The antifreeze peptide AFP6 from the polar fish Pseudopleuronectus ame
ricanus has been expressed in and secreted by the yeast Saccharomyces
cerevisiae as a biologically active molecule. The gene for the 37 amin
o acid long peptide has been chemically synthesized using yeast prefer
red codons. Subsequently, the gene has been cloned into an episomal ex
pression vector as well as in a multicopy integration vector, which is
mitotically more stable. The expression is under the control of the i
nducible GAL7 promoter. The enzyme alpha-galactosidase has been invest
igated as a carrier protein to facilitate expression and secretion of
AFP. In order to reach increased expression levels, tandem repeats of
the AFP gene (up to eight copies) have been cloned. In most cases the
genes are efficiently expressed and the products secreted. The express
ion level amounts to approximately 100 mg/l in the culture medium. In
a number of genetic constructs the genes are directly linked and expre
ssed as AFP multimers. In other constructs linker regions have been in
serted between the AFP gene copies, that allow the peptide to be proce
ssed by specific proteinases, either from the endogenous yeast proteol
ytic system or from a non-yeast source. The latter requires a separate
processing step after yeast cultivation to obtain mature AFP. In all
these cases proteolytic processing is incomplete, generating a heterog
eneous mixture of mature AFP, carrier and chimeric protein, and/or a m
ixture of AFP-oligomers. The antifreeze activity has been demonstrated
for such mixtures as well as for AFP multimers.