SKIN-TYPE ANTIFREEZE PROTEIN FROM THE SHORTHORN SCULPIN, MYOXOCEPHALUS-SCORPIUS - EXPRESSION AND CHARACTERIZATION OF A M-R-9,700 RECOMBINANT PROTEIN

A cDNA clone encoding a presumptive antifreeze protein was isolated fr om a skin library from shorthorn sculpin, Myoxocephalus scorpius. The alone encodes a 92-residue mature polypeptide (sssAFP-2) without any s ignal and prosequence, which suggests an intracellular localization. I t is the largest alanine-rich, alpha-helical type I antifreeze protein known. A recombinant fusion protein containing an N-terminal-linked H is-tag was produced and purified from Escherichia coli. This protein i s alpha-helical at 0 degrees C and exhibits significant antifreeze act ivity. Northern blot and reverse transcription-polymerase chain reacti on analyses indicate that sssAFP-2 mRNA has limited tissue distributio n and is present in peripheral tissues such as skin and dorsal fin, bu t is notably absent in the liver. These studies reinforce recent evide nce that indicate that the external tissues of cold water marine fishe s are major organs for antifreeze protein synthesis and are likely the first line of defense against the threat of freezing.