Dual‐cysteine tag‐directed fluorescence platform for high‐throughput screening of <i>E. coli</i> protein expression systems

Abstract Methods for accurate and efficient protein quantification are crucial in the fields of biomanufacturing, synthetic biology and protein development for optimizing related bioprocesses. Herein, we developed a fluorescent probe, FL‐NO 2 , composed of fluorescein linked to dinitroethylene groups, enabling site‐specific covalent labeling of cysteine residues. This probe exhibits high specificity for CPGC‐tagged proteins, and the impacts of the location of the inserted dual‐Cysteine tag, as well as the introduction of a flexible GGGGS linker, on the protein activity and the labeling efficiency were investigated to extend the practical applicability of this fluorescent tool. Using FL‐NO 2 as a fluorescence‐guided expression platform for in situ protein detection during bacterial fermentation allowed the key parameter optimization. Moreover, this fluorescence‐guided platform enabled high‐throughput screening of promoter libraries overcoming the labor‐intensive limitations of traditional protein expression analysis. This work provides a platform that facilitates the development of in situ protein quantification and high‐throughput protein engineering techniques.