Camel heavy-chain antibodies: diverse germline V(H)H and specific mechanisms enlarge the antigen-binding repertoire. Eur Mol Biol Organ J 19:921–930
Deschacht N, De Groeve K, Vincke C, Raes G, De Baetselier P, Muyldermans S (2010) A novel promiscuous class of camelid single-domain antibody contributes to the antigen-binding repertoire. J Immunol 184:5696–5704
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Romão, E., Poignavent, V., Vincke, C., Ritzenthaler, C., Muyldermans, S., Monsion, B. (2018). Construction of High-Quality Camel Immune Antibody Libraries. In: Hust, M., Lim, T. (eds) Phage Display. Methods in Molecular Biology, vol 1701. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7447-4_9
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DOI: https://doi.org/10.1007/978-1-4939-7447-4_9
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