title: TIPI: TEV protease-mediated induction of protein instability.
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.
authors.affiliation: Philipps Universität Marburg, Fachbereich 17, Biologie - Genetik, Marburg, Germany.
authors.forename: Christof
authors.initials: C
authors.lastname: Taxis
authors.forename: Michael
authors.initials: M
authors.lastname: Knop
mesh_terms: D001665:Binding Sites; D010450:Endopeptidases / Q000378:metabolism*; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D055550:Protein Stability; D011506:Proteins / Q000378:metabolism*; D059748:Proteolysis; D054875:Ubiquitination; D015003:Yeasts
chemical_list: D011506:Proteins; D010450:Endopeptidases; C099488:TEV protease; D046988:Proteasome Endopeptidase Complex; C061553:ATP dependent 26S protease