Taxis Christof, Stier Gunter, Spadaccini Roberta, Knop Michael
EMBL, Cell Biology and Biophysics Unit, Meyerhofstr. 1, Heidelberg, Germany.
Mol Syst Biol. 2009;5:267. doi: 10.1038/msb.2009.25. Epub 2009 Apr 28.
Methods that allow for the manipulation of genes or their products have been highly fruitful for biomedical research. Here, we describe a method that allows the control of protein abundance by a genetically encoded regulatory system. We developed a dormant N-degron that can be attached to the N-terminus of a protein of interest. Upon expression of a site-specific protease, the dormant N-degron becomes deprotected. The N-degron then targets itself and the attached protein for rapid proteasomal degradation through the N-end rule pathway. We use an optimized tobacco etch virus (TEV) protease variant combined with selective target binding to achieve complete and rapid deprotection of the N-degron-tagged proteins. This method, termed TEV protease induced protein inactivation (TIPI) of TIPI-degron (TDeg) modified target proteins is fast, reversible, and applicable to a broad range of proteins. TIPI of yeast proteins essential for vegetative growth causes phenotypes that are close to deletion mutants. The features of the TIPI system make it a versatile tool to study protein function in eukaryotes and to create new modules for synthetic or systems biology.
能够对基因或其产物进行操控的方法在生物医学研究中成果斐然。在此，我们描述一种通过基因编码调控系统来控制蛋白质丰度的方法。我们开发了一种可附着于目标蛋白质N端的休眠N-降解子。在位点特异性蛋白酶表达时，休眠的N-降解子会被去保护。然后，N-降解子会将自身及附着的蛋白质靶向，通过N端规则途径进行快速蛋白酶体降解。我们使用一种优化的烟草蚀纹病毒（TEV）蛋白酶变体并结合选择性靶标结合，以实现对N-降解子标记蛋白质的完全快速去保护。这种方法，即对经TIPI-降解子（TDeg）修饰的靶标蛋白质进行TEV蛋白酶诱导的蛋白质失活（TIPI），快速、可逆，且适用于多种蛋白质。对酵母营养生长所必需的蛋白质进行TIPI会导致接近缺失突变体的表型。TIPI系统的这些特性使其成为研究真核生物蛋白质功能以及为合成生物学或系统生物学创建新模块的通用工具。
