Protein expression in Pichia pastoris: recent achievements and perspectives for heterologous protein production

Abstract

Pichia pastoris is an established protein expression host mainly applied for the production of biopharmaceuticals and industrial enzymes. This methylotrophic yeast is a distinguished production system for its growth to very high cell densities, for the available strong and tightly regulated promoters, and for the options to produce gram amounts of recombinant protein per litre of culture both intracellularly and in secretory fashion. However, not every protein of interest is produced in or secreted by P. pastoris to such high titres. Frequently, protein yields are clearly lower, particularly if complex proteins are expressed that are hetero-oligomers, membrane-attached or prone to proteolytic degradation. The last few years have been particularly fruitful because of numerous activities in improving the expression of such complex proteins with a focus on either protein engineering or on engineering the protein expression host P. pastoris. This review refers to established tools in protein expression in P. pastoris and highlights novel developments in the areas of expression vector design, host strain engineering and screening for high-level expression strains. Breakthroughs in membrane protein expression are discussed alongside numerous commercial applications of P. pastoris derived proteins.

Fig. 1

General considerations for heterologous gene expression in P. pastoris. Expression plasmids harbouring the gene(s) of interest (GOI) are linearized prior to transformation. Selectable markers (e.g., Amp^R) and origin of replication (Ori) are required for plasmid propagation in E. coli. The expression level of the protein of interest may depend on (i) the chromosomal integration locus, which is targeted by the 5′ and 3′ homologous regions (5′HR and 3′HR), and (ii) on the gene copy number. A representative promoter (P) and transcription terminator (TT) pair are shown. Proper signal sequences will guide recombinant protein for intracellular or secretory expression, and will govern membrane integration or membrane anchoring

Fig. 2

Novel ‘Pichia Pool’ plasmid sets for intracellular and secretory expression. a General features of pXYZ vector for intracellular expression. Letters refer to the choice of promoters (X), selection markers (Y), and restriction enzymes (Z) for linearization. Available elements are shown in boxes. The vector backbone harbours an ampicillin resistance marker and origin of replication for maintenance of the plasmid in E. coli. The GOI is EcoRI–NotI cloned directly after the promoter of choice. The Kozak consensus sequence for yeast (i.e., CGAAACG), should be restored between the EcoRI cloning site and the start codon of the GOI in order to achieve optimal translation. In addition, sequence variation within this region will allow fine-tuning translation initiation efficiency. Expression in P. pastoris is driven either by the methanol inducible AOX1 or the constitutive GAP promoter. Positive clones can be selected for by antibiotic resistance (i.e., to Zeocin™ or geneticin sulphate) or by selection for His or Arg prototrophy. Selection marker expression is uniformly driven by the ARG4 promoter–terminator pair. b Plasmid pAaZBgl from ‘Pichia Pool’ is shown as an example of a vector made for secretory expression encoding S. cerevisiae α-MF signal sequence in front of the GOI cloning site. The Kex2 processing site AAAAGA should be restored between the XhoI cloning site and the fusion point of the GOI