Yeast are a single-celled eukaryotic organisms that combine high levels of recombinant protein production with eukaryotic post-translational modifications (PTMs). Some advantages of using yeast over other systems for protein expression include:

  • Relatively inexpensive setup and running costs
  • Very high levels of recombinant protein production
  • Able to perform many PTMs e.g. N-glycosylation
  • Simplified downstream processing for secreted proteins
  • Amenable to large-scale fermentation

Yeast Expression Hosts

There are two strains commonly used for protein production in yeast: Saccharomyces cerevisiae and Pichia pastoris.P. pastoris has a number of advantages over S. cerervisiae that make it the preferred host cell for expression: a strong, inducible promoter, high growth rates and PTM patterns more similar to higher eukaryotes.

P. pastoris is a methylotrophic yeast, capable of using methanol as the sole source of carbon. In the presence of methanol, the alcohol oxidase gene (AOX1) is highly up-regulated and can account for up to 30% of total cell protein. Current expression systems exploit this feature and use the AOX1 promoter to drive recombinant protein expression in the presence of methanol.

P. pastoris does not secrete many native proteins. Consequently, by using the alpha-mating factor secretion signal peptide and exploiting the secretory pathway, downstream processing can be greatly simplified. Secreting recombinant proteins also avoids the need to disrupt the tough yeast cell wall for release of intracellular proteins.

Some common P. pastoris expression strains are listed below.

Strain Features
PichiaPink Strain 1 Adenine auxotroph
PichiaPink Strain 2 Adenine auxotroph, Protease A knockout
PichiaPink Strain 3 Adenine auxotroph, Protease B knockout
PichiaPink Strain 4 Adenine auxotroph, Protease A and Protease B knockout
GS115r Histidine auxotroph
KM71H Strain uses weaker AOX2 promoter

Some vectors commonly used for expression in P. pastoris are listed below.

Strain Selection Features
pPink-αHC Nutrient-based (Adenine) Complements ade2 deletion, secretion via α-mating factor
pPICZα-A,B,C Zeocin Secretion via α-mating factor, 6xHis and C-myc C-term tags
pPICZ-A,B,C Zeocin 6xHis and C-myc C-term tags
pHIL-S1 Nutrient-based (Histidine) Complements HIS4 deletion, secretion via PHO1 secretion signal

Post-translational Modifications (PTMs)

Yeast are capable of performing a basic subset of PTMs including N-glycosylation, the most common modification. The N-glycan structures produced by yeast are less complex compared to higher eukaryotic systems (e.g. insect and mammalian cells), and contain large amounts of mannose. These high mannose structures can be removed with the aid of enzymes such as N-Glycosidase F (PNGase).

Recent advances in genetic engineering have led to the development of yeast strains that produce mammalian-like N-glycosylation structures. These strains carry a relatively large subset of genes of mammalian origin that aid in building more complex structures and trafficking of the modifying enzymes within the cell.