Simple preparation method of ubiquitin or ubiquitin-like-AMC probe

Abstract

The invention belongs to the technical field of protein probe synthesis, and relates to a simple preparation method of a ubiquitin or ubiquitin-like-AMC probe, ubiquitin hydrazide or ubiquitin-like hydrazide is used as a raw material, and the ubiquitin or ubiquitin-like-AMC probe is synthesized by directly carrying out ammonolysis on the ubiquitin or ubiquitin-like-AMC molecule after the protein hydrazide is oxidized; the ubiquitin hydrazide is Ub (1-74)-NHNH2; the ubiquitin-like hydrazide is NEDD8 (1-74)-NHNH2 or ISG15-C78S-(2-155)-NHNH2, and the structural formula of the ubiquitin-like hydrazide is shown in the description. The preparation method is simple, high in reaction conversion efficiency, simple in synthesis steps, low in synthesis cost and suitable for large-scale production and application.

Description

Technical Field:

[0001] The present invention relates to a simple preparation method of ubiquitin or ubiquitin-like-AMC probe, belonging to the technical field of protein probe tool synthesis. Background Art:

[0002] Ubiquitin protein (Ub) is a small protein with 76 amino acids existing in all eukaryotes. Its main function is to label proteins for degradation by the 26S proteasome. The ubiquitination process plays a crucial role in cells. By participating in the clearance of misfolded or damaged proteins, it regulates life processes such as the cell cycle, DNA damage repair, immune and inflammatory regulation, signal transduction and regulation. Ubiquitin-like (Ubl) has a similar tertiary structure to ubiquitin and can also be modified onto substrate proteins to regulate various cellular processes. For example, ISG15 plays an indispensable role in diseases such as antiviral, immune regulation, and cancer, and NEDD8 is widely involved in processes such as neural development, embryogenesis, and immune regulation.

[0003] Ub-AMC, as a ubiquitin protein probe, has currently been widely used in deubiquitinating enzyme activity assays and high-throughput screening of deubiquitinating enzyme inhibitors. Deubiquitinating enzymes recognize and hydrolyze Ub-AMC, simultaneously releasing the fluorescent reporter group 7-amino-4-methylcoumarin (AMC). By monitoring the change in fluorescence intensity of the system, the enzymatic process can be reflected. Similarly, Ubl-AMC probes can also be used for deubiquitin-like enzyme activity assays and high-throughput screening of deubiquitin-like enzyme inhibitors. Currently, the synthesis methods of ubiquitin protein probe Ub-AMC and ubiquitin-like protein probe Ubl-AMC mainly include the following several:

[0004] The article (Angew Chem Int Ed. 2022, 61, e202206205) makes mature ubiquitin or ubiquitin-like protein or its variant contact with a glycylated tool molecule in the presence of protease Lb pro to obtain a biochemical tool with deubiquitinating or deubiquitin-like enzyme activity. This strategy needs to act in an active protein environment. When the tool molecule is a conventional amino molecule, the yield can reach 95%. When the tool molecule is Gly-Gly-AMC, due to the limited solubility of the small molecule Gly-Gly-AMC in aqueous solution, the yield decreases. The yield of the ubiquitin and ubiquitin-like AMC probes shown in its related patent (CN 114181993 A) is about 40%.

[0005] The article (RSC Adv. 2016, 6, 47926 - 47930) proposed an effective two - step ligation strategy for the chemical synthesis of Ub - AMC. By relying on the one - pot ligation - desulfurization of double - stranded hydrazide - based NCL coupling, and simultaneously using norleucine (Nle) instead of methionine helps to improve the purity of the synthesized peptide segment without generating oxidation by - products. However, this method requires the synthesis of ubiquitin fragments through polypeptide synthesis, with cumbersome steps, a low final overall yield, and is not applicable to the synthesis of AMC probes for other ubiquitin - like proteins.

[0006] The article (J. Pept. Sci. 2014, 20, 102 - 107) achieved the ligation of Ub(1 - 75) - NHNH2 with glycine - appended - AMC through a protein - hydrazide - based native chemical ligation method, and then obtained the ubiquitin probe Ub - AMC by removing the appended group. This method is not only cumbersome to operate, with a high synthesis cost, but also can only achieve the synthesis of the ubiquitin probe Ub - AMC. Summary of the Invention:

[0007] The object of the present invention is to propose a simple and efficient method for obtaining ubiquitin or ubiquitin - like - AMC probes. Using ubiquitin hydrazide or ubiquitin - like hydrazide as raw materials, based on the direct ammonolysis of the oxidation of protein hydrazide by Gly - Gly - AMC molecules to synthesize ubiquitin or ubiquitin - like - AMC probes.

[0008] In order to achieve the above object, the present invention proposes a simple preparation method for ubiquitin or ubiquitin - like - AMC probes, and the specific steps are as follows:

[0009] Dissolve 1 equivalent of ubiquitin hydrazide or ubiquitin - like hydrazide in PBS buffer, and add an aqueous sodium nitrite solution until the sodium nitrite content in the system is 10 equivalents. React at - 20 °C for 30 minutes; after the reaction is completed, add an equal - volume DMSO solution containing 200 equivalents of Gly - Gly - AMC molecules, and adjust the pH to 7.0, and react at room temperature for 30 minutes; after the reaction is completed, use semi - preparative high - performance liquid chromatography to purify the above reaction solution, collect the purified solution, and lyophilize to obtain the target product ubiquitin or ubiquitin - like - AMC probe.

[0010] The ubiquitin hydrazide described in the present invention is Ub(1 - 74) - NHNH2; the ubiquitin - like hydrazide is Nedd8(1 - 74) - NHNH2 or ISG15 - C\(_7\)8S-(2 - 155) - NHNH2.

[0011] The PBS buffer described in the present invention contains 6M guanidine hydrochloride and 0.1M disodium hydrogen phosphate, with pH = 1.0 - 5.0.

[0012] Compared with the prior art, the features and advantages of the present invention are:

[0013] The present invention uses ubiquitin hydrazide or ubiquitin-like hydrazide as raw materials, and directly synthesizes ubiquitin or ubiquitin-like-AMC probes by ammonolysis of protein hydrazide oxidation with Gly-Gly-AMC molecules. The preparation method is simple, the reaction conversion efficiency is high, the synthesis steps are simple, the synthesis cost is low, and it is suitable for large-scale production applications. Description of the Drawings:

[0014] Figure 1 It is the high performance liquid chromatography (HPLC) diagram of ubiquitin hydrazide and the product during the synthesis of the ubiquitin probe Ub-AMC.

[0015] Figure 2 It is the high performance liquid chromatography (HPLC) diagram of the ubiquitin probe Ub-AMC.

[0016] Figure 3 It is the mass spectrometry diagram of the ubiquitin probe Ub-AMC.

[0017] Figure 4 It is the high performance liquid chromatography (HPLC) diagram of the ubiquitin-like probe NEDD8-AMC.

[0018] Figure 5 It is the mass spectrometry diagram of the ubiquitin-like probe NEDD8-AMC.

[0019] Figure 6 It is the high performance liquid chromatography (HPLC) diagram of the ubiquitin-like probe ISG15-C78S-AMC.

[0020] Figure 7 It is the mass spectrometry diagram of the ubiquitin-like probe ISG15-C78S-AMC.

[0021] Figure 8 It is the schematic diagram of the synthesis principle of the ubiquitin or ubiquitin-like-AMC probe. Detailed Embodiments:

[0022] To facilitate the understanding of the present invention, the following further illustrates the implementation process of the present invention through specific examples in combination with the drawings. These descriptions are only to further illustrate the features and advantages of the present invention, rather than limiting the claims of the invention.

[0023] Example 1:

[0024] This example relates to the preparation method of ubiquitin hydrazide or ubiquitin-like hydrazide, and the specific steps include:

[0025] (1) Protein expression: Pick a monoclonal colony of Escherichia coli transformed with ubiquitin Ub(1-76) protein into 10 mL of LB medium containing the corresponding resistance, pick a monoclonal colony of Escherichia coli transformed with NEDD8(1-76) protein into another 10 mL of LB medium containing the corresponding resistance, pick a monoclonal colony of Escherichia coli transformed with ISG15-C78S-His protein into yet another 10 mL of LB medium containing the corresponding resistance, and pick a monoclonal colony of Escherichia coli transformed with Lb pro protein into still another 10 mL of LB medium containing the corresponding resistance; incubate the above 4 kinds of bacterial solutions at 37 °C with shaking at 220 rpm for 14-16 hours, and then amplify each of the 5 incubated bacterial solutions by a volume ratio of 1:100 into 5 1-L fresh LB media containing the corresponding resistance, and continue to incubate at 37 °C with shaking at 220 rpm until the OD 600 absorbance value reaches 1.0, and add IPTG (isopropyl-β-D-thiogalactoside) with a final concentration of 400 μM to each and continue to incubate at 16 °C with shaking at 180 rpm for 16 hours to induce the prokaryotic expression of each protein;

[0026] (2) Protein purification: Centrifuge each bacterial solution after protein expression induction, discard the supernatant, and resuspend the obtained bacterial cells sufficiently with lysis buffer (50 mM HEPEs, 150 mM NaCl, pH = 7.4) (the bacterial cells obtained from each 1-L LB medium are resuspended in 20 mL of lysis buffer), and then use an ultrasonic cell disruptor for lysis. Next, centrifuge the lysed bacterial solution at 14000 rpm at 4-8 °C for 30 minutes using a high-speed refrigerated centrifuge;

[0027] For ISG15-C78S-His and Lb pro, incubate the centrifuged supernatant with a nickel column respectively, then wash with low-concentration imidazole and elute with high-concentration imidazole to obtain purified ISG15-C78S-His protein solution and Lb pro protein solution respectively;

[0028] For Ub(1-76), add perchloric acid to the centrifuged supernatant according to a volume ratio of 200:1 to precipitate impurity proteins. Then centrifuge at 14000 rpm at 4-8 °C at high speed for 30 minutes, collect the supernatant to obtain a purified Ub protein solution;

[0029] For NEDD8(1-76), its expression form is insoluble inclusion bodies. It is necessary to discard the supernatant and dissolve the precipitate with a certain volume of 8 M urea solution at pH 8.0, sonicate for 20 minutes, and then centrifuge at 14000 rpm at 4-8 °C at high speed for 30 minutes to collect the supernatant to obtain a purified NEDD8 protein solution;

[0030] Transfer each of the above-mentioned preliminarily purified protein solutions into a corresponding dialysis bag with a molecular weight cut-off of 3000 Da, and dialyze it in a dialysis buffer (50 mM HEPEs, 150 mM NaCl, pH = 7.4) for 12 hours. Then, replace the fresh dialysis buffer and dialyze again for 12 hours. Then, filter each dialyzed protein solution through a 0.22-μm filter membrane, and perform molecular sieve separation using a Superdex 75 10 / 300 GL size exclusion chromatography column (manufactured by Cytiva) to obtain each target protein solution. Rapidly freeze each target protein solution in liquid nitrogen and store it in a -80°C refrigerator for later use.

[0031] The sequence of the Ub(1-76) protein of the present invention is:

[0032] MQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFAGKQLEDGRTLSDYNIQKESTLHLVLRLRGG (SEQ ID NO.1);

[0033] The sequence of the NEDD8(1-76) protein of the present invention is:

[0034] MLIKVKTLTGKEIEIDIEPTDKVERIKERVEEKEGIPPQQQRLIYSGKQMNDEKTAADYKILGGSVLHLVLALRGGGGLRQ (SEQ ID NO.2);

[0035] The sequence of the ISG15-C78S-His protein of the present invention is:

[0036] MGWDLTVKMLAGNEFQVSLSSSMSVSELKAQITQKIGVHAFQQRLAVHPSGVALQDRVPLASQGLGPGSTVLLVVDKSDEPLSILVRNNKGRSSTYEVRLTQTVAHLKQQVSGLEGVQDDLFWLTFEGKPLEDQLPLGEYGLKPLSTVFMNLRLRGGSSHHHHHH (SEQ ID NO.3);

[0037] The sequence of the Lb pro protein of the present invention is:

[0038] MGSSHHHHHHHHGSSMELTLYNGEKKTFYSRPNNHDNCWLNAILQLFRYVEEPFFDWVYSSPENLTLEAIKQLEDLTGLELHEGGPPALVIWNIKHLLHTGIGTASRPSEVCMVDGTDMCLADFHAGIFLKGQEHAVFACVTSNGWYAIDDEDFYPWTPDPSDVLVFVPYDQEPLNGEWKAK (SEQ ID NO.4);

[0039] (3) Preparation of ubiquitin hydrazide or ubiquitin-like hydrazide

[0040] (a) Preparation of ubiquitin hydrazide Ub(1-74)-NHNH2: Concentrate the purified Ub(1-76) protein to 10 mg / mL. Add 60 μL of hydrazine hydrate (85%) to each milliliter of the protein concentrate. After mixing evenly, adjust the pH of the solution to 8.0, and add the Lb pro protein solution from step (2) to a final concentration of 4 μM. Place the reaction solution in a water bath at 37°C and monitor it by HPLC every 1 h. After the reaction is completed, purify the reaction solution using semi-preparative high-performance liquid chromatography, collect the purified solution, and lyophilize it to obtain ubiquitin hydrazide Ub(1-74)-NHNH2;

[0041] (b) Preparation of ubiquitin-like hydrazide NEDD8(1-74)-NHNH2: Concentrate the purified NEDD8(1-76) protein to 10 mg / mL. Add 60 μL of hydrazine hydrate (85%) to each milliliter of the protein concentrate. After mixing evenly, adjust the pH of the solution to 8.0, and add the Lb pro protein solution from step (2) to a final concentration of 4 μM. Place the reaction solution in a water bath at 37°C and monitor it by HPLC every 1 h. After the reaction is completed, purify the reaction solution using semi-preparative high-performance liquid chromatography, collect the purified solution, and lyophilize it to obtain ubiquitin-like hydrazide NEDD8(1-74)-NHNH2;

[0042] (c) Preparation of ubiquitin-like hydrazide ISG15-C78S-(2-155)-NHNH2: Concentrate the purified ISG15-C78S-(2-155) protein to 10 mg / mL. Add 60 μL of hydrazine hydrate (85%) to each milliliter of the protein concentrate. After mixing evenly, adjust the pH of the solution to 8.0, and add the Lb pro protein solution from step (2) to a final concentration of 4 μM. Place the reaction solution in a water bath at 37°C and monitor it by HPLC every 1 h. After the reaction is completed, purify the reaction solution using semi-preparative high-performance liquid chromatography, collect the purified solution, and lyophilize it to obtain ubiquitin-like hydrazide ISG15-C78S-(2-155)-NHNH2;

[0043] Example 2: Synthesis of Ub-AMC

[0044] Dissolve 2 mg of Ub(1-74)-NHNH2 prepared in Example 1 in 200 μL of PBS buffer (containing 6 M guanidine hydrochloride, 0.1 M disodium hydrogen phosphate, pH = 2.3), and add 30 μL of an aqueous sodium nitrite solution with a concentration of 0.1 M. React at -20 °C for 30 minutes; after the reaction, add 200 μL of a DMSO (dimethyl sulfoxide) solution containing 15.39 mg of Gly-Gly-AMC molecule (Bide Pharmaceuticals, CAS No.: 191723-65-6), adjust the pH to 7.0, and react at room temperature for 30 minutes; after the reaction is completed, use semi-preparative high performance liquid chromatography to purify the above reaction solution, collect the purified solution, and lyophilize to obtain the target product ubiquitin probe Ub-AMC. The yield is 45%.

[0045] Figure 1 It is the high performance liquid chromatography diagram during the reaction. The product in the diagram is the high performance liquid chromatography of the product measured at 10 min of the reaction.

[0046] The purified Ub-AMC probe was characterized by high performance liquid chromatography and mass spectrometry, and the results are shown in Figure 2 and Figure 3 .

[0047] Example 3: Synthesis of NEDD8-AMC

[0048] Dissolve 2 mg of NEDD8(1-74)-NHNH2 prepared in Example 1 in 200 μL of PBS buffer (containing 6 M guanidine hydrochloride, 0.1 M disodium hydrogen phosphate, pH = 2.3), and add 30 μL of 0.1 M aqueous sodium nitrite solution. React at -20 °C for 30 minutes; after the reaction, add 200 μL of a DMSO solution containing 15.39 mg of Gly-Gly-AMC molecule, adjust the pH to 7.0, and react at room temperature for 30 minutes; after the reaction is completed, use semi-preparative high performance liquid chromatography to purify the above reaction solution, collect the purified solution, and lyophilize to obtain the target product NEDD8-AMC, a ubiquitin-like probe. The yield is 40%.

[0049] The NEDD8-AMC probe was characterized by high performance liquid chromatography and mass spectrometry, and the results are shown in Figure 4 and Figure 5 .

[0050] Example 4: Synthesis of ISG15-C78S-AMC

[0051] Dissolve 2 mg of ISG15-C78S-(2-155)-NHNH2 prepared in Example 1 in 200 μL of PBS buffer (6 M guanidine hydrochloride, 0.1 M disodium hydrogen phosphate, pH = 2.3), add 30 μL of 0.1 M sodium nitrite aqueous solution, and react at -20 °C for 30 minutes; after the reaction is completed, add 200 μL of DMSO solution containing 15.39 mg of Gly-Gly-AMC molecules, adjust the pH to 7.0, and react at room temperature for 30 minutes; after the reaction is completed, use semi-preparative high performance liquid chromatography to purify the above reaction solution, collect the purified solution, and lyophilize to obtain the target product ubiquitin-like probe ISG15-C78S-AMC. The yield is 46%.

[0052] The ISG15-C78S-AMC probe was characterized by high performance liquid chromatography and mass spectrometry, and the results are shown in Figure 6 and Figure 7 .

Claims

1. A method for preparing a ubiquitin or ubiquitin-like-AMC probe, characterized in that, The specific steps are as follows: Dissolve ubiquitin hydrazide or ubiquitin-like hydrazide in PBS buffer, add an aqueous solution of sodium nitrite, and react at -20 °C for 30 minutes; after the reaction is completed, add a DMSO solution containing Gly-Gly-AMC molecules, adjust the pH to 7.0, and react at room temperature for 30 minutes; after the reaction is completed, use semi-preparative high-performance liquid chromatography to purify the above reaction solution, collect the purified solution, and lyophilize to obtain the target product ubiquitin or ubiquitin-like-AMC probe.

2. The preparation method of the ubiquitin or ubiquitin-like-AMC probe according to claim 1, characterized in that The ubiquitin hydrazide is Ub(1-74)-NHNH2; the ubiquitin-like hydrazide is NEDD8(1-74)-NHNH2 or ISG15-C78S-(2-155)-NHNH2.

3. The preparation method of the ubiquitin or ubiquitin-like-AMC probe according to claim 1, characterized in that, The PBS buffer contains 6 M guanidine hydrochloride and 0.1 M disodium hydrogen phosphate, pH = 1.0 - 5.0.

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