A genetically encoded biosensor roKate for monitoring the redox state of the glutathione pool

About authors

1 Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Moscow, Russia

2 The Research Institute for Translational Medicine, Pirogov Russian National Research Medical University, Moscow, Russia

Correspondence should be addressed: Dmitry S. Bilan
Miklouho-Maclay, 16/10, Moscow, 117997; moc.liamg@nalib.s.d

About paper

Funding: this work was supported by the Russian Foundation for Basic Research (Project mol_a_dk No.16-34-60175).

Author contribution: Shokhina AG was responsible for the experimental part of the study. Belousov VV and Bilan DS supervised the study and prepared this manuscript.

Received: 2018-12-26 Accepted: 2019-03-02 Published online: 2019-03-15

Genetically encoded fluorescent sensors are exploited to study a variety of biological processes in living organisms in real time. In recent years, a whole family of biosensors has been developed, serving to visualize changes in the glutathione redox state. The aim of our experiment was to design a biosensor based on the red fluorescent protein mKate2 for measuring the 2GSH/GSSG ratio. A pair of cysteine amino acid residues were introduced into the structure of the fluorescent protein using site-directed mutagenesis. These residues form a disulfide bridge when the surrounding glutathione pool is oxidized, affecting the spectral characteristics of the protein. Our biosensor, which we called roKate, was tested in vitro on an isolated protein. Specifically, we examined the spectral characteristics, pH and the redox potential of the sensor. Additionally, the performance of roKate was evaluated using the culture of living mammalian cells. The fluorescent signal emitted by the sensor was very bright and remarkably stable under pH conditions varying in the physiological range. Irreversibly oxidized in mammalian cells, roKate stands out from other members of this biosensor family. This biosensor should be preferred in the experiments when the time between the manipulations with the biological object and the subsequent analysis of the induced effect is substantial, as is the case with long sample preparation.

Keywords: genetically encoded fluorescent sensor, glutathione, 2GSH/GSSG ratio, roKate