Antifreeze Protein in Antarctic Marine Diatom, Chaetoceros neogracile

The antifreeze protein gene (Cn-AFP) from the Antarctic marine diatom, Chaetoceros neogracile was cloned and characterized. The full-length Cn-AFP cDNA contained an open reading frame of 849 bp and the deduced 282 amino acid peptide chain encodes a 29.2 kDa protein, which includes a signal peptide of 30 amino acids at the N terminus. Both the Cn-AFP coding region with and without the signal sequence were cloned and expressed in Escherichia coli. Recombinant Cn-AFPs were shown to display antifreeze activities based on measuring the thermal hysteresis and modified morphology of single ice crystals. Recombinant mature Cn-AFP showed 16-fold higher thermal hysteresis activity than that of pre-mature Cn-AFP at the same concentration. The ice crystal shape changed to an elongated hexagonal shape in the presence of the recombinant mature Cn-AFP, while single ice crystal showed a circular disk shape in absence of Cn-AFP. Northern analysis demonstrated a dramatic accumulation of Cn-AFP transcripts when the cells were subjected to freezing stress. This rapid response to freeze stress, and the antifreeze activity of recombinant Cn-AFPs, indicates that Cn-AFP plays an important role in low temperature adaptation.