A rapid approach for in locus overexpression of Trypanosoma brucei proteins Article Swipe
Hana Váchová
,
Glenda Alquicer
,
Miroslava Šedinová
,
Jana Šáchová
,
Miluše Hradilová
,
Vladimír Varga
·
YOU?
·
· 2020
· Open Access
·
· DOI: https://doi.org/10.1016/j.molbiopara.2020.111300
YOU?
·
· 2020
· Open Access
·
· DOI: https://doi.org/10.1016/j.molbiopara.2020.111300
Altering amounts of a protein in a cell has become a crucial tool for understanding its function. In many organisms, including the protozoan parasite Trypanosoma brucei, protein overexpression has been achieved by inserting a protein-coding sequence into an overexpression vector. Here, we have adapted the PCR only based system for tagging trypanosome proteins at their endogenous loci such that it in addition enables a tetracycline-inducible T7 RNA polymerase-mediated protein overexpression. Hence, this approach bypasses the need for molecular cloning, making it rapid and cost effective. We validated the approach for ten flagellum-associated proteins with molecular weights ranging from 40 to over 500 kDa. For a majority of the recombinant proteins a significant (3-50 fold) increase in the cellular amount was achieved upon induction of overexpression. Two of the largest proteins studied, the dynein heavy chains, were significantly overexpressed, while two were not. Our data suggest that this may reflect the extent of the T7 RNA polymerase processivity on the trypanosome genomic DNA. We further show that the overexpression is informative as to cellular functions of the studied proteins, and that these cultures can serve as an excellent source for purification of the overexpressed proteins. We believe that this rapid in locus overexpression system will become a valuable tool to interrogate cellular functions and biochemical activities of trypanosome proteins.
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- Type
- article
- Language
- en
- Landing Page
- https://doi.org/10.1016/j.molbiopara.2020.111300
- OA Status
- hybrid
- Cited By
- 4
- References
- 49
- Related Works
- 10
- OpenAlex ID
- https://openalex.org/W3043116201
All OpenAlex metadata
Raw OpenAlex JSON
- OpenAlex ID
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https://openalex.org/W3043116201Canonical identifier for this work in OpenAlex
- DOI
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https://doi.org/10.1016/j.molbiopara.2020.111300Digital Object Identifier
- Title
-
A rapid approach for in locus overexpression of Trypanosoma brucei proteinsWork title
- Type
-
articleOpenAlex work type
- Language
-
enPrimary language
- Publication year
-
2020Year of publication
- Publication date
-
2020-07-16Full publication date if available
- Authors
-
Hana Váchová, Glenda Alquicer, Miroslava Šedinová, Jana Šáchová, Miluše Hradilová, Vladimír VargaList of authors in order
- Landing page
-
https://doi.org/10.1016/j.molbiopara.2020.111300Publisher landing page
- Open access
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YesWhether a free full text is available
- OA status
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hybridOpen access status per OpenAlex
- OA URL
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https://doi.org/10.1016/j.molbiopara.2020.111300Direct OA link when available
- Concepts
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Biology, Trypanosoma brucei, RNA, Molecular biology, Cloning (programming), Cell biology, Gene, Computational biology, Genetics, Computer science, Programming languageTop concepts (fields/topics) attached by OpenAlex
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4Total citation count in OpenAlex
- Citations by year (recent)
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2025: 1, 2022: 2, 2021: 1Per-year citation counts (last 5 years)
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49Number of works referenced by this work
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10Other works algorithmically related by OpenAlex
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