A fluorescent probe for specifically and rapidly labeling a double-cysteine tag protein, a preparation method and applications thereof

Abstract

The application discloses a fluorescent probe for specifically and rapidly marking a double-cysteine tag protein, a preparation method and application, and the probe can emit a fluorescent signal only when reacting with two cysteines simultaneously. The newly designed fluorescent probe can rapidly react with a recombinant protein containing a specific double-cysteine-containing peptide tag, and can mark POI in a site-specific manner. The fluorescent probe has a structure as shown in formula I, is named as 3,7-dihydroxy-5,5-dimethyl-4,6-bis((E)-2-nitrovinyl)-3'H,5H spiro[di-benzo[b,E]silicon-10,1'-isobenzofuran]-3'-ketone, and is abbreviated as Si-FLNO2.

Description

Technical Field

[0001] This invention relates to a probe, its preparation method, and its application; more specifically, it relates to a fluorescent probe for specifically and rapidly labeling biscysteine-tagged proteins, its preparation method, and its application. Background Technology

[0002] Fluorescent labeling of proteins of interest (POIs) using genetically encoded fluorescent proteins or fusing peptide tags to POIs is a widely used method in chemical biology for studying protein expression, localization, and transport in living cells and organisms. However, relatively large fusion tags can spatially interfere with the folding, function, and even localization of POIs. In recent years, strategies using small-sized tags to bind fluorescent probes have been extensively reported; however, many probes in existing techniques suffer from poor water solubility, low sensitivity, long response times, poor specificity, high fluorescence background, and biotoxicity in biological applications. Therefore, it is essential to develop a fluorescent probe capable of rapidly binding small-sized tags to specifically and rapidly label biscysteine-tagged proteins, along with its preparation method and applications. Summary of the Invention

[0003] The purpose of this invention is to overcome the shortcomings of existing technologies and provide a fluorescent probe for the specific and rapid labeling of dicysteine-tagged proteins. This probe only emits a fluorescent signal when it reacts with both cysteine ​​residues simultaneously. This newly designed fluorescent probe reacts rapidly with recombinant proteins containing specific dicysteine ​​(di-Cys) peptide tags, enabling site-specific labeling of POIs.

[0004] This invention also provides a method for preparing and applying a fluorescent probe for specifically and rapidly labeling cysteine-tagged proteins. The preparation method is simple and easy to implement, and the reaction conditions are mild.

[0005] The technical concept and principle of this invention are as follows: Research has shown that a 3,7-dihydroxy-5,5-dimethyl-4,6-bis((E)-2-nitrovinyl)-silicone anthracene ring derivative probe can selectively react with cysteine ​​and di-Cys recombinant proteins, causing changes in their fluorescence. Furthermore, within the concentration range of 0-0.3 mg / mL di-Cys protein, the fluorescence intensity at 610 nm shows a good linear relationship with the protein concentration. This invention is the first to prepare a 3,7-dihydroxy-5,5-dimethyl-4,6-bis((E)-2-nitrovinyl)-silicone anthracene ring derivative and the first to use it for the selective detection of cysteine ​​and rapid labeling of intracellular di-Cys recombinant proteins, thus solving the problems existing in the prior art.

[0006] The technical solution of the present invention is as follows:

[0007] The present invention provides a fluorescent probe for the specific and rapid labeling of cysteine-tagged proteins, having a structure as shown in Formula I, and named 3,7-dihydroxy-5,5-dimethyl-4,6-bis((E)-2-nitrovinyl)-3'H,5H-spiro[dibenzo[b,E]silyl-10,1'-isobenzofuran]-3'-one, abbreviated as Si-FLNO2.

[0008]

[0009] The method for preparing the fluorescent probe for specifically and rapidly labeling biscysteine-tagged proteins described above in this invention includes the following steps: reacting 3,7-dihydroxy-5,5-dimethyl-3'-oxo-3'H,5H-spiro[dibenzo[b,e]silyl-10,1'-isobenzofuran]-4,6-dicarboxaldehyde in a nitrobenzene mixed solution to generate a solid, which is 3,7-dihydroxy-5,5-dimethyl-4,6-bis((E)-2-nitrovinyl)-3'H,5H-spiro[dibenzo[b,E]silyl-10,1'-isobenzofuran]-3'-one, which is the fluorescent probe for specifically and rapidly labeling biscysteine-tagged proteins.

[0010] The method for preparing the fluorescent probe for specifically and rapidly labeled biscysteine-tagged proteins described above in this invention further includes the following technical solution: the reaction conditions are 70-80℃ with stirring for at least 72 hours; after the reaction, purification is performed, followed by vacuum drying to obtain the fluorescent probe for specifically and rapidly labeled biscysteine-tagged proteins. A further technical solution involves purification after the reaction, specifically as follows: the reaction solution is filtered under reduced pressure, and the crude product is further purified on a silica gel column using dichloromethane / methanol.

[0011] The present invention relates to the application of the fluorescent probe for the specific and rapid labeling of dicysteine-tagged proteins described above in the specific and rapid labeling of dicysteine-tagged proteins. This probe can be used for the rapid and specific labeling of di-Cys recombinant proteins.

[0012] The present invention has the following beneficial effects:

[0013] 1) The fluorescent probe of this invention is dispersed in PBS buffer solution at pH 7.4 and has almost no fluorescence. After undergoing a 1,4-Michael addition reaction with di-Cys, the spiro ring is opened due to electron transfer, the conjugated structure is increased, the absorption changes, and the fluorescence becomes significant.

[0014] 2) The fluorescence response method of the present invention has a strong fluorescence response to Cys, but a weak fluorescence response to amino acids such as lysine, tyrosine, tryptophan, glutamic acid, valine, and aspartic acid, and has good selectivity and specificity.

[0015] 3) The fluorescence response method of this invention has high detection sensitivity and can rapidly label di-Cys recombinant proteins, with a detection limit of 0-0.3 mg / mL for di-Cys. Compared with commercially available fluorescent dyes (fluorescein-5-maleimide), under the same test conditions, this probe exhibits stronger fluorescence specificity, higher signal-to-noise ratio, and better stability for Cys-containing proteins. This probe possesses the excellent performance of fluorescent dyes specifically labeling di-Cys recombinant proteins and can be widely used for the labeling and detection of di-Cys recombinant proteins.

[0016] 4) The fluorescent probe of this invention has low toxicity to organisms and can perform fluorescence imaging of di-Cys recombinant proteins in bacteria. Attached Figure Description

[0017] Figure 1 The fluorescence spectra of the fluorescent probe of the present invention reacting with different amino acids in Example 2 are shown.

[0018] Figure 2 The fluorescence increment diagram is shown after the fluorescent probe of the present invention in step 3 reacts with Cys.

[0019] Figure 3 This is a graph showing the change in fluorescence intensity over time after the fluorescent probe of the present invention reacts with Cys in Example 3.

[0020] Figure 4 Fluorescence increment diagram of the fluorescent probe of the present invention and the polypeptide containing di-Cys tag in Example 4.

[0021] Figure 5 The image shows the 1H-NMR spectrum of the fluorescent probe of the present invention in Example 1.

[0022] Figure 6 This is a high-resolution mass spectrum of the fluorescent probe of the present invention in Example 1.

[0023] Figure 7 This is a gel electrophoresis image of the fluorescent probe of the present invention reacting with the protein in Example 5.

[0024] Figure 8 This is a fluorescence image of the di-Cys recombinant protein in cells, obtained by the fluorescent probe of the present invention in Example 6. Detailed Implementation

[0025] The present invention will now be described in further detail with reference to the accompanying drawings and embodiments.

[0026] Example 1: Preparation of a fluorescent probe for the rapid labeling of di-cysteine-tagged proteins, i.e., a fluorescent probe for the rapid labeling of di-Cys recombinant proteins.

[0027] 430 mg of 3,7-dihydroxy-5,5-dimethyl-3'-oxo-3'H,5H-spiro[dibenzo[b,e]silyl-10,1'-isobenzofuran]-4,6-dicarboxaldehyde, 77 mg of ammonium acetate, and 284 mg of anhydrous sodium sulfate were dissolved in 15 mL of nitromethane and stirred in an oil bath at 75 °C for 72 h. After the reaction was completed, the reaction solution was concentrated under reduced pressure, and the crude product was further purified by dichloromethane / methanol on a silica gel column. Vacuum drying yielded 140 mg of an orange solid, which was the fluorescent probe for the specific rapid labeling of biscysteine-tagged proteins. 1 See the H-NMR spectrum and high-resolution mass spectrum. Figure 5 , Figure 6 The molecular weight of the obtained pure fluorescent probe was 516.10.

[0028] Process route in this embodiment:

[0029]

[0030] Example 2: Spectroscopic properties of the reaction between the probe of the present invention and various amino acids

[0031] Weigh 5.16 mg of the fluorescent probe prepared in Example 1 for rapid and specific detection of di-Cys recombinant protein, and prepare a 1 mM CH3CN solution in 10 mL as the stock solution.

[0032] Add 30 μL of the above stock solution to PBS buffer, then add the analytes Cys, Ala, Arg, Asn, Asp, Glu, Gln, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, and Val to bring the final concentration of the analytes to 100 μM and the final concentration of the fluorescent probe to 10 μM. Measure the fluorescence emission spectrum immediately at an excitation wavelength of 590 nm. The slit widths for excitation and emission are 5 / 2.5 nm. The resulting fluorescence spectra are shown below. Figure 1 As shown.

[0033] The above results indicate that:

[0034] (1) The fluorescent probe prepared in Example 1 has almost no fluorescence in solution, but with the addition of Cys, the fluorescence emission of the probe at 610 nm is enhanced by about 70 times.

[0035] (2) The fluorescent probe prepared in Example 1 has high selectivity and specificity for Cys, and under the above conditions, it can distinguish Cys from Ala, Arg, Asn, Asp, Glu, Gln, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, and Val.

[0036] Example 3: Spectral properties of the fluorescent probe of the present invention reacting with Cys products

[0037] 30 μL of the stock solution from Example 1 was added to PBS buffer (pH 7.4), and different equivalents of Cys were added to bring the final concentration of the fluorescent probe to 10 μM. The Cys concentrations were 0 μM, 2 μM, 4 μM, 6 μM, 8 μM, 10 μM, 12 μM, 14 μM, 16 μM, 18 μM, and 20 μM, respectively. The fluorescence emission spectrum was measured immediately after the Cys addition. The excitation wavelength was 590 nm; the slit widths for excitation and emission were 5 / 2.5 nm. The resulting fluorescence intensity spectrum increments are shown in [Figure number missing]. Figure 2 .

[0038] 30 μL of the stock solution from Example 1 was added to PBS buffer (pH 7.4), and Cys was added to bring the final concentration of the fluorescent probe to 10 μM and the Cys concentration to 100 μM. Immediately after the addition of Cys, the fluorescence kinetics curve was measured. The kinetics curve was measured using an excitation wavelength of 590 nm and an emission wavelength of 610 nm; the excitation and emission slit widths were 5 / 2.5 nm. The obtained kinetic curves are shown in [Figure number missing]. Figure 3 .

[0039] Example 4: Spectral properties of the fluorescent probe of the present invention and di-Cys-containing polypeptide products

[0040] 30 μL of the stock solution from Example 1 was added to PBS buffer (pH 7.4), and different equivalents of di-Cys-containing peptides 15C2C, 15C4C, 15C6C, and 15C8C were added to bring the final concentration of the fluorescent probe to 10 μM, with peptide concentrations of 0 μM, 1 μM, 2 μM, 3 μM, 4 μM, 5 μM, 6 μM, 7 μM, 8 μM, 9 μM, and 10 μM, respectively. The fluorescence emission spectrum was measured immediately after the addition of the di-Cys-containing peptides. The excitation wavelength was 590 nm; the slit widths for excitation and emission were 5 / 2.5 nm. The resulting fluorescence intensity spectrum increments are shown in the figure. Figure 4 Figure (a) shows the fluorescence titration spectrum of peptide 15C2C, Figure (b) shows the fluorescence titration spectrum of peptide 15C4C, Figure (c) shows the fluorescence titration spectrum of peptide 15C6C, and Figure (d) shows the fluorescence titration spectrum of peptide 15C8C.

[0041] The experimental results show that the fluorescence intensity increases with increasing concentration of di-Cys-containing peptide after the reaction, indicating that it can be used for labeling di-Cys-containing peptides. The fluorescent probe itself is non-fluorescent, and the addition of di-Cys peptide causes fluorescence enhancement, reaching its maximum value when the amount of di-Cys peptide is equal to that of the probe.

[0042] Example 5: Imaging of recombinant proteins containing di-Cys tags using the probe of the present invention.

[0043] The recombinant protein EG5C-1 containing the di-Cys tag was expressed in *E. coli*. Recombinant EG5C-1 with di-Cys tags at the C-terminus and N-terminus was diluted to 0.1 mg / mL and mixed with the stock solution from Example 1 at a 1:1 ratio to form probe-protein adducts. Separation and analysis were performed using 12% SDS-PAGE. After boiling the mixture of adduct and loading buffer for 5 minutes, 15 μL was loaded into each well, and the electrophoresis voltage was 120 V. Gel fluorescence imaging was performed in a Tanon-5200Multi gel imaging system under green light excitation. As a control, Coomassie brilliant blue staining was performed, followed by imaging under 302 nm transmitted light. The resulting protein gel electrophoresis analysis is shown in [Figure number missing]. Figure 7 .

[0044] Example 6: Imaging of di-Cys-tagged recombinant proteins in E. coli using the probe of the present invention.

[0045] 10 μM of the stock solution from Example 1 was added to 1 mL of E. coli fermentation broth expressing the di-Cys-tagged recombinant protein Xyn, and co-incubated with the bacterial culture for 1 h. After centrifugation, the supernatant was discarded, and the broth was resuspended in water. The E. coli expressing the di-Cys-tagged recombinant protein was imaged using a confocal microscope. The experimental results are as follows: Figure 8 As shown.

[0046] In addition to the embodiments described above, the present invention may have other implementations. All technical solutions formed by equivalent substitution or equivalent transformation fall within the protection scope claimed by the present invention.

Claims

1. A fluorescent probe for specific and rapid labeling of biscysteine-tagged proteins, characterized in that, The probe has the structure shown in Formula I and is named 3,7-dihydroxy-5,5-dimethyl-4,6-bis((E)-2-nitrovinyl)-3'H,5Hspiro[dibenzo[b,E]silyl-10,1'-isobenzofuran]-3'-one, abbreviated as Si-FLNO2 2. A method for preparing a fluorescent probe for specifically and rapidly labeling biscysteine-tagged proteins as described in claim 1, characterized in that, Includes the following steps: Reaction of 3,7-dihydroxy-5,5-dimethyl-3'-oxo-3'H,5H-spiro[dibenzo[b,e]silyl-10,1'-isobenzofuran]-4,6-dicarboxaldehyde in a nitromethane mixture yields a solid, 3,7-dihydroxy-5,5-dimethyl-4,6-bis((E)-2-nitrovinyl)-3'H,5H-spiro[dibenzo[b,E]silyl-10,1'-isobenzofuran]-3'-one, which is a fluorescent probe for the specific and rapid labeling of biscysteine-tagged proteins.

3. The method for preparing a fluorescent probe for specifically and rapidly labeling biscysteine-tagged proteins according to claim 2, characterized in that, The reaction conditions are 70-80℃ with stirring for more than 72 hours; after the reaction is completed, the sample is purified and then vacuum dried to obtain a fluorescent probe that specifically and rapidly labels cysteine-tagged proteins.

4. The method for preparing a fluorescent probe for specifically and rapidly labeling biscysteine-tagged proteins according to claim 3, characterized in that, After the reaction is completed, purification is performed. The specific purification steps are as follows: the reaction solution is filtered under reduced pressure, and the crude product is further purified on a silica gel column using dichloromethane / methanol.

Images