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 mode l organism for elucidating the mechanisms of critical biological processes such as cell-cycle control, DNA replication, and stress-mediated signal tra nsduction has fostered the development and utilization of expression system s for gene function analysis. Using the promoter of the ctr4(+) copper tran sporter gene from S. pombe, we created a series of vectors, named pctr4(+)- X, which regulate the expression of heterologous genes as a function of cop per availability. In this system, the addition of copper ions at levels tha t are non-toxic to yeast cells represses gene expression, while copper depr ivation strongly induces gene expression. Conveniently, changes of growth m edium or carbon sources are not required to shut down or induce gene expres sion. The Cu-starvation-mediated inducible expression system is rapid, prod ucing heterologous proteins within 3 h, with sustained expression of protei ns that persists for several hours. The pctr4(+)-X expression vectors harbo r unique restriction sites constructed in-frame to DNA sequences encoding f or epitope tags, which facilitate the detection or purification of the hete rologous proteins using commercially available antibodies and affinity colu mns. Furthermore, the pctr4(+)-X copper-regulatable protein expression vect ors have been constructed with three different selectable markers, offering more versatility for studying gene function in fission yeast. (C) 2001 Els evier Science B.V. All rights reserved.