Conjugation of type I antifreeze protein to polyallylamine increases thermal hysteresis activity

Antifreeze proteins (AFPs) are ice binding proteins found in some plants, insects, and Antarctic fish allowing them to survive at subzero temperatures by inhibiting ice crystal growth. The interaction of AFPs with ice crystals results in a difference between the freezing and melting temperatures, termed thermal hysteresis, which is the most common measure of AFP activity. Creating antifreeze protein constructs that reduce the concentration of protein needed to observe thermal hysteresis activities would be beneficial for diverse applications including cold storage of cells or tissues, ice slurries used in refrigeration systems, and food storage. We demonstrate that conjugating multiple type I AFPs to a polyallylamine chain increases thermal hysteresis activity compared to the original protein. The reaction product is approximately twice as active when compared to the same concentration of free proteins, yielding 0.5 °C thermal hysteresis activity at 0.3 mM protein concentration. More impressively, the amount of protein required to achieve a thermal hysteresis of 0.3 °C is about 100 times lower when conjugated to the polymer (3 μM) compared to free protein (300 μM). Ice crystal morphologies observed in the presence of the reaction product are comparable to those of the protein used in the conjugation reaction.