title: A novel GAL recombinant yeast strain for enhanced protein production.
abstract: A novel strain of Saccharomyces cerevisiae in which the GAL1 gene was replaced with the GAL4 gene has been designed. The GAL1 gene encodes galactokinase (Gal1p), an enzyme that phosphorylates galactose. Gal4p activates genes necessary for galactose metabolism and is among the best characterized transcription activators. Here we describe a GAL recombinant strain that contains the GAL4 gene fused to the natural GAL1 promoter in addition to the normal constitutively expressed chromosomal GAL4 gene. To evaluate whether both gratuitous induction and regulated overexpression of the positive regulator improve protein production, low- and multi-copy expression vectors containing the GAL1 promoter fused to the structural gene for green fluorescent protein (GFP) were introduced into wild-type, gal1 and GAL recombinant strains. In yeast containing the multi-copy plasmid there was an approximately 3.3-fold increase in GFP production in the gal1 mutant strain. Moreover, in the resulting GAL recombinant cells a 4.6-fold increase in fluorescence relative to the wild-type was observed. The GAL recombinant strain should therefore prove useful for maximal expression of heterologous genes driven by a galactose-inducible promoter.
authors.affiliation: National Institute of Chemistry, Laboratory for Biosynthesis and Biotransformation, Hajdrihova 19, SI-1000 Ljubljana, Slovenia. mateja.novak@ki.si
authors.forename: Mateja Novak
authors.initials: MN
authors.lastname: Stagoj
authors.forename: Aleksandra
authors.initials: A
authors.lastname: Comino
authors.forename: Radovan
authors.initials: R
authors.lastname: Komel
mesh_terms: D004268:DNA-Binding Proteins; D015966:Gene Expression Regulation, Fungal; D056892:Mediator Complex; D010957:Plasmids / Q000235:genetics; D014176:Protein Biosynthesis / Q000235:genetics*; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics*; D013045:Species Specificity; D015534:Trans-Activators / Q000235:genetics*; D014157:Transcription Factors / Q000235:genetics
chemical_list: D004268:DNA-Binding Proteins; C058337:GAL11 protein, S cerevisiae; C052130:GAL4 protein, S cerevisiae; D056892:Mediator Complex; D029701:Saccharomyces cerevisiae Proteins; D015534:Trans-Activators; D014157:Transcription Factors