• Codon optimisation and gene synthesis
• Subcloning of gene in yeast expression vector
• Transform construct into E coli strain
• Prepare and linearise plasmid and transform 3-5 yeast strains (Hefe)
• Small scale expression, screening & expression pilot
• Scale up culture and purification
• Affinity purification (Ni2+, GST, etc)
• QC: SDS-PAGE analysis, western blot, purity, concentration
• Project report
Lead time: 9-10 weeks
Pichia pastoris expresses a very low amount of secreted proteins meaning the majority of proteins in the media represent the recombinant protein of interest. This property clearly simplifies the downstream protein purification process.
Pichia pastoris can be grown at high cell density. Thus,this host organism allows for high yield protein production.
Pichia pastoris possesses the complete post-translational machinery characteristic of eukaryotic cells. Thus, this host organism is able to perform phosphorylations and glycosylations (different from those observed in mammalian cells protein expression).
To conclude, Pichia pastoris protein expression is the first choice for the production of secreted proteins.