Pichia pastoris (P. pastoris) Cell Lines
Pichia pastoris (P. pastoris) is a widely used protein expression host for the production of biopharmaceuticals and industrial enzymes. P. pastoris belongs to methylotrophic yeasts, which share a common pathway to metabolize one-carbon compounds as carbon and energy sources. The species of methylotrophic yeasts, P. pastoris (recently reclassified as Komagataella pastoris) and H. polymorpha (also named Pichiaangusta) have been widely employed and become a substantial workhorse for biotechnology including heterologous protein production.
Figure: General considerations for heterologous gene expression in P. pastoris (Appl Microbiol Biotechnol, 2014)
Methylotrophic yeasts have two major features: (1) they are capable of growing to high cell densities even in unsophisticated fermentation process; (2) their high demand for methanol-oxidizing enzymes endows them with very strong and strictly regulated promoters. These features make it possible that methylotrophic yeasts can be used not only in the process development for the commercial production of feed protein (single cell protein) but also as production systems for recombinant proteins. The widely used P. pastoris and H.polymorpha have different genetics in alcohol oxidases expression: P. pastoris expresses AOX1 and AOX2 while H. polymorpha only expresses MOX. Besides P pastoris and H.polymorpha, P.methanolica and C. boidinii are also used as expression systems.
Just as shown in the figure, some points should be considered when P. pastoris is used to clone and express heterologous proteins. These considerations include the selection of host strain, the choice of promoter, transcription terminator (TT), markers combination and the application for either intracellular or secreted expression.
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Host strains: The widely used commercial available strains are mainly 5 classes, wild-type strains (e.g. X-33), auxotrophic strains (e.g. GS 115, KM71), protease-deficient strains (e.g. SMD 1168), glyco-engineered strains (e.g. SuperMan5) and some other strains. Notably, engineered P. pastoris has been able to secrete recombinant proteins with uniform human N-linked glycans. In natural and recombinant proteins, glycosylation is one of the most common PTMs (post-translational protein modifications), which impacts protein folding, solubility, stability, trafficking, bioavailability, immunogenicity and functional activity.The use of engineered P. pastoris broadens the applications of microbial systems in antibody expression.
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Vectors: The promoters for protein expression in P. pastorisinclude inducible promoters (AOX1, FLD1, ADH1, GUT1, etc.) and constitutive promoters (GAP, TEF1, etc.). For P. pastoris, HIS4 (auxotrophic markers) and zeocin resistance (dominant markers) are the most popularly markers used. The heterologous proteins can be intracellular or secreted expression. P. pastorishas the ability to secrete high titres of proteins into culture media. The prominently used secretion signals are derived from P. pastoris endogenous acid phosphatase(PHO1), S. cerevisiaeα-mating factor (α -MF) and S. cerevisiae invertase(SUC2).
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Creative Biolabs provides the following microbial systems for various proteins expression:
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➢ Bacterial systems: E.coli; Bacillus, Lactoccocus lactis, etc.
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➢ Yeast systems: Saccharomyces cerevisiae, Pichia pastoris, H. polymorpha ,etc.
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➢ Filamentous fungi
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➢ Unicellular algae
References:
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Danilo Porro and Diethard Mattanovich. Recombinant protein production in yeasts.Methods Mol Biol. 2012;824:329-58.
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Mudassar Ahmad, et al. Protein expression in Pichia pastoris: recent achievementsand perspectives for heterologous protein production. Appl Microbiol Biotechnol. 2014 Jun;98(12):5301-17.
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Dongxing Zha. Production of Monoclonal Antibodiesin Glycoengineered Pichia pastoris. Antibody expression and production (Mohamed AI-Rubeai editor): 77-98.
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