Expression of a Winter Flounder Antifreeze Protein in E. coli and its Effect on Ice Nucleation

Abstract

Ice-binding proteins are known to interact with ice to depress freezing point non-colligatively or to inhibit ice recrystallization; however, their effects on ice nucleation are not fully understood. The antifreeze proteins of fish are among the best-studied ice-binding proteins, and AFP6 found in the winter flounder plasma is a small alpha-helical antifreeze protein that depresses the freezing point. In this study, recombinant AFP6 was expressed intracellularly in E.coli as fusion protein, then cleaved to produce monomeric active AFP6. A mutant AFP6 without antifreeze activity was produced in the same manner. AFP6 and its fusion protein precursor exhibited similar antifreeze activity, whereas the mutant AFP had no measurable antifreeze activity. AFP6 and its fusion protein precursor were both found to inhibit silver iodide-induced ice nucleation. Conversely, AFP6 was observed to promote uninduced ice nucleation at lower temperatures, while its fusion protein precursor and the AFP6 mutant did not.