Expression of Recombinant Proteins in Yeast - - BioPharm International


Expression of Recombinant Proteins in Yeast

Recombinant genes are transferred to Pichia pastoris by shuttle vectors designed with the promoter, a cloning site for gene insertion, regulatory elements for maintenance in E. coli, and regulatory elements for maintenance and expression in Pichia. The vector is integrated into the Pichia genome by homologous recombination, either at the promoter site (AOX or GAP), or at the site of an auxotrophic gene (i.e., His4 in the commonly used GS115 strain). Thus, integration of a plasmid would allow selection on media devoid of a given amino acid such as histadine. Alternatively, vectors are available with the Sh ble gene, allowing for selection in media containing Zeocin. Zeocin is toxic and should be avoided in the manufacture of clinical use materials. Secretion signals are available for Pichia that will allow accumulation of recombinant proteins in culture medium with the signal sequences removed during secretion. The secretion signal from the Saccharomyces α-factor is the most common, but the signal sequence of the acid phosphatase (PHO1) gene is also available.14

The site of insertion of a recombinant gene will determine the phenotype of an otherwise wild-type recombinant Pichia strain. A vector cloned into a selection gene, such as His4, will grow normally on methanol, a Mut+ phenotype. There are two genes that encode AOX: AOX1, from which most AOX is derived, and AOX2, from which about 15% of AOX is derived.15 The AOX1 promoter is used to drive recombinant expression. If the vector is integrated into the host AOX1 site, the ability of the strain to utilize methanol is significantly diminished, leading to a slow methanol utilization or MutS phenotype. If both the AOX1 and AOX2 genes are knocked out, then the strain cannot utilize methanol and has a Mut phenotype. The non-wild type strains will sometimes express higher levels of product and have different fermentation strategies that can simplify the process.14,18

Stratton et al. provide methods for growth and induction of product from each phenotype and advantages for each.18 An important note is that Pichia growth is rapid on glycerol or glucose, but these carbon sources repress expression from the AOX1 promoter. The primary advantage of the Mut+ strain is the rapid growth on methanol and high rate of production of foreign protein. The disadvantage is sensitivity to high methanol concentrations and a need to regulate the amount of methanol fed, typically requiring biomass accumulation using glycerol or glucose and a subsequent shift to methanol. The MutS strains are much less sensitive to methanol concentration but grow much more slowly on methanol. Mut strains cannot utilize methanol as a carbon source, and another source is needed, but can use methanol as an inducer. Thus, feeding a limiting amount of carbon source and maintaining a low level of methanol will allow for culture growth and product production. A side benefit of the use of lower amounts of methanol is related to the safety aspects of methanol, which is flammable and combustible. Large-scale fermentation will require a significant amount of methanol and proper measures must be in place to ensure safety. A license from Research Corporation Technologies is necessary for the use of Pichia pastoris as a production strain for commercial or research purposes.

Recently, a novel system with the capability of producing recombinant glycoproteins in yeast has emerged with glycosylation sequences similar to secreted human glycoproteins produced in mammalian cells. The glycosylation pathway of Pichia pastoris was modified by eliminating endogenous enzymes, which add high mannose chains to N-glycosylation intermediates. In addition, at least five active enzymes, involved in synthesizing humanized oligosaccharide chains, were specifically transferred into P. pastoris. The results by Choi, et al. and by Hamilton et al. demonstrated the proper localization of mannosidases I and II, N-acetylglucosaminyltransferases I and II, and UDP-GlcNAc transporter into recombinant strains of P. pastoris lacking the Och1p alpha-1-6-mannosyltransferase, which initiates yeast but not human type glycosylation.19,20

The ability to produce large quantities of humanized glycoproteins in yeast could offer advantages in that glycosylated structures could be highly uniform and easily purified. In addition, cross-contamination with mammalian viruses and other mammalian host glycoproteins may be eliminated by using fed-batch production in yeast with much shorter fermentation times than mammalian cells.

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