Stanford Katherine R, Ajmo Joanne M, Bahia Parmvir K, Hadley Stephen H, Taylor-Clark Thomas E
Department of Molecular Pharmacology & Physiology, Morsani College of Medicine, University of South Florida, 12901 Bruce B Downs Blvd, Tampa, FL, 33612, USA.
BMC Res Notes. 2018 Nov 22;11(1):827. doi: 10.1186/s13104-018-3929-x.
Redox-sensitive green fluorescent protein (roGFP) is a genetically-encoded redox-sensitive protein used to detect cellular oxidative stress associated with reactive oxygen species production. Here we replaced the cysteine at position 147 of roGFP1 (variant of roGFP) with selenocysteine in order to increase redox sensitivity of the redox reporter.
Expression of roGFP1 selenoprotein (roGFP1-Se147) in HEK293 cells required the presence of a selenocysteine insertion sequence and was augmented by co-expression with SBP2. roGFP1-Se147 demonstrated a similar excitation and emission spectra to roGFP1. Although expression of roGFP1-Se147 was limited, it was sufficient enough to perform live cell imaging to evaluate sensitivity to oxidation and reduction. roGFP1-Se147 exhibited a 100-fold increase in sensitivity to oxidation with HO in comparison to roGFP1 as well as a 20-fold decrease in the EC of HO. Furthermore, roGFP1-Se147, unlike roGFP1, was able to detect oxidation caused by the mitochondrial electron transport complex III inhibitor antimycin A. Unfortunately roGFP-Se147 exhibited a diminished dynamic range and photoinstability.
氧化还原敏感型绿色荧光蛋白（roGFP）是一种基因编码的氧化还原敏感蛋白，用于检测与活性氧产生相关的细胞氧化应激。在此，我们将roGFP1（roGFP的变体）第147位的半胱氨酸替换为硒代半胱氨酸，以提高氧化还原报告基因的氧化还原敏感性。
roGFP1硒蛋白（roGFP1-Se147）在HEK293细胞中的表达需要硒代半胱氨酸插入序列的存在，并且与SBP2共表达可增强其表达。roGFP1-Se147表现出与roGFP1相似的激发和发射光谱。尽管roGFP1-Se147的表达有限，但足以进行活细胞成像以评估其对氧化和还原的敏感性。与roGFP1相比，roGFP1-Se147对HO氧化的敏感性增加了100倍，同时HO的半数有效浓度（EC）降低了20倍。此外，与roGFP1不同，roGFP1-Se147能够检测由线粒体电子传递复合物III抑制剂抗霉素A引起的氧化。不幸的是，roGFP-Se147表现出动态范围减小和光不稳定。
