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How do I get rid of this pesky protein? The Hows and Whys of Protein Degradation

Here, we compare and contrast two techniques for targeted protein degradation: the GFP-Nanobody and Auxin-Inducible Degron Systems.

Targeted Protein Degradation in vivo

Being able to understand how a protein acts in specific tissues and at what time in cellular development are key for developing an understanding of a protein’s function. There are methods to do this at the genomic level such as the Cre/Lox system or RNAi. However, often a more direct approach is needed. This can be achieved through degradation of a target protein. There are several methods to achieve target degradation, but they can be summarized in 3 steps:

1. Tag your protein of interest at the endogenous locus using CRISPR/Cas9 or another editing method.

2. Express a recognition and degradation targeting protein (RAD protein) using a tissue-specific or condition-specific promoter.

3. The RAD protein binds to the tag on the protein of interest and targets the protein of interest for degradation.


Controlled degradation of a target protein can be a powerful tool for scientists as they look to understand the role of a protein at different points of development. Additionally, valuable tools can be built using this technology to precisely control protein expression at key developmental stages. As this technique becomes more widespread, there is abundant potential for new tools and discoveries through controlled protein degradation:

  • Avoiding maternal protein contribution when studying essential genes during embryonic development.
  • Isoform specific depletion to understand the roles of different isoforms.
  • Identifying tissue-specific protein functions.
  • Control of fertility and fecundity.
  • Conditional immobilization or control of mobility.


There are multiple strategies for inducing degradation. Here I will contrast 2 commonly used methods: the GFP-nanobody system and the Auxin Inducible Degron (AID) system.

GFP-Nanobody System

This method uses a single-domain antibody fragment, known as a nanobody, as a tag for recognition. Nanobodies, consisting of only a single peptide chain, are much smaller than traditional antibodies and can be easily encoded for expression in the genome. To use GFP as the tag, the anti-GFP nanobody is fused to a component of E3 ubiquitin ligase complex. When this fusion protein is expressed, it binds to the GFP tag and ubiquitinates the target protein, which is subsequently degraded by the proteosome.

  • Protein degradation can be monitored using GFP
  • Can use existing GFP-tagged strains
  • Spatial and temporal control of degradation depending on the GFP-nanobody expression
  • GFP tag can interfere with native protein function
  • Can’t be used in the germline

Auxin-Inducible Degron (AID) system

The Auxin Inducible Degron (AID) system has been adapted for use in C. elegans from the auxin hormone signaling pathway involved in plant growth regulation. Target proteins are tagged with a 44 amino acid degron sequence. This is recognized by the F-box protein TIR1, which is a part of an E3 ubiquitin ligase complex called the SCF complex. However, TIR1 only binds to the degron sequence in the presence of auxin. Auxin is easily taken up into the worm and egg, but with no effect on wild-type C. elegans growth, development, or behavior. The AID system provides spatial control of degradation by expressing the TIR1 under tissue-specific promoters, and provides temporal control of degradation with the application of auxin at a specific time.

  • Small tag
  • Spatial and temporal control
  • Can be used in the germline
  • Degron tag can interfere with native protein function
  • Second method needed to validate protein degradation (antibody staining)


Both of these methods have the advantage that a single tagged protein can be combined with multiple different tissue-specific expression patterns of the RAD protein for precise evaluation of the effect of depleting target protein. With any targeted degradation method, it is important to monitor the quantity of target protein, as different levels and rates of depletion have been observed depending on the protein abundance, localization, and accessibility.

Degradation tagging of proteins is a powerful genetic tool that is only beginning to be fully utilized to uncover protein function.  It’s a good tool to keep in mind when digging deeper into the genetic pathways that make our favorite worm tick.

Curious to learn more about how InVivo Biosystems can help you incorporate inducible protein degradation into your next project?

About The Author

Trisha Brock

Dr. Brock is the Director of Research & Technical Delivery at InVivo Biosystems. She has been creating and studying transgenic C. elegans for 17 years including training at Washington State University, University of Utah, and Harvard. At InVivo Biosystems, Dr. Brock brings technical expertise in the application of genetics and genetic engineering to create and use animal models for biomedical research.

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