Abstract
Reverse genetics approaches require methods to inactivate a specific protein. One possibility is to modify the target protein with a degradation signal (degron). Degrons are short, transferable sequences that confer protein instability. They target proteins for degradation either constitutively or after activation, e.g., by phosphorylation, presence of a binding partner, or conformational rearrangements in the substrate. In this chapter, we describe a synthetic way to activate a degron. It employs the generation of an N-degron by cleavage of a substrate with the site-specific tobacco etch virus (TEV) protease. Subsequently, the substrate is targeted for degradation by the ubiquitin-proteasome system. This TEV protease-induced protein instability system provides a powerful approach to generate conditional mutants for synthetic biology or for the investigation of protein functions in a specific cellular context.
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Acknowledgments
We thank A. Kaufmann for helpful comments on the manuscript.
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Taxis, C., Knop, M. (2012). TIPI: TEV Protease-Mediated Induction of Protein Instability. In: Dohmen, R., Scheffner, M. (eds) Ubiquitin Family Modifiers and the Proteasome. Methods in Molecular Biology, vol 832. Humana Press. https://doi.org/10.1007/978-1-61779-474-2_43
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DOI: https://doi.org/10.1007/978-1-61779-474-2_43
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