High-level secretion of recombinant proteins from S. cerevisiae by co-expression of genes from 2-micron vectors.
Chris Finnis, Gill Shuttleworth, Darrell Sleep
Molecular Biology Department, Delta Biotechnology Ltd., 59 Castle Boulevard, Nottingham, NG7 1FD, United Kingdom
Delta Biotechnology has developed stable 2-micron vectors for the industrial production of recombinant human albumin (Recombumin ® ) and albumin fusion proteins (albufuse TM ) from S. cerevisiae at up to 5 g/L soluble protein in culture supernatants. These whole 2-micron vectors comprise solely yeast DNA and the cDNA of interest, which was inserted at a unique Sna BI-site near the origin of replication. Insertion of large DNA fragments at this Sna BI-site can lead to instability. However, the 2-micron plasmid is functionally crowded and contains few 'silent' regions for the insertion of additional DNA. Insertion of DNA at alternative sites frequently destabilises the plasmid by interfering with the replication and partitioning mechanism. A second insertion site has now been identified that allows additional DNA to be inserted with minimal impact on plasmid stability. This second site has been used for the stable co-expression of genes from whole 2-micron vectors. For example, a chaperone gene has been co-expressed from this site to assist the high level secretion of recombinant proteins. This resulted in a 15-fold increase in recombinant protein expression in shake flask culture and a corresponding titre of approximately 4 g/L fully functional protein in high cell density fermentation. This demonstrates that S. cerevisiae is a viable alternative to Pichia pastoris for the commercial production of recombinant proteins.