Construction and characterization of protein-based cysteine nanosensor for the real time measurement of cysteine level in living cells

Cysteine plays a critical role in maintaining normal human metabolism, redox homeostasis, and immune regulation. Despite its functional versatility, cysteine metabolism in the human body is not well understood because of the lack of a robust tool for real-time measurement of cysteine at the cellular and sub-cellular level. In the present study, a genetically encoded nanosensor was developed using Cj0982 protein of Campylobacter jejuni, Enhanced Cyan Fluorescent Protein (ECFP) and Venus. The Cj0982 was sandwiched between ECFP and Venus for the construction of the nanosensor, named as Cys-FS (Cysteine-Fluorescent-Sensor). The Cys-FS is pH stable, specific to cysteine and has an affinity of 1.2 x 10(-5) M. A range of affinity mutants were also developed with a cumulative cysteine detection range from 800 nM to 3.5 mM. The Cys-FS nanosensor was expressed in bacterial, yeast and mammalian cells, and the dynamics of cysteine level was measured in living cells using the confocal microscopy. The results showed that the Cys-FS nanosensor successfully monitored the dynamics of cysteine in both prokaryotic and eukaryotic systems without disrupting the cell. Thus, this study presents a novel nanosensor that can measure cysteine in living cells. This nanosensor is minimally invasive and non-toxic. (C) 2019 Elsevier B.V. All rights reserved.