Targeted protein degradation using deGradFP in Trypanosoma brucei

Wellcome Open Research

article

Midori Ishii¹  Bungo Akiyoshi¹ 

[1] Department of Biochemistry, University of Oxford

关键词: Trypanosoma brucei;    targeted protein degradation;    deGradFP;    degron;    kinetoplastid;    kinetochore;   

DOI  :  10.12688/wellcomeopenres.17964.2

学科分类：内科医学

来源: Wellcome

【 摘 要 】

Targeted protein degradation is an invaluable tool in studying the function of proteins. Such a tool was not available inTrypanosoma 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, inT. 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 inT. brucei, deGradFP can serve as a powerful tool to rapidly deplete proteins of interest, especially those with low turnover rates.

【 授权许可】

CC BY