Structure-Based Design of Supercharged, Highly Thermoresistant Antibodies

Mutation of surface residues to charged amino acids increases resistance to aggregation and can enable reversible unfolding. We have developed a protocol using the Rosetta computational design package that supercharges proteins while considering the energetic implications of each mutation. Using a homology model, a single-chain variable fragment antibody was designed that has a markedly enhanced resistance to thermal inactivation and displays an unanticipated approximate to 30-fold improvement in affinity. Such supercharged antibodies should prove useful for assays in resource-limited settings and for developing reagents with improved shelf lives.