Freeze-thaw-induced aggregation of bovine gamma globulin was efficiently inhibited by an intrinsically disordered plant protein dehydrin

Immunoglobulin, which is widely used in the formulation of protein drugs, is prone to aggregation due to freezing. The aggregated immunoglobulin exhibits decreased immune-reactivity and increasing immunogenicity. Accordingly, large amounts of excipients are added to immunoglobulin drugs to prevent aggregation. In the present study, we found that an Arabidopsis dehydrin (AtHIRD11), which is a stress-related intrinsically disordered protein, could efficiently inhibit the cryoaggregation of bovine gamma globulin (BGG). AtHIRD11 was 3 to 4 orders of magnitude more efficient than general protectants such as sugars and amino acids at the molar levels. The K-segment, which is a conserved sequence of dehydrin, was one of the protective sites of AtHIRD11. Amino acid substitution analysis indicated that the hydrophobic amino acids contributed to the cryoprotective activity of the K-segment. Moreover, the activity was roughly correlated with the hydropathy scores of hydrophobic amino acids. BGG and the K-segment individually migrated in size exclusion chromatography, showing that the K-segment did not bind to BGG in solution. This suggests that dehydrin may prevent the cryoaggregation of BGG via the K-segment through a transient hydrophobic interaction. Dehydrin may be utilized as an effective stabilizer of immunoglobulin to minimize aggregation under freezing conditions.