A Hypersensitive Genetically Encoded Fluorescent Indicator (roGFP2-Prx1) Enables Continuous Measurement of Intracellular H2O2 during Cell Micro-cultivation.

Abstract

Hydrogen peroxide (H2O2) is a toxic oxidant produced as a byproduct of several biological processes. At too high levels of hydrogen peroxide cells will experience oxidative stress, leading to a cellular response to decrease its levels and to protect the cells. Previously, methods used to study and quantify intracellular H2O2 have been limited by both sensitivity and specificity. However, an increasing number of genetically encoded fluorescent indicators (GEFIs) are becoming available, which can specifically detect low levels of intracellular hydrogen peroxide. In this study, we use such a biosensor designed to monitor cytosolic H2O2 levels in the budding yeast Saccharomyces cerevisiae during continuous cultivation and in the absence of a fluorescence microscope. The fluorescent biosensor contains a peroxiredoxin protein fused to an engineered GFP molecule expressed from a commonly used yeast plasmid (pRS416-TEF1). The peroxiredoxin-based fluorescent indicator reduces H2O2, ultimately resulting in a GFP signal being emitted by the sensor. Here, we apply this biosensor to study cytosolic H2O2 levels in S. cerevisiae strains with and without recombinant protein production.