Tuning degradation to achieve specific and efficient protein depletion

J David Barrass, Gonzalo I Mendoza-Ochoa, Isabella E Maudlin, Emanuela Sani, Jean D Beggs

Research output: Contribution to journalArticlepeer-review

Abstract

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.

Original languageEnglish
Article numbere59874
JournalJournal of Visualized Experiments (JoVE)
Issue number149
DOIs
Publication statusPublished - 20 Jul 2019

Keywords

  • immunology and infection
  • issue 149
  • AID
  • auxin
  • β-estradiol
  • degron
  • protein depletion
  • regulated gene expression
  • Saccharomyces cerevisiae

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