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-Micheal addition reaction of the sulfhydryl to unsaturated ketone and releases fluorescence. FL-DTspecifically 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 inE. 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.
Keywords: dicysteine-tagged peptide, fluorescent probe, rapid labelling, expression conditions screening, xylanase
Introduction
Biomanufacturing of enzymes, proteins, therapeutics, and chemicals based on engineered cells or bacterium has been an emerging area that shows great importance. For instance, xylanase, which is mostly produced in the bacterial system, has enormous applications including waste-water treatment, pre-bleaching in the paper and pulp industry, bioconversion of biomass to biofuels, and other high-value final products, as well promoting the texture of nutrients in the food industry.1, 2 The expression of biomanufacturing from laboratory to industry with reliable scale-up remains a tremendous economic and technical barrier, which requires substantial efforts in researching and optimizing these bioprocesses. The conventional protein quantification methods include Lowry method,3 Bradford method,4 Bicinchoninic acid (BCA) method,5 etc.,6 which are all time-consuming and economically inefficient due to involving cell breaking and protein purification processes. Thus, it is of great importance to develop convenient and rapid methods to monitor biomanufacturing productivity and this would facilitate the optimization of the bioprocesses.
Fluorescent labeling of a specific protein of interest (POI) with genetically encoded fluorescent proteins or fusing the POI to an enzyme tag are widely used approaches in chemical biology to study the expression, localization, and trafficking of the protein in live cells and organisms.7, 8 However, the relatively large size of fused tags can sterically disturb the folding, functions, or even the localization of the POI.9, 10 Alternatively, Tsien and coworkers developed small organic fluorophores, termed FlAsH and ReAsH, that are activated by binding to specific tetracystein motifs (e.g. CCPGCC).11-13 This method allows the visualization of intracellular trafficking of recombinant proteins without the washing step.14, 15 However, these arsenic probes have an ambiguously toxic impact on living cells,16, 17 along with the uncertainty resulting from reversibility,13, 16, 18 limiting their applications. Nevertheless, the small molecule protein-labeling strategy with a non-toxic fluorogenic agent would be promising to monitor enzymatic productivity.
Our previous work reported that the Michael addition reaction of thiol to α , β -unsaturated ketone in a 2-cyclopentenone modified fluorescein releases the etherified phenolic group (the ring-open of spiro group due to the nucleophilic attack of thiol), resulting in a fluorescence enhancement (Scheme 1A).19 However, the monothiol responsive probe lacks specificity towards POIs.20, 21 Herein, we report a new fluorescein-based protein-labeling agent (FL-DT), which is modified by two 2-cyclopentenone, making the probe emit fluorescent signals only when it reacts with two thiols simultaneously. This newly designed FL-DT reacts with a specific di-Cys containing peptide tag rapidly and shows promises in monitoring the production of POIs determining the total amount of protein expressed in E. coli system in a fast and highly specific way (Scheme 1B and 1C).