The antifreeze peptide AFP6 from the polar fish Pseudopleuronectus americanus has been expressed in and secreted by the yeast Saccharomyces cerevisiae as a biologically active molecule. The gene for the 37 amino acid long peptide has been chemically synthesized using yeast preferred codons. Subsequently, the gene has been cloned into an episomal expression vector as well as in a multicopy integration vector, which is mitotically more stable. The expression is under the control of the inducible GAL7 promoter. The enzyme alpha-galactosidase has been investigated 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 expression level amounts to approximately 100 mg/l in the culture medium. In a number of genetic constructs the genes are directly linked and expressed as AFP multimers. In other constructs linker regions have been inserted between the AFP gene copies, that allow the peptide to be processed by specific proteinases, either from the endogenous yeast proteolytic 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 heterogeneous mixture of mature AFP, carrier and chimeric protein, and/or a mixture of AFP-oligomers. The antifreeze activity has been demonstrated for such mixtures as well as for AFP multimers.
An enzyme-linked immunosorbent assay (ELISA) is described for the quantitative analysis of soya protein in meat products. The assay is applicable to raw and sterilized products and is not dependent on soya variety and type of soya ingredient (concentrate, isolate, etc.). Therefore, the assay can be applied without knowledge of product composition, heat pretreatment and other processing conditions. The ELISA is specific for soya; no interference of other product components has been found. The detection limit of the ELISA is 0.5% soya protein. Qualitative analysis by immunoblotting is possible at much lower concentrations. The antibodies used are specific for sodium dodecyl sulphate (SDS) denatured soya protein subunits. The products are extracted with SDS and 2-mercaptoethanol, which guarantees optimum recovery of the protein components. Product extracts can be analysed directly by ELISA without removal of the SDS by using nitrocellulose as a solid phase.
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