Yeast Expression Systems

Yeasts are single cell microbial eukaryotes that have been used in food production by humans for thousands of years. The classic Baker's yeast, Saccharomyces cerevisiae was the first yeast to be used for recombinant protein production, but in the last 10 years Pichia pastoris has rapidly gained popularity and is a common choice today (Mattanovich et al., 2012).

Other yeasts that are increasingly used for heterologous protein production are Kluyveromyces lactis and Pichia angusta (formerly known as Hansenula polymorpha) (van Ooyen et al., 2006; Gellissen et al., 2005).

For the expression of heterologous protein in S. cerevisiae there are options for both episomal and integrative vectors. In both cases the promoters that direct expression of the gene of interest is either constitutive (usually from the glycolytic pathway) or inducible (PGAL1/PGAL10). Episomal vectors in S. cerevisiae make use of 2μ, which is a 6.3 kbp plasmid that can be extrachromosomally maintained at 50-100 copies in the nucleus.

Pichia pastoris and P. angusta are both methylotrophic yeasts and expression systems based on the strong promoters induced by methanol have been developed for both systems (Cregg et al., 2000; Gellissen et al., 2005). The PAOX1 promoter for alcohol oxidase 1 in P. pastoris is very strong and can drive expression of heterologous proteins up to several grams per liter.

Using methanol for induction has some technical safety complications since it is flammable and toxic. Other gene regulatory sequences have been adapted for use in recombinant protein production in P. pastoris and both inducible (PFLD; acetamide) and constitutive (PGAP) promoters are available (Cos et al., 2006).

Protein expression in the methylotrophic yeasts are always driven from vectors that have been integrated into chromosomal DNA by homologous recombination. Strains which have vectors stably integrated are commonly selected through antibiotic selection with Zeocin or with ade2 auxotrophs (commercialized by Life Technologies as Pichia Pink™).

Cytoplasmic proteins can be expressed successfully in P. pas- toris although it is more frequently used to secrete proteins. Complex eukaryotic proteins with multiple disulfide bridges have been expressed in P. pastoris. To enable secretion the pre- pro sequence from the a-mating factor of S. cerevisiae is introduced at the N-terminus of the protein to be expressed. The methylotropic yeasts are especially well suited for high- density cultivation and, in bioreactors, can reach over 120 g of dry weight per liter.

As eukaryotes, yeasts are capable of most posttranslational modifications including glycosylation. Depending on the use of the protein product, care should be taken since there are significant differences in the type of glycans added to proteins by yeast compared to mammalian cells.

Glycans added to the nitrogen of asparagine in the sequence Asn-Xaa-Ser/Thr (N- glycosylation) are similar to what is seen in mammalian cells. But in yeasts the produced N-glycosylation structures are different from what is seen in mammalian cells and are very rich in mannose, an effect termed hypermannosylation. In S. cerevisiae these chain are much longer (50-150) than what is usually seen in P. pastoris (8-17). Glycosylation of the O-type on serines/threonines also occur in P. pastoris but is less characterized.

Besides the yeasts described above, other unicellular microbial eukaryotes used for recombinant protein production are filamentous fungi such as Aspergillus spp. and Trichoderma spp. (Ward, 2012) and protozoans including Leishmania tar- entolae (Niimi, 2012). Their use for heterologous protein production in research laboratories has so far been limited. The interested reader can find more information about these systems in the suggested further reading.

 






Date added: 2024-06-13; views: 89;


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