A thiamine-regulatable epitope-tagged protein expression system in fission yeast

Abstract

Schizosaccharomyces pombe, the fission yeast, has been a popular and useful model system for investigating the mechanisms of biological processes for a long time. To facilitate purification, localization, and functional analysis of gene products, a wide range of expression vectors have been developed. Several of these vectors utilize the inducible/repressible promoter systems and enable the episomal expression of proteins as fusion proteins with epitope tags attached to their N terminus or C terminus. This chapter provides a detailed protocol for expression of the epitope-tagged proteins from thiamine-regulatable nmt promoter in fission yeast. The yeast culture conditions and procedures for yeast transformation, expression induction, preparation of whole-cell extracts, and analysis of epitope-tagged protein expression by Western blotting are described. © 2012 Springer Science+Business Media, LLC.