title: Selection and characterisation of binders based on homodimerisation of immunoglobulin V(H) domains.
abstract: The antigen-binding surface of antibodies is formed by the heterodimerisation of the two variable domains of the light (V(L)) and heavy (V(H)) chains. We have previously described the spontaneous formation of V(H) dimers (VHD) in both bacteria and mammalian cells. The self-association of a single domain produces a homo-VHD, in which the two identical V(H) domains generate a unique symmetric surface for antigen binding that is never found in the normal V(L)/V(H) antibody binding site. We developed a phagemid vector for the construction of phage display libraries in which a cysteine residue, introduced at the C-terminus of the only V(H) cloned, allowed display of homo-VHDs. Panning of the library on different proteins yielded antigen specific binders against lysozyme, glutathione S-transferase and streptavidin. A lysozyme specific homo-VHD was further characterised with an apparent affinity determined to be 216+/-6.6 nM. Importantly, the results showed that its binding activity was fully dependent on the dimerisation of both identical V(H) domains.
authors: International Centre for Genetic Engineering and Biotechnology, Area Science Park, Padriciano 99, 34012 Trieste, Italy. jin@icgeb.org
authors: Hulin
authors: H
authors: Jin
authors: Jorge
authors: J
authors: Sepúlveda
authors: Oscar R
authors: OR
authors: Burrone
mesh_terms: D000595:Amino Acid Sequence; D000918:Antibody Specificity; D001483:Base Sequence; D001666:Binding Sites, Antibody; D015153:Blotting, Western; D003545:Cysteine / Q000235:genetics / Q000378:metabolism; D019281:Dimerization; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D007143:Immunoglobulin Heavy Chains / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009113:Muramidase / Q000378:metabolism; D019151:Peptide Library; D010957:Plasmids; D017434:Protein Structure, Tertiary
chemical_list: D007143:Immunoglobulin Heavy Chains; D019151:Peptide Library; D009113:Muramidase; D003545:Cysteine