title: Regulated overproduction of the GAL4 gene product greatly increases expression from galactose-inducible promoters on multi-copy expression vectors in yeast.
abstract: High-level, galactose-inducible expression originating from GAL promoters in Saccharomyces cerevisiae is mediated by highly specific interactions between the GAL4-coded protein and nucleotide sequences. The potential utility of recombinant GAL promoter/foreign gene constructions for the regulatable and high-level expression of foreign proteins in yeast is well recognized. However, the utility of this system is limited severely in the case of multiple copies of such constructions due to the very low level of the GAL4-coded protein and to the loss of inducibility which occurs if levels of the GAL4 protein are amplified constitutively. To surmount these limitations, we have constructed a novel yeast strain which overproduces the GAL4 protein in a regulated fashion. This 'integrant' strain contains an integrated copy of a hybrid gene consisting of the galactose-inducible GAL10 promoter fused to the GAL4 structural gene. In the absence of galactose, the integrant strain and the isogenic 'non-integrant' parental strain show only a basal level of transcription from the constitutively active chromosomal GAL4 gene. However, following the addition of galactose to the culture medium, the 'integrant' strain synthesizes at least 20-fold more GAL4 mRNA and substantially more GAL4 protein than the 'non-integrant' strain. A high-copy-number expression vector containing the GAL10 promoter and alpha mating factor pre-pro leader fused to the structural gene for Epstein-Barr virus gp350 was introduced into both types of cells. The resulting transformed 'integrant' cells produced approximately five-fold more gp350 mRNA and ten-fold more gp350-related proteins than the transformed 'non-integrant' cells following galactose induction. This 'integrant' strain should prove generally useful for the maximal, regulated expression in yeast of structural genes driven by a galactose-inducible promoter.
authors.affiliation: Department of Virus and Cell Biology, Merck Sharp & Dohme Research Laboratories, West Point, PA 19486.
authors.forename: L D
authors.initials: LD
authors.lastname: Schultz
authors.forename: K J
authors.initials: KJ
authors.lastname: Hofmann
authors.forename: L M
authors.initials: LM
authors.lastname: Mylin
authors.forename: D L
authors.initials: DL
authors.lastname: Montgomery
authors.forename: R W
authors.initials: RW
authors.lastname: Ellis
authors.forename: J E
authors.initials: JE
authors.lastname: Hopper
mesh_terms: D004274:DNA, Recombinant; D005690:Galactose; D005786:Gene Expression Regulation; D005800:Genes, Fungal; D005814:Genes, Viral; D005818:Genetic Engineering; D005822:Genetic Vectors; D006023:Glycoproteins / Q000235:genetics; D004854:Herpesvirus 4, Human / Q000235:genetics; D011401:Promoter Regions, Genetic; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D014764:Viral Proteins / Q000235:genetics
chemical_list: D004274:DNA, Recombinant; D006023:Glycoproteins; D014764:Viral Proteins; D005690:Galactose