J. Biol. Chem., Vol. 266, Issue 12, 7339-7344, Apr, 1991
Inhibition of recrystallization in ice by chimeric proteins containing antifreeze domains
GM Mueller, RL McKown, LV Corotto, C Hague and GJ Warren
DNA Plant Technology Corporation, Oakland, California 94608.
Using synthetic DNA, we assembled a gene encoding a protein identical in
sequence to one of the antifreeze proteins produced by the fish
Pseudopleuronectes americanus (winter flounder). To address the
relationship between structure and function, we also assembled genes
encoding proteins varying in sequence and length. The synthetic genes were
cloned into a bacterial expression vector to generate translational fusions
to the 3' end of a truncated staphylococcal protein A gene; the chimeric
proteins encoded by these fusions, varying only in their antifreeze
domains, were isolated from Escherichia coli. The antifreeze domains
conferred the ability to inhibit ice recrystallization, which is
characteristic of naturally occurring antifreeze proteins, on the chimeric
proteins. The chimeric proteins varied in their effectiveness of inhibiting
ice recrystallization according to the number of 11-amino acid repeats
present in the antifreeze moiety. A protein with only two repeats lacked
activity, while the inhibitory activity increased progressively for
proteins containing three, four, and five repeats. Some activity was lost
upon removal of either the salt bridge or the carboxyl-terminal arginine,
but surprisingly, not when both features were absent together.
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