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

Background

In this protocol, we discuss the use of a hypersensitive hydrogen peroxide (H₂O₂) sensor, roGFP2-Prx1, in the yeast Saccharomyces cerevisiae. S. cerevisiae is a broadly used industrial yeast species but also a popular model organism to study eukaryotic cellular biology. Within biological systems, this includes the generation and buildup of unwanted toxins like H₂O₂. When oxygen is incompletely reduced to water, reactive oxygen species such as H₂O₂ are produced. If the intracellular level of H₂O₂ becomes too high, the cell will endure oxidative stress, which drains resources and increases maintenance costs (Jamieson, 1998; Malhotra et al., 2008; Dever et al., 2016). Oxidative phosphorylation in the mitochondria is responsible for a major part of the H₂O₂ generated in eukaryotic cells (Malhotra et al., 2008). However, another important source of H₂O₂ is the production of proteins destined for secretion, and more specifically their folding. The H₂O₂ resulting from secretory protein production is formed in the iterative process of the making and breaking of disulfide bonds before the proteins reach their final folded state. Especially for recombinant protein production, it is often the goal to achieve high protein titers and yields, which means a high flow through the secretory pathway and the folding machinery in the endoplasmic reticulum (ER). Increased secretory protein folding will lead to an elevated production of H₂O₂, and therefore potentially oxidative stress.

For many years, methods for measurement of intracellular reactive oxygen species (ROS), including H₂O₂, consisted of different ROS-sensitive dyes. These dyes are known for their undefined specificity and poor resolution, both temporally and spatially (Morgan et al., 2016). A new technology circumventing these problems was introduced in the form of genetically encoded fluorescent indicators (GEFIs) (Lukyanov and Belousov, 2014). GEFIs are fluorescent biosensors that enable real-time monitoring of several cellular processes but also of relevant compounds like H₂O₂ (Lukyanov and Belousov, 2014). The basis for the specific GEFI used in this protocol is a fusion of two proteins, which form a redox pair when together. One of these is a highly H₂O₂-reactive peroxiredoxin e.g., yeast Prx1, and the other a green fluorescent protein, roGFP2. RoGFP2 is an engineered enhanced GFP that has two cysteines in close proximity to the chromophore. Upon the making or breaking of a disulfide bond between those two cysteines, there will be a change in the protonation state of the chromophore, which changes the excitation wavelength (Schwarzländer et al., 2016). With the reduction of H₂O₂, the two cysteines in Prx1 form a disulfide bond that will next react into a mixed disulfide bond between Prx1 and the roGFP2 molecule; this finally resolves into an internal disulfide bond within roGFP2, thereby changing the excitation wavelength. A schematic overview of this mechanism is shown in Figure 1. RoGFP2 has excitation peaks at 488 nm in the reduced form and at 400 nm in the oxidized form that carries the internal disulfide bond (Schwarzländer et al., 2016). By dividing the signal measured at 400 nm by the signal measured at 488 nm, an oxidized:reduced ratio can be determined. At a basal level of endogenous H₂O₂ under non-stressed conditions, the sensor maintains an oxidized:reduced ratio near 1:1 (Schwarzländer et al., 2016). Furthermore, the sensor is ratiometric, and therefore any potential differences in sensor abundance between strains and/or conditions are expected not to affect measurements. The functionality and sensitivity of these sensors in cells have been tested by the direct addition of exogenous H₂O₂, but also a reducing agent (DTT), to the media (Morgan et al., 2016; Gast et al., 2021). Importantly, the roGFP2-Prx1 sensor can detect both oxidizing and reducing changes in the extracellular environment.

Here, we present a protocol on the use of the roGFP2-Prx1 sensor for in vivo measurements of cytosolic H₂O₂ during continuous micro-cultivation of S. cerevisiae in a BioLector (Mp2 Labs). This protocol was successfully used to detect changes in cytosolic H₂O₂ levels in S. cerevisiae due to recombinant protein production (Gast et al., 2021). The sensors are expected to be adequate to measure H₂O₂ produced from sources other than recombinant protein production as well.

Figure 1. H₂O₂ oxidizes cysteines in Prx1-roGFP2 into a disulfide bond altering sensor fluorescence. (1) Prx1 in the genetically-encoded fluorescent indicator reduces H₂O₂ to water by making an internal disulfide bond. (2) One of the electrons of a cysteine in the roGFP2 reacts with one of the cysteines in the disulfide bond, resulting in a new disulfide bond between Prx1 and roGFP2. (3) The second cysteine in roGFP2 reacts with the other cysteine in the disulfide bond between Prx1 and roGFP2, resulting in a new disulfide bond between the two cysteines in roGFP2. (4) The disulfide bond between the cysteines in roGFP2 changes the equilibrium of different forms of the chromophore, resulting in a shift of the wavelength of maximal excitation of roGFP2 from 488 to 400 nm.