A Saccharomyces cerevisiae autoselection system for optimised recombinant protein expression.
Yeasts are attractive organisms for recombinant protein production. They combine highly developed genetic systems and ease of use with reductions in time and costs. We describe an autoselection system for recombinant protein expression in Saccharomyces cerevisiae which increases yields 5-10-fold compared to conditional selection for expression plasmids. Multicopy expression plasmids encoding essential MOB1 or CDC28 genes are absolutely necessary for the viability of host cells with mob1 or cdc28 deletions in their genomes. Such plasmids are stably maintained, even in rich medium, so optimising biomass production and yields of recombinant protein. Plasmid copy numbers are also increased by limiting selective MOB1 and CDC28 gene expression prior to induction. GST- or 6His-tagged proteins are produced for affinity purification and are expressed from a conditional GAL1-10 promoter to avoid potentially toxic effects of recombinant proteins on growth. Autoselection systems for expressing single or pairs of proteins are described. We demonstrate the versatility of this system by expressing proteins from a number of organisms and include several large and problematic products. The in vitro reconstruction of a step in mitotic regulation shows how this expression system can be successfully applied to the detailed analysis of complex metabolic pathways.
Geymonat
Marco
M
Division of Stem Cell Biology and Developmental Genetics, National Institute for Medical Research, Mill Hill, London NW7 1AA, UK.
Spanos
Adonis
A
Sedgwick
Steven G
SG
D000818:Animals; D017489:CDC28 Protein Kinase, S cerevisiae / Q000235:genetics; D018797:Cell Cycle Proteins / Q000235:genetics; D015966:Gene Expression Regulation, Fungal; D006801:Humans; D051379:Mice; D010750:Phosphoproteins / Q000235:genetics; D010957:Plasmids; D011401:Promoter Regions, Genetic; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000302:isolation & purification; D045473:Severe acute respiratory syndrome-related coronavirus / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics; D014764:Viral Proteins / Q000096:biosynthesis / Q000235:genetics
D018797:Cell Cycle Proteins; C110689:MOB1 protein, S cerevisiae; D010750:Phosphoproteins; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D014764:Viral Proteins; D017489:CDC28 Protein Kinase, S cerevisiae