ABSTRACT
This protocol describes a method for creation of a highly diverse and functional synthetic phage-displayed repertoire of fully human domain antibodies (dAbs). The repertoire is based on two human frameworks (one VH and one Vκ) that express well in bacteria and are frequently used in human antibodies. To achieve this, we first build dAb libraries, containing full synthetic diversity at key positions in the complementarity-determining regions (CDRs). We then use an antigen-independent preselection of this primary dAb repertoire on generic ligands of the VH and the Vκ scaffolds (namely, the bacterial superantigens, protein A and L) to enrich for folded dAbs. Finally, the CDRs of these preselected dAbs are randomly recombined to further expand genetic diversity. The resulting phage repertoire is in excess of 10(10) clones and is largely populated by correctly folded (over 50%) functional dAbs.
Subject(s)
Cell Surface Display Techniques/methods , Peptide Library , Single-Domain Antibodies/chemistry , Single-Domain Antibodies/genetics , Amino Acid Sequence , Base Sequence , Complementarity Determining Regions/chemistry , Complementarity Determining Regions/genetics , Gene Library , Gene Order , Genetic Variation , Genetic Vectors , Humans , Immunoglobulin Heavy Chains/chemistry , Immunoglobulin Heavy Chains/genetics , Immunoglobulin Variable Region/chemistry , Immunoglobulin Variable Region/genetics , Molecular Sequence Data , Oligonucleotides/chemistry , Polymerase Chain Reaction/methods , Single-Domain Antibodies/immunology , Staphylococcal Protein A/immunology , Staphylococcal Protein A/metabolism , Templates, Genetic , Transformation, BacterialABSTRACT
We have combined phage display and array screening for the rapid isolation of pairs of interacting polypeptides. Our strategy, named SAC (selection by avidity capture), is based on the avidity effect, the formation of highly stable complexes formed by multivalent interactions; in our case, between a receptor (multivalently displayed on phage) and a ligand (coexpressed as a multimeric fusion protein). Capture of the long-lived interaction complex allows the isolation of phage bearing cognate interaction pairs, as we demonstrate for a range of interactions, including Ab-antigen pairs and the rapamycin-dependent interaction of FKBP-12 and FRAP. Cognate phage are enriched by SAC up to 1000-fold and interacting pairs can be identified by array screening. Application of SAC to Ab-antigen interactions as a model system yielded over 140 specific Abs to a single antigen and 92 Abs to three different fetal human brain antigens in a single round of SAC each. Our results suggest that SAC should prove useful for the identification and study of receptor-ligand interactions in particular among extracellular proteins, as well as for the rapid generation of specific Abs to multiple antigens.
