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