Expression and secretion of antifreeze peptides in the yeast Saccharomyces cerevisiae

Abstract

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.

Heterologous Expression of Human Cholecystokinin in Saccharomyces cerevisiae

“…Thus, it was not surprising that pro-CCK processing was abolished at the Arg 105 -Arg 106 residues in a kex2 mutant. As seen in other instances where Kex2 has been responsible for post-translational processing, not all of the proprotein was processed at this site (Miyajima et al, 1986;Thim et al, 1986;Zsebo et al, 1986;Moody et al, 1987;Driedonks et al, 1995). Notably, even when low amounts of pro-CCK were passed through the yeast secretory pathway, as was the case when prepro-CCK was expressed in yeast, there were still relatively high quantities of C-terminally extended CCK secreted.…”

Bactericidal Effects of a Fusion Protein of Llama Heavy-Chain Antibodies Coupled to Glucose Oxidase on Oral Bacteria

“…Episomal S. cerevisiae expression vectors were derived from pUR2741 (9). For the production of GOx, the GOx gene from A. niger (11) was modified.…”

Stable, High-Level Expression of a Type I Antifreeze Protein inEscherichia coli

“…Recrystallization inhibition activity was assayed by a variation of the splat assay (18,29). A 1-l droplet of rAFP solution, water, or buffer control was spread between two coverslips and placed in the chamber of a Linkam HFS91 cold stage attached to a Zeiss photomicroscope.…”

“…The apparent simplicity of structure of the type I AFPs makes them particularly attractive for such purposes, enabling relatively easy genetic manipulation as well as experimental (4,10 -12) and theoretical (13)(14)(15)(16) analysis of their ice-binding properties. However, their small size (M r 3000 -5000) and lack of globular tertiary structure have apparently rendered this class of protein susceptible to degradation when expressed in heterologous host systems such as Escherichia coli, yeast, Drosophila, and plants (17)(18)(19)(20)(21). This has necessitated their expression as fusion proteins or with signal sequences directing their secretion into the culture medium (18,(21)(22)(23).…”

“…However, their small size (M r 3000 -5000) and lack of globular tertiary structure have apparently rendered this class of protein susceptible to degradation when expressed in heterologous host systems such as Escherichia coli, yeast, Drosophila, and plants (17)(18)(19)(20)(21). This has necessitated their expression as fusion proteins or with signal sequences directing their secretion into the culture medium (18,(21)(22)(23). Although such fusion proteins have generally tended to retain AFP activity it has been necessary to engineer suitable chemical or proteolytic cleavage sites and undertake further processing and purification steps to generate the mature form of the AFP.…”