A simple and unified protocol to purify all seven Escherichia coli RNA polymerase sigma factors.

Autor: Kędzierska B; Department of Bacterial Molecular Genetics, Faculty of Biology, University of Gdańsk, Gdańsk, Poland., Stodolna A; Department of Bacterial Molecular Genetics, Faculty of Biology, University of Gdańsk, Gdańsk, Poland., Bryszkowska K; Department of Bacterial Molecular Genetics, Faculty of Biology, University of Gdańsk, Gdańsk, Poland., Dylewski M; Department of Bacterial Molecular Genetics, Faculty of Biology, University of Gdańsk, Gdańsk, Poland., Potrykus K; Department of Bacterial Molecular Genetics, Faculty of Biology, University of Gdańsk, Gdańsk, Poland. katarzyna.potrykus@ug.edu.pl.
Jazyk: angličtina
Zdroj: Journal of applied genetics [J Appl Genet] 2024 Sep; Vol. 65 (3), pp. 615-625. Date of Electronic Publication: 2024 May 06.
DOI: 10.1007/s13353-024-00870-3
Abstrakt: RNA polymerase sigma factors are indispensable in the process of bacterial transcription. They are responsible for a given gene's promoter region recognition on template DNA and hence determine specificity of RNA polymerase and play a significant role in gene expression regulation. Here, we present a simple and unified protocol for purification of all seven Escherichia coli RNA polymerase sigma factors. In our approach, we took advantage of the His 8 -SUMO tag, known to increase protein solubilization. Sigma factors were first purified in N-terminal fusions with this tag, which was followed by tag removal with Ulp1 protease. This allowed to obtain proteins in their native form. In addition, the procedure is simple and requires only one resin type. With the general protocol we employed, we were able to successfully purify σ D , σ E , σ S , and σ N . Final step modification was required for σ F , while for σ H and σ FecI , denaturing conditions had to be applied. All seven sigma factors were fully functional in forming an active holoenzyme with core RNA polymerase which we demonstrated with EMSA studies.
(© 2024. The Author(s).)
Databáze: MEDLINE
Externí odkaz: