Skin-type antifreeze protein from the shorthorn sculpin, Myoxocephalus scorpius. Expression and characterization of a Mr 9, 700 recombinant protein.
A cDNA clone encoding a presumptive antifreeze protein was isolated from a skin library from shorthorn sculpin, Myoxocephalus scorpius. The clone encodes a 92-residue mature polypeptide (sssAFP-2) without any signal and prosequence, which suggests an intracellular localization. It is the largest alanine-rich, alpha-helical type I antifreeze protein known. A recombinant fusion protein containing an N-terminal-linked His-tag was produced and purified from Escherichia coli. This protein is alpha-helical at 0 degreesC and exhibits significant antifreeze activity. Northern blot and reverse transcription-polymerase chain reaction analyses indicate that sssAFP-2 mRNA has limited tissue distribution and is present in peripheral tissues such as skin and dorsal fin, but is notably absent in the liver. These studies reinforce recent evidence that indicate that the external tissues of cold water marine fishes are major organs for antifreeze protein synthesis and are likely the first line of defense against the threat of freezing.
Structural Biology and Biochemistry Division, Hospital for Sick Children, and Departments of Biochemistry and Laboratory Medicine and Pathobiology, University of Toronto, Toronto M5G 1L5, Ontario, Canada.
W K
WK
Low
M
M
Miao
K V
KV
Ewart
D S
DS
Yang
G L
GL
Fletcher
C L
CL
Hew
D000595:Amino Acid Sequence; D000818:Animals; D021322:Antifreeze Proteins, Type I; D001483:Base Sequence; D002942:Circular Dichroism; D018076:DNA, Complementary / Q000235:genetics; D029941:Fish Proteins; D005412:Flatfishes / Q000502:physiology*; D005615:Freezing; D015723:Gene Library; D006020:Glycopeptides / Q000096:biosynthesis / Q000235:genetics*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011487:Protein Conformation; D012333:RNA, Messenger / Q000032:analysis; D011994:Recombinant Proteins / Q000096:biosynthesis; D012621:Seasons; D017422:Sequence Analysis, DNA; D012879:Skin Physiological Phenomena / Q000235:genetics*; D014018:Tissue Distribution
D021322:Antifreeze Proteins, Type I; D018076:DNA, Complementary; D029941:Fish Proteins; D006020:Glycopeptides; D012333:RNA, Messenger; D011994:Recombinant Proteins; C114600:sssAFP-2 protein, Myoxocephalus scorpius