Utilizing redox-sensitive GFP fusions to detect in vivo redox changes in a genetically engineered prokaryote.

Autor: Reuter WH; New England Biolabs, 240 County Road, Ipswich, MA, 01938, USA., Masuch T; New England Biolabs, 240 County Road, Ipswich, MA, 01938, USA; Ruhr University Bochum, Institute of Biochemistry and Pathobiochemistry, Microbial Biochemistry, Universitätsstr. 150, 44780, Bochum, Germany., Ke N; New England Biolabs, 240 County Road, Ipswich, MA, 01938, USA., Lenon M; New England Biolabs, 240 County Road, Ipswich, MA, 01938, USA., Radzinski M; The Hebrew University of Jerusalem, The Alexander Silberman Institute of Life Sciences, Safra Campus Givat Ram, Jerusalem, 91904, Israel., Van Loi V; Institute for Biology-Microbiology, Freie Universität Berlin, 14195, Berlin, Germany., Ren G; New England Biolabs, 240 County Road, Ipswich, MA, 01938, USA., Riggs P; New England Biolabs, 240 County Road, Ipswich, MA, 01938, USA., Antelmann H; Institute for Biology-Microbiology, Freie Universität Berlin, 14195, Berlin, Germany., Reichmann D; The Hebrew University of Jerusalem, The Alexander Silberman Institute of Life Sciences, Safra Campus Givat Ram, Jerusalem, 91904, Israel., Leichert LI; Ruhr University Bochum, Institute of Biochemistry and Pathobiochemistry, Microbial Biochemistry, Universitätsstr. 150, 44780, Bochum, Germany., Berkmen M; New England Biolabs, 240 County Road, Ipswich, MA, 01938, USA. Electronic address: berkmen@neb.com.
Jazyk: angličtina
Zdroj: Redox biology [Redox Biol] 2019 Sep; Vol. 26, pp. 101280. Date of Electronic Publication: 2019 Jul 20.
DOI: 10.1016/j.redox.2019.101280
Abstrakt: Understanding the in vivo redox biology of cells is a complex albeit important biological problem. Studying redox processes within living cells without physical disruption or chemical modifications is essential in determining the native redox states of cells. In this study, the previously characterized reduction-oxidation sensitive green fluorescent protein (roGFP2) was used to elucidate the redox changes of the genetically engineered Escherichia coli strain, SHuffle. SHuffle cells were demonstrated to be under constitutive oxidative stress and responding transcriptionally in an OxyR-dependent manner. Using roGFP2 fused to either glutathione (GSH)- or hydrogen peroxide (H 2 O 2 )- sensitive proteins (glutaredoxin 1 or Orp1), the cytosolic redox state of both wild type and SHuffle cells based on GSH/GSSG and H 2 O 2 pools was measured. These probes open the path to in vivo studies of redox changes and genetic selections in prokaryotic hosts.
(Copyright © 2019 The Authors. Published by Elsevier B.V. All rights reserved.)
Databáze: MEDLINE
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