Targeted protein degradation using deGradFP in Trypanosoma brucei [version 1; peer review: 4 approved]
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...
| Main Authors: | , |
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| Format: | Article |
| Language: | English |
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Wellcome
2022-06-01
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| Series: | Wellcome Open Research |
| Subjects: | |
| Online Access: | https://wellcomeopenresearch.org/articles/7-175/v1 |
| _version_ | 1811344786477547520 |
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| author | Bungo Akiyoshi Midori Ishii |
| author_facet | Bungo Akiyoshi Midori Ishii |
| author_sort | Bungo Akiyoshi |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-04-13T19:52:24Z |
| format | Article |
| id | doaj.art-f44ec073b8d645a0bae059055a3ffbe3 |
| institution | Directory Open Access Journal |
| issn | 2398-502X |
| language | English |
| last_indexed | 2024-04-13T19:52:24Z |
| publishDate | 2022-06-01 |
| publisher | Wellcome |
| record_format | Article |
| series | Wellcome Open Research |
| spelling | doaj.art-f44ec073b8d645a0bae059055a3ffbe32022-12-22T02:32:28ZengWellcomeWellcome Open Research2398-502X2022-06-01719908Targeted protein degradation using deGradFP in Trypanosoma brucei [version 1; peer review: 4 approved]Bungo Akiyoshi0https://orcid.org/0000-0001-6010-394XMidori Ishii1Department of Biochemistry, University of Oxford, Oxford, OX1 3QU, UKDepartment of Biochemistry, University of Oxford, Oxford, OX1 3QU, UKTargeted 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.https://wellcomeopenresearch.org/articles/7-175/v1Trypanosoma brucei targeted protein degradation deGradFP degron kinetoplastid kinetochoreeng |
| spellingShingle | Bungo Akiyoshi Midori Ishii Targeted protein degradation using deGradFP in Trypanosoma brucei [version 1; peer review: 4 approved] Wellcome Open Research Trypanosoma brucei targeted protein degradation deGradFP degron kinetoplastid kinetochore eng |
| title | Targeted protein degradation using deGradFP in Trypanosoma brucei [version 1; peer review: 4 approved] |
| title_full | Targeted protein degradation using deGradFP in Trypanosoma brucei [version 1; peer review: 4 approved] |
| title_fullStr | Targeted protein degradation using deGradFP in Trypanosoma brucei [version 1; peer review: 4 approved] |
| title_full_unstemmed | Targeted protein degradation using deGradFP in Trypanosoma brucei [version 1; peer review: 4 approved] |
| title_short | Targeted protein degradation using deGradFP in Trypanosoma brucei [version 1; peer review: 4 approved] |
| title_sort | targeted protein degradation using degradfp in trypanosoma brucei version 1 peer review 4 approved |
| topic | Trypanosoma brucei targeted protein degradation deGradFP degron kinetoplastid kinetochore eng |
| url | https://wellcomeopenresearch.org/articles/7-175/v1 |
| work_keys_str_mv | AT bungoakiyoshi targetedproteindegradationusingdegradfpintrypanosomabruceiversion1peerreview4approved AT midoriishii targetedproteindegradationusingdegradfpintrypanosomabruceiversion1peerreview4approved |