Expression of Recombinant Proteins in Yeast

Yeast systems have been a staple for producing large amounts of proteins for industrial and biopharmaceutical use for many years. Yeast can be grown to very high cell mass densities in well-defined medium. Recombinant proteins in yeast can be over-expressed so the product is secreted from the cell and available for recovery in the fermentation solution. Proteins secreted by yeasts are heavily glycosylated at consensus glycosylation sites. Thus, expression of recombinant proteins in yeast systems historically has been confined to proteins where post-translations glycosylation patterns do not affect the function of proteins. Several yeast expression systems are used for recombinant protein expression, including Sacharomyces, Scizosacchromyces pombe, Pichia pastoris and Hansanuela polymorpha.

In this article,* we discuss some of the technologies and aspects associated with biomanufacturing of therapeutics in yeast systems and highlight some techniques that are used in either basic research or clinical manufacturing, but are not typically utilized for both.

Yeast Expression Vectors

The expression of human proteins in prokaryotes has limitations in that prokaryotes do not have compartmentalized secretion system like eukaryotes and hence post-translational modifications like glycosylation do not occur. In cases like antibodies, these limiations also affect the proper folding of the proteins and hence their applicability. As a result, these proteins have to be made in higher eukaryotic mammalian cell systems. But mammalian systems are expensive and may render a very low yield. As a result, alternative systems using lower eukaryotes like yeasts Saccharomyces cerevisiae, Schizosaccharomyces pombe, and methylotropic yeasts like Pichia pastoris, and Pichia methanolica have gained importance. These systems do not glycosylate the same way as that of human glycosylation. For example, S. cerevisiae produces high mannose structures, and has been useful in producing properly folded active and soluble multi-subunit proteins.

As in any prokaryote, yeast expression systems also require an origin of replication or integration, a strong promoter, and a selection marker. Expression of recombinant proteins in S. cerevisiae can be done using three types of vectors: integration vectors (YIp), episomal plasmids (YEp), and centromeric plasmids (YCp).

YIp Vectors. The YIp integrative vectors are vectors that do not replicate autonomously, but integrate into the genome at low frequencies by homologous recombination. Integration of circular plasmid DNA by homologous recombination leads to a copy of the vector sequence flanked by two direct copies of the yeast sequence. Typically, YIp vectors integrate as a single copy. However, methods to integrate multiple copies and stable cell lines with up to 15-20 copies of recombinant gene integrations have been developed for over-expressing specific genes.2 YIp plasmids with two yeast segments, such as YFG1 and the URA3 marker, have the potential to integrate at either of the genomic loci, whereas vectors containing repetitive DNA sequences, such as Ty elements or rDNA, can integrate at any of the multiple sites within genome.2

YEp Vectors. The YEp yeast episomal plasmid vectors replicate autonomously because of the presence of a segment of the yeast 2 μm plasmid that serves as an origin of replication (2 μm ori). The 2 μm ori is responsible for the high copy-number and high frequency of transformation of YEp vectors. Most YEp plasmids are relatively unstable and even under conditions of selective growth, only 60 to 95 percent of the cells retain the YEp plasmid. The copy number of most YEp plasmids ranges from 10 to 40 copies per cell. Although this system is used for small scale expression studies, the use of YEp vectors in large-scale manufacturing is not advisable.

YCp Vectors. YCp yeast centromere plasmid vectors are autonomously replicating vectors containing centromere sequences (CEN), and autonomously replicating sequences (ARS). The YCp vectors are typically present at very low copy numbers from 1 to 3 per cell. These vectors are also relatively unstable and not very useful in high level expression but are used as regular cloning vectors (e.g., pYC2, pBM272).

Selection Markers