Fast protein-depletion system utilizing tetracycline repressible promoter and N-end rule in yeast

• Verfasst von: Gnanasundram, Sivakumar Vadivel [VerfasserIn]
• Verfasst von: Koš, Martin [VerfasserIn]
• Titel: Fast protein-depletion system utilizing tetracycline repressible promoter and N-end rule in yeast
• Verf.angabe: Sivakumar Vadivel Gnanasundram and Martin Koš
• Jahr: 2015
• Jahr des Originals: 2014
• Umfang: 7 S.
• Fussnoten: Published online ahead of print on December 24, 2014 ; Gesehen am 10.06.2020
• Titel Quelle: Enthalten in: Molecular biology of the cell
• Ort Quelle: Bethesda, Md. : American Society for Cell Biology, 1992
• Jahr Quelle: 2015
• Band/Heft Quelle: 26(2015), 4, Seite 762-768
• ISSN Quelle: 1939-4586
• DOI: doi:10.1091/mbc.E14-07-1186
• Datenträger: Online-Ressource
• Sprache: eng
• K10plus-PPN: 1700314475

Abstract

A protein depletion by promoter shutoff or protein destabilization is an important tool in investigation of functions of essential genes. Various approaches using different repressible promoters, inducible degrons, or their combinations were developed. While successful, the current techniques have a drawback in that they require fusion of a large degradation tag to the target protein and/or a change in growth conditions to repress the promoter. We describe efficient protein depletion using the combination of a metabolically inert tetracycline repressible promoter with tetracycline aptamer and constitutive target protein destabilization by means of ubiquitin fusion. The target protein does not require a tag, and its elimination is several fold faster compared with standard promoter shutoff systems. A depletion time of <40 min was sufficient to achieve a robust phenotype.