Isolation and characterization of an antifreeze protein precursor from transgenic Drosophila: evidence for partial processing.
Transgenic Drosophila melanogaster that contain a winter flounder antifreeze protein (AFP) gene fused to the transcriptional and translational control sequences of the host heat shock protein 70 gene express the transgene under heat shock conditions. They secrete into the hemolymph small quantities of a protein that reacts with antisera to AFP and is of a similar size to the proAFP precursor. To facilitate purification and characterization of this precursor, transformed fly lines homozygous for inserts on the 2nd, 3rd and X chromosomes were crossed together to generate a line with five and six AFP genes present in males and females, respectively. AFP production in the multi-gene line was approximately equal to the sum of that observed in the three starting lines and was just sufficient to perturb the growth of ice crystals. The AFP component was purified from heat-denatured hemolymph of this line by cation- and anion-exchange chromatography, followed by reverse-phase HPLC. Edman degradation sequencing of the purified protein showed that its N-terminus began two amino acids in from the predicted signal peptide cleavage point. An additional amino acid sequence was present that began two amino acids further into the 'pro' sequence. These AFP products are consistent with processing of the proAFP in Drosophila by a type IV dipeptidyl aminopeptidase, as has been suggested for processing in flounder.
Peters
I D
ID
Department of Biochemistry, Queen's University, Kingston, Canada.
Rancourt
D E
DE
Davies
P L
PL
Walker
V K
VK
D000595:Amino Acid Sequence; D000818:Animals; D030801:Animals, Genetically Modified; D021301:Antifreeze Proteins; D003433:Crosses, Genetic; D018819:Dipeptidyl Peptidase 4; D004152:Dipeptidyl-Peptidases and Tripeptidyl-Peptidases; D004331:Drosophila melanogaster / Q000235:genetics / Q000378:metabolism*; D005260:Female; D005432:Flounder / Q000235:genetics; D008297:Male; D008969:Molecular Sequence Data; D011498:Protein Precursors / Q000235:genetics / Q000302:isolation & purification*; D021382:Protein Sorting Signals; D011506:Proteins / Q000235:genetics / Q000302:isolation & purification*
D021301:Antifreeze Proteins; D011498:Protein Precursors; D021382:Protein Sorting Signals; D011506:Proteins; C079434:antifreeze protein precursor; D004152:Dipeptidyl-Peptidases and Tripeptidyl-Peptidases; D018819:Dipeptidyl Peptidase 4