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).