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Gene. 2007 Sep 15;399(2):120-8. Epub 2007 May 22.

A Saccharomyces cerevisiae autoselection system for optimised recombinant protein expression.

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1
Division of Stem Cell Biology and Developmental Genetics, National Institute for Medical Research, Mill Hill, London NW7 1AA, UK.

Abstract

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.

PMID:
17566670
DOI:
10.1016/j.gene.2007.05.001
[Indexed for MEDLINE]
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