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Updated: Jul 27, 2026

Targeting Cysteine Thiols for in Vitro Site-specific Glycosylation of Recombinant Proteins
Published on: October 4, 2017
An engineered cysteine sensor optimized for high-throughput screening identifies regulators of intracellular thiol
Rachel P M Abrams1, Rebecca G Donahue1, Jessica Ma1
1Helen Diller Family Comprehensive Cancer Center, University of California, San Francisco, San Francisco, CA, USA.
None:
Dysregulation of cysteine-dependent processes is implicated in many diseases, including cancer. Despite the importance of cysteine in crucial cellular functions, including protein synthesis, redox balance, and glutathione production, a lack of efficient assays to measure cellular cysteine has limited efforts to identify agents that affect physiological cysteine levels. We employed circular permutation to engineer a fluorescent sensor that changes conformation upon cysteine binding. Biochemical experiments showed that this sensor is selective for cysteine, operating in the 10 μM-10 mM range. To demonstrate the sensor's applicability, we performed high-throughput screens for compounds that reduce cellular cysteine. Liquid chromatography of cell extracts validated the effect of two hit compounds, and mechanistic investigations showed that one was dependent on the anticancer target, xCT. Future application of this sensor in cell biology and drug discovery will advance understanding of cysteine metabolism and drive the development of therapeutics that restore cysteine homeostasis.
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