New England Biolabs, 240 County Road, Ipswich, MA, 01938, USA.
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.
Redox Biol. 2019 Sep;26:101280. doi: 10.1016/j.redox.2019.101280. Epub 2019 Jul 20.
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 (HO)- sensitive proteins (glutaredoxin 1 or Orp1), the cytosolic redox state of both wild type and SHuffle cells based on GSH/GSSG and HO pools was measured. These probes open the path to in vivo studies of redox changes and genetic selections in prokaryotic hosts.
了解细胞内的氧化还原生物学是一个复杂但重要的生物学问题。在不进行物理破坏或化学修饰的情况下研究活细胞内的氧化还原过程，对于确定细胞的天然氧化还原状态至关重要。在这项研究中，先前表征的氧化还原敏感绿色荧光蛋白（roGFP2）被用于阐明基因工程大肠杆菌菌株 SHuffle 的氧化还原变化。研究表明，SHuffle 细胞处于持续的氧化应激状态，并以 OxyR 依赖的方式进行转录反应。使用与谷胱甘肽 (GSH) 或过氧化氢 (HO) 敏感蛋白（谷氧还蛋白 1 或 Orp1）融合的 roGFP2，基于 GSH/GSSG 和 HO 池测量了野生型和 SHuffle 细胞的细胞浆氧化还原状态。这些探针为在原核宿主中进行氧化还原变化和遗传选择的体内研究开辟了道路。
