The effect of charge mutations on the stability and aggregation of a human single chain Fv fragment

James Austerberry, Rana Dajani, Stanislava Panova, Dorota Roberts, Alexander Golovanov, Alain Pluen, Christopher F van der Walle, Shahid Uddin, Jeremy Derrick, James Warwicker, Robin Curtis

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The aggregation propensities of a series of single-chain variable fragment (scFv) proteins harbouring supercharged sequences, salt bridges and lysine/arginine-enriched motifs were characterised as a function of pH and ionic strength to isolate the electrostatic contributions. Recent improvements in aggregation predictors rely on using knowledge of native-state protein-protein interactions. Consistent with previous findings, electrostatic contributions to native protein-protein interactions correlate with aggregate growth pathway and rates. However, strong reversible self-association observed for selected mutants under native conditions did not correlate with aggregate growth, indicating ‘sticky’ surfaces that are exposed in the native monomeric state are inaccessible when aggregates grow. We find that even though similar native-state protein-protein interactions occur for the arginine and lysine-enriched mutants, aggregation propensity is increased for the former and decreased for the latter, providing evidence that lysine suppresses protein-protein interactions under these conditions. The supercharged mutants follow the behaviour observed for basic proteins under acidic conditions; where excess net charge decreases conformational stability and increases nucleation rates, but conversely reduces aggregate growth rates due to increased intermolecular electrostatic repulsion. The results highlight the limitations of using native-state protein-protein interactions as predictors for aggregation propensity and provide guidance on how to engineer stabilizing charged mutations.
Original languageEnglish
Pages (from-to)18-30
JournalEuropean Journal of Pharmaceutics and Biopharmaceutics
Early online date1 Feb 2017
Publication statusPublished - 1 Jun 2017


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