Expression and characterization of an active and thermally more stable recombinant antifreeze polypeptide from ocean pout, Macrozoarces americanus, in Escherichia coli: improved expression by the modification of the secondary structure of the mRNA.
The cDNA clone coding for the ocean pout antifreeze polypeptide (AFP) was modified to improve translation of its mRNA in Escherichia coli. A recombinant AFP (rAFP), MetLys-AFP-Lys, was expressed successfully using the lambda PL promoter, and constituted 1-2% of total bacterial proteins. The rAFP was purified to homogeneity from the soluble fractions of bacterial extracts. Its identity was confirmed by amino acid analysis, automated Edman degradation, immuno-blot and activity measurements. Although the rAFP is indistinguishable from the authentic AFP in its secondary structure, thermal hysteretic activity and the alteration of ice crystal structure, it is, however, thermally more stable (approximately 4.5 degrees C increase in Tm) and is more effective in inhibiting ice growth along the a-axis. These investigations indicate that the extra amino acids in rAFP significantly improve the thermal stability and ice-binding activity of the polypeptide.
Li
X M
XM
Research Institute, Hospital for Sick Children, Toronto, Ontario, Canada.
Trinh
K Y
KY
Hew
C L
CL
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D021301:Antifreeze Proteins; D001483:Base Sequence; D002942:Circular Dichroism; D004926:Escherichia coli / Q000235:genetics; D005399:Fishes / Q000235:genetics*; D005615:Freezing; D015964:Gene Expression Regulation, Bacterial; D006023:Glycoproteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D007053:Ice; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D011401:Promoter Regions, Genetic / Q000235:genetics; D012333:RNA, Messenger / Q000737:chemistry / Q000235:genetics*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification
D000596:Amino Acids; D021301:Antifreeze Proteins; D006023:Glycoproteins; D007053:Ice; D012333:RNA, Messenger; D011994:Recombinant Proteins