A novel copper-regulated promoter system for expression of heterologous proteins in Schizosaccharomyces pombe.
The increasing use of the fission yeast Schizosaccharomyces pombe as a model organism for elucidating the mechanisms of critical biological processes such as cell-cycle control, DNA replication, and stress-mediated signal transduction has fostered the development and utilization of expression systems for gene function analysis. Using the promoter of the ctr4(+) copper transporter gene from S. pombe, we created a series of vectors, named pctr4(+)-X, which regulate the expression of heterologous genes as a function of copper availability. In this system, the addition of copper ions at levels that are non-toxic to yeast cells represses gene expression, while copper deprivation strongly induces gene expression. Conveniently, changes of growth medium or carbon sources are not required to shut down or induce gene expression. The Cu-starvation-mediated inducible expression system is rapid, producing heterologous proteins within 3 h, with sustained expression of proteins that persists for several hours. The pctr4(+)-X expression vectors harbor unique restriction sites constructed in-frame to DNA sequences encoding for epitope tags, which facilitate the detection or purification of the heterologous proteins using commercially available antibodies and affinity columns. Furthermore, the pctr4(+)-X copper-regulatable protein expression vectors have been constructed with three different selectable markers, offering more versatility for studying gene function in fission yeast.
Bellemare
D R
DR
Département de Biochimie, Université de Sherbrooke, J1H 5N4, Sherbrooke, QC, Canada.
Sanschagrin
M
M
Beaudoin
J
J
Labbé
S
S
D027682:Cation Transport Proteins; D003300:Copper / Q000494:pharmacology*; D000079923:Copper Transporter 1; D004305:Dose-Response Relationship, Drug; D005786:Gene Expression Regulation / Q000187:drug effects; D005822:Genetic Vectors / Q000235:genetics; D008565:Membrane Proteins / Q000235:genetics; D020928:Mitogen-Activated Protein Kinases / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011506:Proteins / Q000235:genetics*; D012568:Schizosaccharomyces / Q000235:genetics* / Q000254:growth & development; D029702:Schizosaccharomyces pombe Proteins
D027682:Cation Transport Proteins; D000079923:Copper Transporter 1; D008565:Membrane Proteins; D011506:Proteins; D029702:Schizosaccharomyces pombe Proteins; D003300:Copper; D020928:Mitogen-Activated Protein Kinases; C095409:sty1 protein, S pombe