Tuning Degradation to Achieve Specific and Efficient Protein Depletion

Autor: J David, Barrass, Gonzalo I, Mendoza-Ochoa, Isabella E, Maudlin, Emanuela, Sani, Jean D, Beggs
Rok vydání: 2019
Předmět:
Zdroj: Journal of visualized experiments : JoVE. (149)
ISSN: 1940-087X
Popis: The plant auxin binding receptor, TIR1, recognizes proteins containing a specific auxin-inducible degron (AID) motif in the presence of auxin, targeting them for degradation. This system is exploited in many non-plant eukaryotes, such that a target protein, tagged with the AID motif, is degraded upon auxin addition. The level of TIR1 expression is critical; excessive expression leads to degradation of the AID-tagged protein even in the absence of auxin, whereas low expression leads to slow depletion. A β-estradiol-inducible AID system was created, with expression of TIR1 under the control of a β-estradiol inducible promoter. The level of TIR1 is tunable by changing the time of incubation with β-estradiol before auxin addition. This protocol describes how to rapidly deplete a target protein using the AID system. The appropriate β-estradiol incubation time depends on the abundance of the target protein. Therefore, efficient depletion depends on optimal timing that also minimizes auxin-independent depletion.
Databáze: OpenAIRE