title: A C-terminal interdomain disulfide bond significantly stabilizes the Fc fragment of IgG.
abstract: We describe the stabilization of human IgG1 Fc by an engineered interdomain disulfide bond at the C-terminal end of the molecule. Covalently interconnecting the C-termini of the CH(3) domains led to an increase of the melting temperatures by 5.6 and 9.1 °C respectively as compared to CH(3) domains in the context of the wild-type Fc. Combined with a recently described additional intradomain disulfide bond, both novel disulfide bonds led to an increase of the Tm by about 18.1 °C to 100.7 °C. The interdomain disulfide bond had no impact on the thermal stability of the CH(2) domain. Far- and near-UV CD spectroscopy showed very similar overall CD profiles, indicating that secondary and tertiary structure of the Fc was not negatively affected. When introduced into an Fc fragment that had been engineered to bind to Her2/neu via a novel antigen binding site located at the C-terminus of the CH(3) domain, the novel inter- and intra-domain bonds also brought about a significant increase in thermostability. Using them in combination, the Tm of the CH(3) domain was raised by 18 °C and thus restored to the Tm of the wild-type CH(3) domain. Importantly, antigen binding of the modified Fc was not affected by the engineered disulfide bonds.
authors.affiliation: University of Natural Resources and Life Sciences, Vienna, Department of Biotechnology, Christian Doppler Laboratory for Antibody Engineering, Muthgasse 18, 1190 Vienna, Austria.
authors.forename: Gordana
authors.initials: G
authors.lastname: Wozniak-Knopp
authors.forename: Florian
authors.initials: F
authors.lastname: Rüker
mesh_terms: D000595:Amino Acid Sequence; D002152:Calorimetry, Differential Scanning; D003001:Cloning, Molecular / Q000379:methods; D004220:Disulfides / Q000737:chemistry*; D006801:Humans; D007141:Immunoglobulin Fc Fragments / Q000737:chemistry* / Q000235:genetics*; D007074:Immunoglobulin G / Q000737:chemistry* / Q000235:genetics*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010843:Pichia / Q000235:genetics; D011489:Protein Denaturation; D015202:Protein Engineering / Q000379:methods*; D055550:Protein Stability; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D013696:Temperature
chemical_list: D004220:Disulfides; D007141:Immunoglobulin Fc Fragments; D007074:Immunoglobulin G