Cystine-rich fish antifreeze is produced as an active proprotein precursor in fall armyworm cells.
Recombinant cystine-rich fish antifreeze protein (AFP) was produced by fall armyworm cells from a baculovirus expression vector containing sea raven AFP cDNA. The natural signal sequence encoded in the cDNA directed secretion of the antifreeze into the medium, from where it was recovered and purified to homogeneity. The M(r) of the exported protein (16k), as determined by SDS-PAGE, was larger than that (14k) of mature AFP isolated from sea raven serum. Sequencing of the recombinant AFP showed that it had a 17-amino-acid extension N-terminal to the 129-amino-acid mature AFP that began where signal peptide cleavage should occur according to current algorithms. Recombinant proAFP had antifreeze activity equivalent to that of the mature AFP, which indicates that the disulfide bonds were correctly formed and that the ice-binding site on the antifreeze is not sterically hindered by the 17-amino-acid N-terminal extension.
Duncker
B P
BP
Department of Biology, Queen's University, Kingston, Ontario, Canada.
Gauthier
S Y
SY
Davies
P L
PL
D000595:Amino Acid Sequence; D000818:Animals; D021301:Antifreeze Proteins; D021341:Antifreeze Proteins, Type II; D001483:Base Sequence; D002352:Carrier Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D002460:Cell Line; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D005399:Fishes / Q000378:metabolism*; D005615:Freezing; D006023:Glycoproteins / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D007700:Kinetics; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D014176:Protein Biosynthesis; D011498:Protein Precursors / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D018411:Spodoptera; D014162:Transfection
D021301:Antifreeze Proteins; D021341:Antifreeze Proteins, Type II; D002352:Carrier Proteins; D017931:DNA Primers; D006023:Glycoproteins; D011498:Protein Precursors; D011994:Recombinant Proteins; C089666:antifreeze protein, sea raven