Inhibition of recrystallization in ice by chimeric proteins containing antifreeze domains.
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.
Mueller
G M
GM
DNA Plant Technology Corporation, Oakland, California 94608.
McKown
R L
RL
Corotto
L V
LV
Hague
C
C
Warren
G J
GJ
D021301:Antifreeze Proteins; D001483:Base Sequence; D002678:Chimera; D003460:Crystallization; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D005615:Freezing; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial; D006023:Glycoproteins / Q000235:genetics*; D007053:Ice; D008969:Molecular Sequence Data; D010957:Plasmids
D021301:Antifreeze Proteins; D006023:Glycoproteins; D007053:Ice