Oral Presentation | Open | Published:
Enhanced protein expression through strain selection, gene disruption, improved vector design and co-expression of endogenous chaperones
Microbial Cell Factoriesvolume 5, Article number: S29 (2006)
The use of Saccharomyces cerevisiae as a host system has been limited by the perception of limited secretion capacity, unstable episomal vectors and aberrant glycosylation. Solutions to all of these limitations are now available.
An analysis of a series of haploid laboratory yeast strains revealed significant intra-strain variability and unstable plasmid segregation. By combining classic chemical mutagenesis and selection a family of highly efficient Saccharomyces cerevisiae strains has been developed for the commercial production of biopharmaceutical products. When combined with a stable , high copy number , episomal expression vector system and a strong constitutive promoter, secreted recombinant protein expression titres in excess of 4 g/L were achieved (see Figure 1). Specific genetic modifications to the host were also introduced to increase product yield and control post-translational modifications, such as proteolysis and glycosylation.
The expression vectors have been further enhanced to facilitate the stable co-expression of multiple proteins. When one of these proteins is a chaperone, the titre of co-expressed recombinant transferrin was increased 15-fold. The applicability of this system has been demonstrated with a wide range of heterologous proteins and is scalable from 10 mL shake flask to cGMP manufacture at high cell density fermentation (8,000 L) in a defined synthetic medium; designed to be integrated with cost-efficient downstream processing.
Significant intra-strain variability and unstable episomal plasmid systems have limited the usefulness of Saccharomyces cerevisiae as an industrial host for the production of biopharmaceuticals. However co-enhancement of the episomal vector system and the host strains is not only possible but has led to significant improvements in recombinant protein production.
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