Disulfide bond introduction for general stabilization of immunoglobulin heavy-chain variable domains.
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 beta-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.
Saerens
Dirk
D
Laboratory of Cellular and Molecular Immunology, Vrije Universiteit Brussel, Pleinlaan 2, B-1050 Brussels, Belgium. dsaerens@vub.ac.be
Conrath
Katja
K
Govaert
Jochen
J
Muyldermans
Serge
S
D000595:Amino Acid Sequence; D003545:Cysteine / Q000235:genetics / Q000378:metabolism; D004220:Disulfides / Q000737:chemistry* / Q000378:metabolism*; D007143:Immunoglobulin Heavy Chains / Q000737:chemistry* / Q000145:classification / Q000235:genetics / Q000378:metabolism*; D007135:Immunoglobulin Variable Region / Q000737:chemistry* / Q000145:classification / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D049329:Nanostructures / Q000737:chemistry; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D016415:Sequence Alignment
D004220:Disulfides; D007143:Immunoglobulin Heavy Chains; D007135:Immunoglobulin Variable Region; D003545:Cysteine