title: 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.
authors.affiliation: Institute of Molecular and Cell Biology, University of Tartu, Tartu, Estonia. tiina.tamm.001@ut.ee
authors.forename: Tiina
authors.initials: T
authors.lastname: Tamm
mesh_terms: D015153:Blotting, Western / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel; D000939:Epitopes / Q000235:genetics / Q000378:metabolism*; D015966:Gene Expression Regulation, Fungal / Q000235:genetics*; D005822:Genetic Vectors / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism*; D012568:Schizosaccharomyces / Q000235:genetics / Q000378:metabolism*; D013831:Thiamine / Q000378:metabolism*; D014170:Transformation, Genetic / Q000235:genetics*
chemical_list: D000939:Epitopes; D011994:Recombinant Proteins; D013831:Thiamine