Scheme 1. The mechanism of (A) probe
FL-ST with single reaction site in previous work and (B) probe FL-DT
with two reaction sites in this work. (C) The design of fluorogenic
peptide-tag labelling strategy with FL-DT.
Experimental section
Experiment and reagents
1H NMR (Ascend 400, 400 MHz) and 13C
NMR (101 MHz) spectra were performed on Bruker spectroscopy. Mass
spectra were measured on Q-Tof mass spectrometer (Agilent 6530).
Fluorescence emission spectra were obtained on RF-5301/PC
fluorophotometer (Shimadzu) and
microplate detector (Bio Tek
Instruments Synergy H1). The construction of recombinant protein was
operated on Mastercycler persona PCR Amplifier (Eppendorf). Purified
protein was obtained from AKTATM Prime protein purification system (GE)
and Ni2+ affinity chromatography (GE).
Enzyme analysis was carried out on
Mini-Protein tetra cell Electrophoresis Apparatus (Tanon). The gel
documentation was collected and analyzed by the
Gel imager (Tanon-5200Multi). The
cell images were observed by the inverted fluorescence microscope
(Olympus IX73P1F). All reagents were used in the experiment directly are
purchased on the market without further purification.
Synthesis
Synthesis of FL-DA
The fluorescent probe FL-DA required for these studies was
synthesized as shown in Scheme S1 . Fluorescein-dialdehyde
(FL-DA ) was obtained according to the previous
work.22, 23 Fluorescein (9.02 mmol, 3.00 g) and
urotropine (45.13 mmol, 6.32 g) were dissolved in trifluoroacetic acid
(25 mL). The resulting mixture was stirred vigorously and refluxed at 90
℃ overnight. After the mixture was cooled to room temperature, 200 mL of
dilute hydrochloric acid (4 mol/L) solution was added to get a turbid
solution and stirred until clear. The solution was extracted with
dichloromethane and saturated brine for several times, then the organic
phase was dried with anhydrous sodium sulfate and collected by reduced
pressure distillation. Chromatography on silica gel column using
dichloromethane as the eluent afforded product as white solid.
Synthesis of FL-DT
FL-DT was synthesized based on the previous work of our group.FL-DA (1.00 mmol, 388 mg), 2-cyclopenten-1-one (4.00 mmol, 328
mg) and imidazole (2.00 mmol, 36 mg) were mixed in 15 mL of
tetrahydrofuran and 10 mL of deionized water. The mixture was stirred at
room temperature for two weeks. After the reaction finished, the crude
solid was collected by reduced pressure distillation and further
purified on a silica gel using dichloromethane/methanol (200:1,V /V ) as the eluent to get a light-yellow solid
Synthesis of short peptides
All short peptides were ordered from
a peptide company (DgPepetides co., ltd), which provided synthesis and
purification. All sequences were
confirmed by mass spectrometry report and high-performance liquid
chromatograph (HPLC) analysis report to ensure that the purity was
higher than 95%.
Fluorescence enhancement test
FL-DT was dissolved in methanol (MeOH) for stock solution (1
mM). The various analytes were prepared for aqueous solution (10 mM).
The probe solutions were diluted with HEPES buffer (10 mM, pH 7.4, 1%
CH3CN) for testing fluorescent
spectra.
Except for Job’s plot and detection of fusion protein, which are carried
out on the Microplate Reader, the rest of the fluorescence signals are
measured on the fluorophotometer.
SDS-PAGE gel electrophoresis and fluorescence imaging
The adducts of probe and protein were separated and analyzed by 12%
SDS-PAGE. The proteins were labelled by probe at a ratio of 1:1 for 2 h.
After mixing with 5× loading buffer and boiling for 5 minutes, the
samples were added to each well with 20 μL. The voltage of
electrophoresis was 120 V, and the gel was imaged by an imaging system
on Gel imager (Tanon-5200Multi) directly (excited under blue light). As
a control, the gel was stained by Coomassie brilliant blue and imaging
under transmitted light at 302 nm.
Construction of expression vectors
The recombinant protein xylanase was
amplified by polymerase chain reaction (PCR) using the genomic DNA
extracted from Bacillus subtilis Lucky9 as template
with primer pair F1
GGCCATATG ATGTTCAAATTCAAAAAAAA with restriction site NdeI and R1
GGCCTCGAG GTACCACACTGTTACGTTAG with restriction site XhoI. PCR
amplification was carried out as follow: 95 °C for 5 min, 35 cycles
including 95 °C for 30 s, 56 °C for 20 s, 72 °C for 90 s, and a final
extension step at 72 °C for 10 min. The product of PCR was determined by
1% agarose gel electrophoresis and purified with PCR Clean up Kit to
recovery. The amplified xylanase gene was inserted into the pET-28a
expression vector after the digestion with NdeI and XhoI and confirmed
by sequencing. Then the tag 10C2C was added at the C-terminal of
xylanase sequence using the primer F2
GGCCATATG ATGTTCAAATTCAAAAAAAA (with cleavage sites of restriction
enzyme NdeI) and R2
GGCCTCGAG GTCTTCTTTGCAACCCGGGCACCAACGGTGGTACCACACTGT (with
cleavage sites of restriction enzyme XhoI) to amplify the sequence of
xylanase-10C2C, and the PCR product
was inserted into pET-28a expression vector after the digestion with
NdeI and XhoI and confirmed by sequencing.
Expression and analysis of recombinant protein
The constructed pET-28a-xylanase-10C2C was transformed into E.
coli BL21 (DE3) by heat shock (42 °C, 90 s). A single colony containing
the recombinant plasmid was picked up from Luria broth (LB) agar plate.
Luria broth media with kanamycin (50 mg/L) was inoculated with a
glycerol stock of E. coli BL21 (DE3) containing constructed
plasmids and grown overnight at 37 °C with shaking of 180 rpm. The
overnight culture was conducted a 1/50 inoculation of 40 mL fresh Luria
broth media containing 50 mg/L kanamycin, then was cultured until the
cells density (OD600) reached 0.4-0.6 (37 °C, 180 rpm).
The induction conditions were optimized, including the inducer
concentration, induction time and induction temperature. The recombined
strain was grown for another 24 h, the cells were harvested by
centrifugation for 10min (12,000 rpm, 4 °C), and the cell pellets were
resuspended in 40 mL phosphate buffer (20 mM, pH 7.5), and lysed by
ultrasonication (5 s, 3 s, total 10 min), then the supernatant of cell
lysis (including cytoplasm and periplasmic fraction) was obtained by
centrifugation for 10 min (12,000 rpm, 4
°C), the pellets by sonication were
also resuspended in 40 mL phosphate buffer. The expression of proteins
was analyzed by 12.5% SDS-PAGE.
Purification and quantitative detection of recombinant protein
After induction, the cell solution was centrifuged to remove the
supernatant, adding 40 mL phosphate buffer (20 mM, pH 7.5) to resuspend
the
cell
pellets for disruption. The crude enzyme was collected by crushing cell
and centrifugation, and purified by affinity chromatography using Ni-NTA
with His-tag in recombinant protein. After recombinant protein N2C was
eluted gradient with imidazole buffer (75-500 mM) and further purified
by gel filtration (Superdex 200) with Tris-NaCl buffer (50 mM, pH 8.0),
the concentrated protein was collected by ultrafiltration with a 10 kDa
filter membrane. The purified recombinant protein was denatured in
boiling water and determined by SDS-PAGE gel using 12.5%
(w/v )
separating gel and 4.0% (w/v ) stacking gel for zymogram
analysis. After electrophoresis, the gel was stained with Coomassie blue
at 65 °C under shaking condition for 30 min. The quantitative detection
of recombinant protein was determined by Bradford method (Coomassie
brilliant Blue G-250 regent) with bovine serum protein as standard.
Living-cell imaging with specific labeling
After fermentation, the cell solution
was centrifuged to remove the supernatant and resuspended with phosphate
buffer (20 mM, pH 7.5). The cell
pellets resuspension was co-incubated with FL-DT (10 μM) at
37
°C for 1 h. After removing the
solution, the cell pellets were resuspended in deionized water and
imaged under an inverted fluorescence microscope.
The microbial flow cytometry
The cell solution was obtained with post-processing as above description
and treated with FL-DT (1:1) at 37 °C for 3 h. Before counting
the single cells with fluorescence change on the flow cytometer via FITC
channel, the samples were diluted to 20,000 cells per 10 μL. N2C and Xyn
were expressed with 0.5 mM IPTG at 30
°C for 20 h. NI is the N2C which was
expressed without IPTG expression at 30 °C for 20 h.
Results and discussion