Regulated expression of proteins in yeast using the MAL61-62 promoter and a mating scheme to increase dynamic range.
The ability to express heterologous genes in yeast has become indispensable for many biological research techniques. Expression systems that can be regulated are particularly useful because they allow an experimenter to control the timing and levels of gene expression. Despite their many advantages, however, surprisingly few conditional expression systems are available for yeast. Moreover, of those that have been described, many are not ideal either because they have high background expression levels, low induced levels, or because they require restrictive growth conditions. Here we describe a conditional expression system that takes advantage of the yeast MAL62 promoter (MAL62p), which can be controlled by adding maltose or glucose to the growth medium to induce or repress transcription, respectively. In addition, we use a mating scheme to dramatically increase the dynamic range of expression levels possible. We show that MAL62p background activity can be effectively eliminated by maintaining expression constructs in a mal(-) yeast strain. High-level expression can be induced in diploids formed by mating the mal(-) strain with a MAL(+) strain. A similar mating scheme may be useful for other conditional expression systems as well. Among other uses, this approach should aid high throughput yeast two-hybrid assays, which rely on maintaining large libraries of expression strains, which are eventually mated to conduct assays for protein interactions. We demonstrate a two-hybrid system in which MAL62p is used in conjunction with the yeast GAL1 promoter to independently regulate expression of both hybrid proteins, and to allow detection of interactions involving toxic proteins.
Finley
Russell L
RL
Center for Molecular Medicine and Genetics, Wayne State University School of Medicine, 540 East Canfield Avenue, Detroit, MI 48201, USA. rfinley@genetics.wayne.edu
Zhang
Huamei
H
Zhong
Jinhui
J
Stanyon
Clement A
CA
D000595:Amino Acid Sequence; D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics; D001483:Base Sequence; D019925:Cyclin A / Q000235:genetics; D019927:Cyclin E / Q000235:genetics; D004171:Diploidy; D004330:Drosophila / Q000235:genetics; D005690:Galactose / Q000494:pharmacology; D005786:Gene Expression Regulation / Q000187:drug effects; D005800:Genes, Fungal / Q000235:genetics*; D005822:Genetic Vectors / Q000235:genetics; D005838:Genotype; D005947:Glucose / Q000494:pharmacology; D006238:Haploidy; D008320:Maltose / Q000494:pharmacology; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins / Q000235:genetics*; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics*; D012697:Serine Endopeptidases / Q000235:genetics; D027981:Symporters; D020798:Two-Hybrid System Techniques; D000099160:LexA Repressor Protein
D001426:Bacterial Proteins; D019925:Cyclin A; D019927:Cyclin E; D005690:Galactose; D005947:Glucose; D008320:Maltose; D009004:Monosaccharide Transport Proteins; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D012697:Serine Endopeptidases; D027981:Symporters; D000099160:LexA Repressor Protein; C067575:maltose transport system, S cerevisiae