Regulated overproduction of the GAL4 gene product greatly increases expression from galactose-inducible promoters on multi-copy expression vectors in yeast
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2023, Protein Expression and PurificationCitation Excerpt :Therefore, in this study, we employed an S. cerevisiae production and purification platform capable of obtaining well-behaved protein samples from both humans and yeast, for the purpose of downstream structure-function studies. The system combines several advantageous plasmid-born properties such as two selection markers, an inducible promoter, and a very high copy number [38], with a modified S. cerevisiae strain harboring the Gal4 transcriptional activator [53]. We included three CTR targets, human hCTR1 and hCTR2 were selected due to their relevance in humans and their role in platinum-based chemotherapy, and CaCTR as an example of a member from an opportunistic fungus with clinical relevance [54].
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2020, Enzyme and Microbial TechnologyCitation Excerpt :Efficient regulation of a multi-gene pathway using the GAL system would require sufficient amount of the transcriptional activator Gal4. As a matter of fact, limited expression level and activity of Gal4 has been found as a limiting factor for transcriptional activation of genes [20]. The availability of Gal4 to the pathway genes can be increased by gene overexpression.
Yeast recombinant production of intact human membrane proteins with long intrinsically disordered intracellular regions for structural studies
2020, Biochimica et Biophysica Acta - BiomembranesCitation Excerpt :This system includes a yeast that features increased plasmid copy numbers from 20 to 200 per cell prior to induction of recombinant protein production, regulated by a galactose inducible promoter. The PAP1500 host strain overexpresses the Gal4 transcription factor concomitantly with recombinant protein production [38]. Target protein genes are codon optimized for S. cerevisiae and produced with a C-terminal yeast-enhanced GFP (yeGFP) TEV-yeGFP-His10-tag, suitable for expression and purification optimization.
Improvement of galactose induction system in Saccharomyces cerevisiae
2011, Journal of Bioscience and BioengineeringNitrile-inducible gene expression in mycobacteria
2009, TuberculosisCitation Excerpt :Negatively-regulated promoters often suffer from high basal levels of transcription and are made more inducible by overexpressing the regulatory protein.1,2 Alternatively, optimal inducibility from positive regulators can be achieved by designing the circuit to include positive feedback in which the regulatory protein is induced simultaneously with the target gene.3 The specific structure of a regulatory circuit can have unexpected effects on gene expression at the single cell level.