Fluorescent labeling of dicysteine‐tagged peptide for monitoring and optimization of protein bio‐production in bacteria

Abstract A rapid and convenient strategy to monitor the productivity of biomanufacturing is essential for the research in optimizing relevant bioprocesses. In this work, we have developed a fluorescein‐derived probe (FL‐DT) that reacts rapidly with thiol groups via 1,4‐Michael addition reaction of the sulfhydryl to unsaturated ketone and releases fluorescence. FL‐DT specifically forms fluorescent adduct with two adjacent thiols in a protein of interest (POI), making the probe a reliable tool for protein quantification. The production of xylanase fused with a short di‐Cys tag was then successfully monitored and quantified with FL‐DT in Escherichia coli system under different protein expression conditions, providing useful information for optimizing the bioprocess. Our work provides a convenient and efficient strategy for POI labeling and monitoring bioproduction.