Figure 4 . (A) Specific
labeling of N2C-expressing cells with FL-DT (10 μM). The
N2C-expressing cells were treated without (a) and with (b)FL-DT , the Xyn-expressing cells were treated without (c) and
with (d) FL-DT . Scale bar: 10 μm. (B) Flow Cytometry data of
N2C (containing 0.5 mM IPTG as inducer), NI (no inducer), Xyn negative
control samples (black line) and the samples treated with FL-DT(red line). (C) Fluorescence spectra of FL-DT (1 μM) with
addition of different equivalents of N2C (0.01-1.00 μM). Photograph ofFL-DT reacted with N2C (0 μM, 0.2 μM and 0.5 μM) visualized
under a handheld UV lamp (inserted figure). Expression concentrations of
recombinant protein with 0.5 mM of IPTG (D) at different temperature for
20 h, and (E) at different time periods at 30 °C.
Conclusion
In summary, we have developed a new fluorescein-derived protein-labeling
agent FL-DT for monitoring the production of POIs in bacterial systems.
The probe releases fluorescence when its α , β -unsaturated
ketone moieties react with two thiols. Further characterizations prove
that FL-DT specifically forms a fluorescent adduct with one peptide tag
containing two adjacent thiol groups within 1 min, offering reliability
in protein quantification. The application of monitoring protein
production is successfully performed using fluorescence microscopy and
flow cytometry in E. coli system that expresses xylanase fused
with a CPGC-containing tag. The quantification of the POI can be
achieved at a high specificity without breaking the cells. Thus, this
new labeling agent provides a convenient tool for rapid POIs labeling
and its applications include but are not limited to monitoring protein
production and optimization of protein expression conditions.