TY - JOUR
T1 - Construction of High-Quality Camel Immune Antibody Libraries
AU - Romão, Ema
AU - Poignavent, Vianney
AU - Vincke, Cécile
AU - Ritzenthaler, Christophe
AU - Muyldermans, Serge
AU - Monsion, Baptiste
PY - 2018
Y1 - 2018
N2 - 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 107-108individual 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 108individual 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.
AB - 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 107-108individual 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 108individual 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.
KW - Golden gate cloning
KW - Immune libraries
KW - Nanobodies
KW - Single-domain antibodies
KW - VHH
UR - http://www.scopus.com/inward/record.url?scp=85033708416&partnerID=8YFLogxK
U2 - 10.1007/978-1-4939-7447-4_9
DO - 10.1007/978-1-4939-7447-4_9
M3 - Article
C2 - 29116505
VL - 1701
SP - 169
EP - 187
JO - Methods in molecular biology (Clifton, N.J.)
JF - Methods in molecular biology (Clifton, N.J.)
SN - 1064-3745
ER -