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Free keywords:
development, protein inactivation, regulated protein inactivation, tissue-specific
Abstract:
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
