Yeast services - General Info - Protein Expression Facility

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 host 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

Some drawbacks of yeast as a host cell expression system include:

PTMs are less complex when compared to higher eukaryotic systems (e.g. insect and mammalian cells)
Yeast’s tough cell wall is cumbersome to disrupt when processing intracellular proteins

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.

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
X-33
GS115r	Histidine auxotroph
KM71H	Strain uses weaker AOX2 promoter

How do I optimise my protein expression in yeast?

Several parameters can impact the success of protein production in the yeast expression system, as described below.

Promoter selection

Selection of the right promoter is critical for successful secreted protein production. Two common promoters used for recombinant protein production in yeast is the highly inducible P_AOX1 and constitutively expressive P_GAP promoters.

The alcohol oxidase 1 promoter (P_AOX1) supports a two-step process for gene regulation, repression and derepression. Commonly, glycerol is used to grow Pichia cells to maximise cell biomass until it is depleted. Methanol, the inducer, is then added into the culture medium for derepression of the promoter which allows for transcription of the gene of interest downstream of AOX1. Although very popular, the use of P_AOX1may not be optimal for commercial-scale fermentation productions due to risks associated with the storage of large quantities the toxic and highly flammable methanol.
The glyceraldehyde-3-phosphate dehydrogenase promoter (P_GAP) takes advantage of selecting a specific carbon source (e.g. glucose, glycerol, maltose methanol, oleic acid, sorbitol, or sucrose) for increasing cell biomass and recombinant target protein expression at the same time.

Clonal variability

Electroporation is the most common method to transform your cloned plasmid DNA into your yeast cell strain of choice. Homologous recombination between your linearised plasmid DNA and the Pichia genome occurs, denoted as a crossover “insertion” event. As this is a chromosomally-integrated system, clonal variability can occur i.e. some clones may have one single crossover event and other clones may have multiple crossovers. It is a general misconception that having more ‘insertions’ may lead to increased target protein expression however due to gene-dosage this varies from protein to protein. Identifying the ‘best” or “optimal” clone is recommended prior to any scale-up productions.

Codon optimisation

Codon optimisation is the process of modifying codons in a gene sequence to match the codon usage bias of the host cell used for expression. This is frequently applied to heterologous proteins expressed in P. pastoris, as codon usage can vary heavily between different organisms. Optimising the codon usage may improve the protein expression.