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Mol Biol Cell. 2015 Feb 15;26(4):762-8. doi: 10.1091/mbc.E14-07-1186. Epub 2014 Dec 24.

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

Author information

1
Biochemistry Center, University of Heidelberg, 69120 Heidelberg, Germany.

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.

PMID:
25540433
PMCID:
PMC4325845
DOI:
10.1091/mbc.E14-07-1186
[Indexed for MEDLINE]
Free PMC Article

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