Improving redox sensitivity of roGFP1 by incorporation of selenocysteine at position 147.

BMC Res Notes

Department of Molecular Pharmacology & Physiology, Morsani College of Medicine, University of South Florida, 12901 Bruce B Downs Blvd, Tampa, FL, 33612, USA.

Published: November 2018

Objective: 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.

Results: 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.

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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6249920PMC
http://dx.doi.org/10.1186/s13104-018-3929-xDOI Listing

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