An antibody single-domain phage display library of a native heavy chain variable region: isolation of functional single-domain VH molecules with a unique interface.
To develop very small antibody-derived recognition units for experimental, medical, and drug design purposes, a heavy chain variable region (VH) single-domain phage-display library was designed and constructed. The scaffold that was used for library construction was a native sequence of a monoclonal antibody with a unique VH/VL interface. There was no need to modify any residues in the VL interface to avoid non-specific binding of VH domain. The library repertoire, consisting of 4x10(8)independent clones, was generated by the randomization of nine amino acid residues in complementary determining region 3. The library was screened by binding to protein antigens, and individual clones were isolated. The VH genes encoding for specific binding clones were rescued and large amounts of soluble and stable single-domain VH protein were made from insoluble inclusion bodies by in vitro refolding and purification. Biochemical and biophysical characterization of the VH protein revealed a highly specific, correctly folded, and stable monomeric molecule. Binding studies demonstrated an affinity of 20 nM. The properties of these molecules make them attractive for clinical, industrial, and research applications, as well as a step toward improvement in the design of small molecules that are based on the hypervariable loops of antibodies.
Faculty of Biology, Technion-Israel Institute of Technology, Technion City, Haifa, 32000, Israel.
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Y
Reiter
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Schuck
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Boyd
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Plaksin
D000595:Amino Acid Sequence; D001435:Bacteriophages / Q000235:genetics*; D001483:Base Sequence; D003001:Cloning, Molecular; D004274:DNA, Recombinant; D007143:Immunoglobulin Heavy Chains / Q000235:genetics* / Q000378:metabolism; D007135:Immunoglobulin Variable Region / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data
D004274:DNA, Recombinant; D007143:Immunoglobulin Heavy Chains; D007135:Immunoglobulin Variable Region