Biosynthesis of winter flounder antifreeze proprotein in E.coli.
A semisynthetic winter flounder antifreeze proprotein (proAFP) coding region was constructed and inserted into a lacZ expression vector. ProAFP was produced from the vector in Escherichia coli as a C-terminal fusion to the first 289 amino acids of beta-galactosidase (beta-gal). The proAFP and beta-gal domains of the beta-gal-proAFP fusion protein were separated by the recognition signal for the blood coagulation protease, factor Xa. Upon induction with isopropylthio-beta-D-galactoside the fusion protein accumulated to levels of 15% of the total protein. The beta-gal-proAFP fusion protein was partially purified by differential centrifugation, but required solubilization prior to factor Xa digestion. The solubilized fusion protein was efficiently and correctly cleaved by factor Xa, after which the proAFP was purified by gel permeation. Bacterial proAFP was indistinguishable from natural proAFP by the criteria of antifreeze activity, amino-terminal sequence (15 cycles), reverse-phase HPLC and SDS-polyacrylamide gel electrophoresis. Circular dichroism measurements showed that proAFP is a composite of random coil and alpha-helical secondary structure, with an alpha-helix content of 44% at 0 degrees C. It seems probable that the C-terminal region of proAFP, which corresponds to the mature AFP protein, is mainly alpha-helical, and that the N-terminal pro-segment is random coiled.
Peters
I D
ID
Department of Biochemistry, Queen's University, Kingston, Ontario, Canada.
Hew
C L
CL
Davies
P L
PL
D000595:Amino Acid Sequence; D000818:Animals; D021301:Antifreeze Proteins; D001483:Base Sequence; D015153:Blotting, Western; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D005412:Flatfishes / Q000378:metabolism*; D005432:Flounder / Q000378:metabolism*; D005615:Freezing; D006023:Glycoproteins / Q000096:biosynthesis* / Q000302:isolation & purification; D008969:Molecular Sequence Data; D010957:Plasmids; D011487:Protein Conformation
D021301:Antifreeze Proteins; D006023:Glycoproteins