Disulfide bond introduction for general stabilization of immunoglobulin heavy-chain variable domains |
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Authors: | Saerens Dirk Conrath Katja Govaert Jochen Muyldermans Serge |
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Institution: | Laboratory of Cellular and Molecular Immunology, Vrije Universiteit Brussel, Pleinlaan 2, B-1050 Brussels, Belgium Vlaams Interuniversitair Instituut voor Biotechnologie, Department of Molecular and Cellular Interactions, Vrije Universiteit Brussel, Pleinlaan 2, B-1050 Brussels, Belgium |
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Abstract: | Several antibody fragment engineering techniques aim at intrinsic stability enhancement, but are not applied in a truly generic way. Here, a strategy is proposed whereby consistent gain in stability is accomplished by introducing a specific disulfide bond between two opposite β-strands in the hydrophobic core of the immunoglobulin heavy-chain variable domain of heavy-chain antibodies (Nanobody). Besides the rational design of a disulfide bond between residues 39 and 87, a Nanobody harboring an extra naturally occurring cystine between residues 54 and 78 was compared to an equivalent Nanobody without that cystine. Both novel disulfide cross-links were introduced in several Nanobodies in various combinations. Interestingly, only the extra naturally occurring cystine consistently increased the conformational and thermal stabilities of wild-type Nanobodies without affecting antigen binding. |
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Keywords: | hPSA human prostate-specific antigen CDR complementarity-determining region GdmHCl guanidine hydrochloride PDB Protein Data Bank Fv variable fragment VH variable domain of the heavy-chain IMGT ImMunoGeneTics information system |
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