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

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.