A rapid approach for in locus overexpression of Trypanosoma brucei proteins

0535707 - ÚMG 2021 RIV NL eng J - Článek v odborném periodiku
Váchová, Hana - Alquicer, Glenda - Šedinová, Miroslava - Šáchová, Jana - Hradilová, Miluše - Varga, Vladimír
A rapid approach for in locus overexpression of Trypanosoma brucei proteins.
Molecular and Biochemical Parasitology. Roč. 239, September (2020), č. článku 111300. ISSN 0166-6851. E-ISSN 1872-9428
Grant CEP: GA MŠMT(CZ) LM2018131; GA ČR GJ16-26444Y
Institucionální podpora: RVO:68378050
Klíčová slova: Overexpressing endogenous proteins * T7 RNA polymerase-mediated transcription * Protein purification * Trypanosomes
Obor OECD: Biochemistry and molecular biology
Impakt faktor: 1.759, rok: 2020
Způsob publikování: Open access
https://www.sciencedirect.com/science/article/pii/S0166685120300645?via%3Dihub

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.
Trvalý link: http://hdl.handle.net/11104/0313655