3.3 Specific recognition of fusion protein
We then moved forward to the application of monitoring the expression of POIs in live cells by employing FL-DT as the protein-labeling agent. Xylanase- expressing E. coli was selected as the model system for this investigation. Since xylanase has no Cys residue, a diCys-containing peptide sequence was fused to the C-terminus of xylanase through protein engineering. Considering the potential steric effect, the easiness, and the cost for protein expression, peptide 10C2C was selected as a tag motif. The recombinant xylanase (N2C) was expressed and purified as described in experimental section. The successful expression of N2C and specific labeling with FL-DTwas confirmed by zymogram analysis with its corresponding band at approximately 25 kDa with distinct fluorescence observed, while the original xylanase (Xyn, about 22 kDa) had no fluorescence response toFL-DT (Figure 3Aand Figure 3B ). Also, no crosslinking was observed (Figure S5 ), demonstrating one FL-DT molecule only bind to one protein. To verify that the enhancement of fluorescence intensity was ascribed to the introduction of di-Cys rather than other amino acids, both Cys residues in the tag were mutated to alanine by constructing a new fused xylanase (NOC) (sequence described in supplementary materials). The results showed that mixing NOC withFL-DT barely generates fluorescence (Figure 3B ). Furthermore, when the thiol groups in N2C pre-reacted with N-methylmaleimide (NMM), the protein lost the ability to activateFL-DT as well (Figure 3B ). These results demonstrated that the FL-DT can respond to the recombinant xylanase that contains a CPGC motif with high selectivity and sensitivity. Additionally, the enzyme activity assays of the purified recombinant proteins were measured by dinitrosalicylic acid reagent (DNS) method based on a standard curve of the Xylose versus optical density (OD540 nm) (Figure 3C ). The results show that the activity of N2C was almost the same as that of the original enzyme, while the N2C enzyme activity after being labeled with FL-DTslightly decreased (Figure 3D ), suggesting that theFL-DT -based protein labeling approach could be a convenient tool for monitoring the production of POIs without impairing the protein function.
To further demonstrate the potential of FL-DT in monitoring the bioproduction of POIs, we then applied FL-DT in a N2C-expressing E. coli system. The FL-DT treated N2C-expressing E. coli cells exhibited enhancing fluorescence within 30 min (Figure S7A ), demonstrating that FL-DTmolecules are able to penetrate the cell membranes and successfully label the expressed N2C in-situ . After staining withFL-DT (10 μM) for one hour under 37 °C, apparent green fluorescence was observed within N2C-expressing E. coli cells under an inverted fluorescence microscope, while the cells containing Xyn showed almost non-fluorescence change (Figure 4A ). In addition, the FL-DT stained E. coli cells were analyzed via flow cytometry. The E. coli cells with a different abundance of expressed N2C (Figure S6 ) were selectively distinguished (Figure 4B ). These results support that FL-DT enables the specific labeling and imaging of a POI in live cells, as well as the screening of cells with high productivity.