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 PAOX1 and constitutively expressive PGAP promoters.
- The alcohol oxidase 1 promoter (PAOX1) 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 PAOX1 may 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 (PGAP) 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.