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Targeted protein degradation using deGradFP in Trypanosoma brucei
Targeted protein degradation is an invaluable tool in studying the function of proteins. Such a tool was not available in Trypanosoma brucei, an evolutionarily divergent eukaryote that causes human African trypanosomiasis. Here, we have adapted deGradFP (degrade green fluorescent protein [GFP]), a p...
Autores principales: | , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
F1000 Research Limited
2022
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9277568/ https://www.ncbi.nlm.nih.gov/pubmed/35865221 http://dx.doi.org/10.12688/wellcomeopenres.17964.2 |
Sumario: | Targeted protein degradation is an invaluable tool in studying the function of proteins. Such a tool was not available in Trypanosoma brucei, an evolutionarily divergent eukaryote that causes human African trypanosomiasis. Here, we have adapted deGradFP (degrade green fluorescent protein [GFP]), a protein degradation system based on the SCF E3 ubiquitin ligase complex and anti-GFP nanobody, in T. brucei. As a proof of principle, we targeted a kinetoplastid kinetochore protein (KKT3) that constitutively localizes at kinetochores in the nucleus. Induction of deGradFP in a cell line that had both alleles of KKT3 tagged with yellow fluorescent protein (YFP) caused a more severe growth defect than RNAi in procyclic (insect form) cells. deGradFP also worked on a cytoplasmic protein (COPII subunit, SEC31). Given the ease in making GFP fusion cell lines in T. brucei, deGradFP can serve as a powerful tool to rapidly deplete proteins of interest, especially those with low turnover rates. |
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