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Selection and characterisation of binders based on homodimerisation of immunoglobulin V H domains
Author(s) -
Jin Hulin,
Sepúlveda Jorge,
Burrone Oscar R.
Publication year - 2003
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/s0014-5793(03)01182-7
Subject(s) - panning (audio) , phage display , lysozyme , streptavidin , phagemid , antibody , microbiology and biotechnology , antigen , chemistry , immunoglobulin light chain , peptide library , cysteine , stereochemistry , biology , peptide sequence , biochemistry , peptide , enzyme , gene , genetics , bacteriophage , escherichia coli , biotin , lens (geology) , paleontology , zoom
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.