Regulatable promoters of Saccharomyces cerevisiae: comparison of transcriptional activity and their use for heterologous expression

Mumberg et al. (1994). Nucleic Acids Res DOI: 10.1093/nar/22.25.5767 Citations: 983

Regulated expression of heterologous genes in yeast needs promoters of known strength at low and high copy. This paper measured lacZ expression from MET25, GAL1, GALL and GALS promoters on CEN and 2-micron plasmids, with 2-micron GAL1 reaching 800 U.

Parts used

MET17 PromoterGAL1 Promoter

Key findings

  • In S. cerevisiae strain YPH499, the MET25 promoter driving lacZ gives beta-galactosidase activity of about 550 U from a 2-micron plasmid (p426MET25) without methionine and about 9 U from a centromeric plasmid (p416MET25) with 1 mM methionine, and the promoter responds to increasing methionine concentration in a logarithmic way (CYC1 terminator, mean of 5 independent colonies).
  • Repression of the MET25 promoter by 1 mM methionine leaves a rest activity of about 10%, estimated with the very stable beta-galactosidase reporter, so MET25 is graded rather than completely repressible. For proteins with a short half-life, only 1% residual activity was found (data not shown in the paper).
  • In S. cerevisiae strain YPH499 grown in glucose (repressing conditions), none of the lacZ expression vectors carrying the GAL1, GALL or GALS promoters on centromeric (p416) or 2-micron (p426) plasmids shows significant beta-galactosidase activity. After a shift of raffinose-grown cells to 4% galactose, activity ranges from about 10 U for the centromeric GALS promoter to about 800 U for the 2-micron GAL1 promoter.
  • The GALL and GALS promoters are deletion variants of the GAL1 promoter that lack either one (GALL) or one and a half (GALS) of the 3 UAS elements required for full galactose induction. They show low but fully repressible activity and are suited to conditional expression of genes that are essential for yeast cells but toxic when overexpressed from the full GAL1 promoter.
  • Combining the MET25, GAL1, GALL and GALS promoters (PCR-cloned as SacI/XbaI fragments upstream of the CYC1 terminator) with centromeric (CEN/ARS, pRS41X) or 2-micron (pRS42X) backbones carrying HIS3, TRP1, LEU2 or URA3 markers yields 32 yeast expression vectors that give regulated expression of heterologous proteins over a range of 2 to 3 orders of magnitude, with a polylinker providing up to 9 unique cloning sites.

Abstract

Dominik Mumberg, Rolf Muller, Martin Funk; Regulatable promoters of Saccharomyces cerevisiae: comparison of transcriptional activity and their use for heterolog