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Protein Engineering vol. 4 no. 8 pp. 995-1002, 1991
© 1991 Oxford University Press

RESEARCH-ARTICLE

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

Xiaomao Li, Khiet Y. Trinh and Choy L. Hew1

Research Institute, The Hospital for Sick Children and the Departments of Biochemistry and Clinical Biochemistry, University of Toronto 100 College Street, Toronto, Ontario, Canada M5G 1L5

1To whom correspondence should be addressed at Department of Clinical Biochemistry

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 XPL 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 ({small tilde}4.5°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.

Keywords: expression plasmid/ice-binding/improved thermal stability

Received October 5, 1990; accepted August 1, 1991.


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[Abstract] [Full Text] [PDF]


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