Fluorescent probe for specifically and rapidly marking double-cysteine tag protein as well as preparation method and application of fluorescent probe

Abstract

The invention discloses a fluorescent probe for specifically and rapidly marking double-cysteine tag protein, a preparation method and application. The probe can emit a fluorescent signal only when reacting with two cysteines at the same time. The newly designed fluorescent probe rapidly reacts with a recombinant protein containing a specific double-cysteine peptide tag, and can carry out site specific labeling on POI (Point of Interest). The fluorescent probe disclosed by the invention has a structure as shown in a formula I, and the name of the fluorescent probe is 3, 7-dihydroxy-5, 5-dimethyl-4, 6-bis ((E)-2-nitrovinyl)-3 'H, 5H spiro [dibenzo [b, E] silicon-10, 1'-isobenzofuran]-3 '-ketone, Si-FLNO2 # imgabs0 # for short, and the name of the fluorescent probe is 3, 7-dihydroxy-5, 5-dimethyl-4, 6-bis ((E)-2-nitrovinyl)-3' H, 5H spiro [dibenzo [b, E] silicon-10, 1 '-isobenzofuran]-3'-ketone.

Description

Technical Field

[0001] The present invention relates to a probe, a preparation method and an application, and more particularly to a fluorescent probe for specifically and rapidly labeling double cysteine-tagged proteins, a preparation method and an application. Background Art

[0002] Fluorescently labeling a protein of interest (POI) with a genetically encoded fluorescent protein or fusing a peptide tag to the POI is a widely used method in chemical biology for studying the expression, localization, and transport of proteins in living cells and organisms. However, relatively large fusion tags may spatially interfere with the folding, function, and even localization of the POI. In recent years, strategies using small-sized tags in combination with fluorescent probes have been widely reported. However, many probes in the prior art have poor water solubility, low sensitivity, long response time, poor specificity, high fluorescence background, and biological toxicity in biological applications. Therefore, it is very necessary to develop a fluorescent probe that can rapidly bind to a small-sized tag to specifically and rapidly label double cysteine-tagged proteins, a preparation method, and an application thereof. Summary of the invention

[0003] The purpose of the present invention is to overcome the shortcomings of the prior art and provide a fluorescent probe for specific and rapid labeling of di-cysteine ​​tagged proteins, which can emit a fluorescent signal only when reacting with two cysteines at the same time. This newly designed fluorescent probe reacts rapidly with recombinant proteins containing specific di-cysteine ​​(di-Cys) peptide tags, and can perform site-specific labeling of POIs.

[0004] The invention also provides a preparation method and application of the fluorescent probe for specific and rapid labeling of double cysteine ​​label proteins. The preparation method is simple and easy, and the reaction conditions are mild.

[0005] The technical concept and principle of the present invention are as follows: It has been found that the 3,7-dihydroxy-5,5-dimethyl-4,6-bis((E)-2-nitrovinyl)-silanthracene ring derivative probe can selectively react with cysteine ​​and di-Cys recombinant proteins and cause changes in their fluorescence, and within the concentration range of 0-0.3 mg / mL di-Cys protein, the fluorescence intensity at 610 nm has a good linear relationship with the protein concentration. The present invention prepares 3,7-dihydroxy-5,5-dimethyl-4,6-bis((E)-2-nitrovinyl)-silanthracene ring derivatives for the first time and uses them for the selective detection of cysteine ​​for the first time, and quickly labels di-Cys recombinant proteins in cells, solving the problems existing in the prior art.

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

[0007] The fluorescent probe for specific rapid labeling of double cysteine ​​tagged proteins of the present invention has a structure as shown in Formula I and is 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 preparation method of the fluorescent probe for specific and rapid labeling of double cysteine ​​tagged proteins of the present invention comprises the following steps: 3,7-dihydroxy-5,5-dimethyl-3'-oxo-3'H, 5H spiro[dibenzo[b,e]silicon-10,1'-isobenzofuran]-4,6-dicarbaldehyde is reacted in a nitroethylene mixed solution to generate a solid of 3,7-dihydroxy-5,5-dimethyl-4,6-bis((E)-2-nitrovinyl)-3'H, 5H spiro[dibenzo[b,E]silicon-10,1'-isobenzofuran]-3'-one, which is a fluorescent probe for specific and rapid labeling of double cysteine ​​tagged proteins.

[0010] The preparation method of the fluorescent probe for specific and rapid labeling of double cysteine-tagged proteins of the present invention has a further technical solution that the reaction conditions are 70-80°C, and the reaction is stirred for more than 72 hours; after the reaction is completed, purification is performed, and vacuum drying is performed to obtain the fluorescent probe for specific and rapid labeling of double cysteine-tagged proteins. A further technical solution is that purification is performed after the reaction is completed, and 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.

[0011] The fluorescent probe for specific and rapid labeling of di-cysteine ​​tagged proteins of the present invention is used for specific and rapid labeling of di-cysteine ​​tagged proteins. The probe can be used for rapid and specific labeling of di-Cys recombinant proteins.

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

[0013] 1) The fluorescent probe of the present invention is dispersed in a PBS buffer solution with a pH of 7.4 and has almost no fluorescence. After a 1,4-Michael addition reaction with di-Cys, the spiro ring opens due to electron transfer, the conjugated structure increases, the absorption changes, and the fluorescence is 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 the present invention has high detection sensitivity and can quickly label di-Cys recombinant proteins. The detection line of di-Cys can reach 0-0.3 mg / mL. Compared with commercial fluorescent dyes (fluorescein-5-maleimide), under the same test conditions, the fluorescence response specificity of this probe to Cys-containing proteins is stronger, the signal-to-noise ratio is higher, and the stability is better. This probe has the excellent performance of a fluorescent dye that specifically labels di-Cys recombinant proteins and can be widely used in the labeling and detection of di-Cys recombinant proteins.

[0016] 4) The fluorescent probe of the present invention has low toxicity to organisms and performs fluorescent imaging of di-Cys recombinant proteins in bacteria. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 1 is the fluorescence spectrum of the fluorescent probe of the present invention reacting with different amino acids in Example 2.

[0018] Figure 2 This is a fluorescence increment diagram after the fluorescent probe of the present invention reacts with Cys in Implementation 3.

[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 a di-Cys tag in Example 4.

[0021] Figure 5 This is the 1H-NMR graph of the fluorescent probe of the present invention in Example 1.

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

[0023] Figure 7 This is the gel electrophoresis diagram after the fluorescent probe of the present invention reacts with protein in Example 5.

[0024] Figure 8 This is a fluorescence image of the fluorescent probe of the present invention in Example 6 imaging di-Cys recombinant protein in cells. DETAILED DESCRIPTION

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

[0026] Example 1 Preparation of a fluorescent probe for specific and rapid labeling of di-cysteine ​​tagged proteins, i.e., a fluorescent probe for 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-dicarbaldehyde, 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 on a silica gel column with dichloromethane / methanol and vacuum dried to obtain 140 mg of an orange solid, which was a fluorescent probe for specific and rapid labeling of double cysteine-tagged proteins ( 1 H-NMR and high-resolution mass spectra are shown in Figure 5 , Figure 6 ). The measured molecular weight of the obtained pure fluorescent probe is 516.10.

[0028] Process route of this embodiment:

[0029]

[0030] Example 2 Spectral properties of the probe of the present invention reacting with various amino acids

[0031] 5.16 mg of the fluorescent probe for rapid and specific detection of di-Cys recombinant protein prepared in Example 1 was weighed and prepared into 10 mL of a 1 mM CH3CN solution as a mother solution.

[0032] 30 μL of the above mother solution was added to PBS buffer, and then the test substances Cys, Ala, Arg, Asn, Asp, Glu, Gln, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, and Val were added respectively, so that the final concentration of the test substance was 100 μM and the final concentration of the fluorescent probe was 10 μM. The fluorescence emission spectrum was immediately tested at an excitation wavelength of 590 nm. The slit width of excitation and emission was 5 / 2.5 nm. The obtained fluorescence spectrum is shown in the figure below. Figure 1 shown.

[0033] The above results show that:

[0034] (1) The fluorescent probe prepared in Example 1 itself has almost no fluorescence in the 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 reaction product of the fluorescent probe of the present invention and Cys

[0037] 30 μL of the mother solution in Example 1 was added to PBS buffer (pH 7.4), and different equivalents of Cys were added to make the final concentration of the fluorescent probe 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. After Cys was added, its fluorescence emission spectrum was measured immediately. The fluorescence emission spectrum was measured with an excitation wavelength of 590 nm; the slit widths of excitation and emission were 5 / 2.5 nm. The obtained fluorescence intensity spectrum increment diagram is shown in Figure 2 .

[0038] 30 μL of the mother solution in Example 1 was added to PBS buffer (pH 7.4), and Cys was added to make the final concentration of the fluorescent probe 10 μM and the Cys concentration 100 μM. After Cys was added, the fluorescence kinetic curve was measured immediately. The kinetic curve was measured with an excitation wavelength of 590 nm and an emission wavelength of 610 nm; the slit widths of the excitation and emission were 5 / 2.5 nm. The obtained kinetic curve is shown in Figure 3 .

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

[0040] 30 μL of the mother solution in Example 1 was added to PBS buffer (pH 7.4), and different equivalents of di-Cys-containing polypeptides 15C2C, 15C4C, 15C6C, and 15C8C were added respectively, so that the final concentration of the fluorescent probe was 10 μM, and the polypeptide concentrations were 0 μM, 1 μM, 2 μM, 3 μM, 4 μM, 5 μM, 6 μM, 7 μM, 8 μM, 9 μM, and 10 μM, respectively. After the di-Cys-containing polypeptide was added, its fluorescence emission spectrum was measured immediately. The fluorescence emission spectrum was measured with an excitation wavelength of 590 nm; the slit width for excitation and emission was 5 / 2.5 nm. The obtained fluorescence intensity spectrum increment graph is shown in Figure 4 Figure (a) shows the fluorescence titration spectrum for polypeptide 15C2C, Figure (b) shows the fluorescence titration spectrum for polypeptide 15C4C, Figure (c) shows the fluorescence titration spectrum for polypeptide 15C6C, and Figure (d) shows the fluorescence titration spectrum for polypeptide 15C8C.

[0041] The experimental results show that the fluorescence intensity after the reaction increases with the concentration of di-Cys-containing peptides, and can be used for labeling di-Cys-containing peptides. The fluorescent probe itself has no fluorescence, and the addition of di-Cys peptides causes the fluorescence enhancement to reach the maximum value when the amount is equal to that of the probe.

[0042] Example 5 Imaging of di-Cys-tagged recombinant proteins using the probe of the present invention

[0043] The recombinant protein EG5C-1 containing a di-Cys tag was expressed in Escherichia coli, and the recombinant EG5C-1 containing di-Cys tags at the C-terminus and N-terminus was diluted to 0.1 mg / mL, and mixed with the mother liquor in Example 1 at a ratio of 1:1 to form a probe and protein adduct, which was separated and analyzed by 12% SDS-PAGE. After boiling the mixture of the adduct and the loading buffer for 5 minutes, 15 μL was loaded into each well, and the electrophoresis voltage was 120 V. Gel fluorescence imaging was taken under green light excitation in a Tanon-5200Multi gel imager. As a control, Coomassie Brilliant Blue staining was used and the image was imaged under 302nm transmitted light. The resulting protein gel electrophoresis analysis diagram is shown in Figure 7 .

[0044] Example 6 Imaging of di-Cys-tagged recombinant proteins by the probe of the present invention in Escherichia coli

[0045] 10 μM of the mother solution in Example 1 was added to 1 mL of E. coli fermentation broth expressing the recombinant protein Xyn containing a di-Cys tag, and incubated with the bacterial broth for 1 hour; after centrifugation, the supernatant was discarded, and the mixture was resuspended in water. The E. coli expressing the recombinant protein containing a di-Cys tag was imaged using a confocal microscope. The experimental results are shown in FIG. Figure 8 shown.

[0046] In addition to the above embodiments, the present invention may also have other implementation modes. Any technical solution formed by equivalent replacement or equivalent transformation falls within the protection scope required by the present invention.

Claims

1. A fluorescent probe for specific and rapid labeling of double cysteine-tagged proteins, characterized in that: The probe has a structure as shown in Formula I, and is 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 2. A method for preparing a fluorescent probe for specific and rapid labeling of double cysteine-tagged proteins as claimed in claim 1, characterized in that: The following steps are involved: 3,7-dihydroxy-5,5-dimethyl-3'-oxo-3'H, 5H spiro[dibenzo[b, e]silyl-10,1'-isobenzofuran]-4,6-dicarbaldehyde is reacted in a nitroethylene mixed solution to generate 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 specific and rapid labeling of double cysteine ​​tagged proteins.

3. The method for preparing a fluorescent probe for specific and rapid labeling of double cysteine-tagged proteins according to claim 2, characterized in that: The reaction conditions are as follows: stirring the reaction at 70-80° C. for more than 72 hours; after the reaction is completed, purification is performed and vacuum drying is performed to obtain a fluorescent probe for specific and rapid labeling of double cysteine-tagged proteins.

4. The method for preparing a fluorescent probe for specific and rapid labeling of double cysteine-tagged proteins according to claim 3, characterized in that: After the reaction is completed, purification is performed, and the specific steps of purification 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.

5. Use of the fluorescent probe for specific and rapid labeling of double cysteine-tagged proteins as claimed in claim 1 for specific and rapid labeling of double cysteine-tagged proteins.

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