Gnanasundram Sivakumar Vadivel, Koš Martin
Biochemistry Center, University of Heidelberg, 69120 Heidelberg, Germany.
Mol Biol Cell. 2015 Feb 15;26(4):762-8. doi: 10.1091/mbc.E14-07-1186. Epub 2014 Dec 24.
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.
通过启动子关闭或蛋白质不稳定来实现蛋白质耗竭是研究必需基因功能的重要工具。人们开发了各种使用不同可抑制启动子、诱导型降解结构域或它们组合的方法。虽然这些方法取得了成功，但目前的技术存在一个缺点，即它们需要将一个大的降解标签与目标蛋白融合，和/或改变生长条件来抑制启动子。我们描述了一种高效的蛋白质耗竭方法，该方法使用代谢惰性的四环素可抑制启动子与四环素适配体相结合，并通过泛素融合实现组成型目标蛋白不稳定。目标蛋白不需要标签，与标准的启动子关闭系统相比，其消除速度快几倍。<40分钟的耗竭时间足以产生明显的表型。
