Kit capable of detecting activity of various E3 ubiquitin ligases

Abstract

The invention discloses a kit capable of detecting activity of various E3 ubiquitin ligases, and discloses a rapid detection kit for protein in-vitro ubiquitination modification, which comprises bombyx mori E1 and E2 of a cloning and prokaryotic expression ubiquitination modification system, ubiquitin molecules, a reaction buffer solution formula and a protein system formula. An in-vitro ubiquitination detection system is constructed, the problems that traditional detection is low in sensitivity, high in cost and insufficient in flexibility are solved, and technical support is provided for efficient and broad-spectrum E3 activity identification.

Description

Technical Field

[0001] The present invention relates to the field of enzyme activity detection, and particularly to a kit capable of detecting the activities of multiple E3 ubiquitin ligases. Background Art

[0002] The ubiquitin-proteasome system is the core mechanism for eukaryotes to regulate protein homeostasis, and precisely controls the localization, activity, and degradation process of target proteins through ubiquitination modification. The ubiquitination modification is completed by a three-step enzymatic cascade reaction of ubiquitin-activating enzyme (E1), ubiquitin-conjugating enzyme (E2), and ubiquitin ligase (E3). Among them, as a key component, the E3 ligase directly determines the specificity of substrate recognition, and catalyzes the transfer of ubiquitin from E2 to the target protein, thereby regulating various physiological processes such as cell proliferation, apoptosis, and DNA repair. Since the abnormal function of E3 is closely related to cancers, neurodegenerative diseases, etc., the technology for detecting its activity has become the core requirement in ubiquitination research and drug development.

[0003] Currently, in vitro ubiquitination activity detection mainly relies on ubiquitination systems derived from mammals or yeasts (such as human E1, E2, and ubiquitin molecules). However, such systems have the following limitations: (1) Heterologous enzyme systems may lead to interspecies compatibility differences, affecting ubiquitination efficiency; (2) The cost of mammalian protein expression is high and the purification is difficult, making it difficult to be applied on a large scale; (3) Existing kits are mostly designed for specific E3 subtypes, lacking the flexibility to adapt to different E2 / E3 combinations. For example, Abcam's E3 autoubiquitination detection kit (ab139469) only contains the E2 enzyme UbcH5a, which is applicable to E3 enzymes such as HDM2, but not to other E3 enzymes (such as RNF43, members of the TRIM family). The combination of these E3 enzymes and UbcH5a will result in a sharp drop in ubiquitination efficiency or no ubiquitination reaction. In addition, traditional detection methods have insufficient sensitivity and are difficult to capture the weak activity of low-abundance E3. For example, the amount of E3 enzyme required in the ubiquitination system is usually greater than 4 μg / mL. These factors have restricted the research and application of new E3 functions.

[0004] The silkworm (Bombyx mori), as a classical model organism, has the advantages of a short life cycle, a clear genetic background, and a mature protein expression system, and is an excellent carrier for developing efficient ubiquitination research tools. Research shows that complete ubiquitination molecular components already exist in the silkworm, including ubiquitin (BmUbiquitin), E1 enzyme (BmE1), E2 enzyme (BmE2), and E3 enzyme (BmE3). Since the E3 activity strictly depends on the synergistic effect of E2, establishing an in vitro ubiquitination detection system based on silkworm homologous components can not only break through the compatibility bottleneck of heterologous systems, but also achieve broad-spectrum and efficient activity analysis by flexibly matching different BmE2s with the E3 to be tested. Summary of the Invention

[0005] In view of this, one of the objectives of the present invention is to provide a kit for detecting the activities of multiple E3 ubiquitin ligases. The present invention constructs a standardized and low-cost core enzyme component library by cloning and prokaryotic expression of BmE1, BmUbiquitin and multiple BmE2s derived from Bombyx mori, and combines an optimized reaction system with a highly sensitive detection method to achieve rapid and quantitative analysis of E3 activity. Another objective of the present invention is to provide the application of the kit for detecting the activities of multiple E3 ubiquitin ligases in detecting the activities of E3 ubiquitin ligases.

[0006] To achieve the above objectives, the present invention provides the following technical solutions:

[0007] 1. A kit for detecting the activities of multiple E3 ubiquitin ligases, the kit comprising recombinant BmE1, recombinant BmUbiquitin-GST or recombinant BmE2; the amino acid sequence of the recombinant BmE1 is as shown in SEQ ID NO.25, the amino acid sequence of the recombinant BmUbiquitin-GST is as shown in SEQ ID NO.36, and the recombinant BmE2s are as shown in SEQ ID NOs.26 to 35.

[0008] Preferably, the kit of the present invention further comprises a reaction solution, and the concentrations of the components of the reaction solution are as follows: 40 mM pH 7.5 Tris-HCl, 5 mM MgCl2, 2 mM ATP, 2 mM DTT (dithiothreitol).

[0009] Preferably, the concentration of the recombinant BmE1 is 2.5 μg / mL, the concentration of the BmUbiquitin-GST is 300 μg

[0010] / mL, and the concentration of the recombinant BmE2 is 0.2 mg / mL.

[0011] 2. The application of the kit for detecting the activities of multiple E3 ubiquitin ligases in detecting the activities of E3 ubiquitin ligases.

[0012] The beneficial effects of the present invention are as follows: A rapid detection kit for in vitro protein ubiquitination modification, including E1, E2 and ubiquitin molecules of the ubiquitination modification system cloned and prokaryotically expressed from Bombyx mori, as well as the reaction buffer formula and the protein system formula. An in vitro ubiquitination detection system is constructed, which solves the problems of low sensitivity, high cost and insufficient flexibility in traditional detection, and provides technical support for the efficient and broad-spectrum identification of E3 activity. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] To make the objectives, technical solutions and beneficial effects of the present invention clearer, the present invention provides the following drawings for illustration:

[0014] Figure 1The protein band of BmE1 after prokaryotic expression and purification was stained with Coomassie Brilliant Blue using SDS-PAGE. The protein band marked with an asterisk is BmE1.

[0015] Figure 2 The protein band of BmUbiquitin-GST after prokaryotic expression and purification was stained with Coomassie Brilliant Blue using SDS-PAGE. The protein band marked with an asterisk is BmUbiquitin-GST.

[0016] Figure 3 Shows the results of in vitro ubiquitination modification of proteins detected by anti-MBP antibody (taking the Bombyx mori BmNPV virus CG30 protein as an example). The BmNPV virus protein CG30 to be tested cooperates with BmE2W, BmE2T, UbcE2F, BmE2J1, BmE2C, and BmE2130 to catalyze ubiquitination, proving that the protein to be tested is an E3 ubiquitin ligase.

[0017] Figure 4 Is the detection sensitivity experiment. Specific Embodiments

[0018] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments, so that those skilled in the art can better understand the present invention and be able to implement it, but the specific embodiments cited do not limit the present invention.

[0019] Example 1: Cloning of Bombyx mori BmE1, 10 BmE2, and BmUbiquitin Genes

[0020] Extract the genomic DNA of Bombyx mori. According to the existing Bombyx mori whole-genome microarray data and the Bombyx mori genome database (http: / / silkworm.swu.edu.cn / silkdb / ), design the upstream and downstream primer pairs for Bombyx mori BmE1, BmUbiquitin genes, and 10 BmE2:

[0021] Table 1. Primers for PCR

[0022]

[0023] Then, using the Bombyx mori genome as a template, perform PCR amplification using PrimeSTAR Max DNA Polymeraseprimerstar (Takara). Store the amplified product at 4°C. After the amplified product is recovered, sequence it to obtain the Bombyx mori BmE1, 10 BmE2, and BmUbiquitin genes.

[0024] Example 2. Construction of Prokaryotic Expression Vectors for Bombyx mori BmE1, 10 Kinds of BmE2, and BmUbiquitin

[0025] After the prokaryotic expression vector pET-30a was digested with KpnI and EcoR I, the extended gene fragments BmE1 (SEQ ID NO.25) and 10 kinds of BmE2 (BmE2W (SEQ ID NO.26), BmE2T (SEQ ID NO.27), UbcE2F (SEQ ID NO.28), BmE2J1 (SEQ ID NO.29), BmE2J2 (SEQ ID NO.30), UbcE2M (SEQ ID NO.31), BmE2G2 (SEQ ID NO.32), BmE2C (SEQ ID NO.33), BmE2130 (SEQ ID NO.34), BmE2X1 (SEQ ID NO.35)) were ligated into the vector. After correct sequencing verification, the corresponding plasmids were stored; after the prokaryotic expression vector pGEX-4T-2 was digested with BamH I and Xho I, the extended gene fragment BmUbiquitin (SEQ ID NO.36) was ligated into the vector. After correct sequencing verification, the corresponding plasmids were stored.

[0026] Example 3. Expression and Purification of Recombinant Proteins BmE1 and BmUbiquitin-GST

[0027] a. High-Level Expression of Recombinant Proteins

[0028] The correctly sequenced expression strains were respectively inoculated into 25 mL of 2×YT liquid medium and cultured overnight at 37°C with 220 rpm; the bacterial solution was transferred to 1 L of 2×YT liquid medium and cultured at 37°C with 220 rpm until the OD600 reached 0.6 - 0.8; the temperature was lowered to 16°C, IPTG (working concentration 5 μg / mL) was added, and the culture was induced at 16°C with 220 rpm for 20 h; centrifuged at 12000×g for 15 min, the supernatant was discarded, and the bacterial cells were collected; 50 mL of binding buffer (20 mM Tris-HCI pH 7.5, 200 mM NaCl) was added to resuspend the bacterial cells, and they were repeatedly frozen and thawed 3 times with liquid nitrogen and sonicated on an ice-water mixture for 30 min. The program was set as follows: power 50%, break for 1 s, stop for 3 s; centrifuged at 15000×g for 25 min, the supernatant was collected and filtered through a 0.45 μM filter membrane.

[0029] b. Nickel Column Affinity Chromatography of Recombinant Proteins

[0030] Discard 20% ethanol in the packing material, and rinse it 3 times with 50 mL of ddH2O; discard the ddH2O in the packing material, add 45 mL of binding buffer to rinse and equilibrate the nickel column. Mix the packing material with the supernatant protein, incubate with rotation at 12 rpm for 4 h, filter out the packing material in the affinity chromatography column, and discard the effluent; Elution of the target protein: Set up eluents with different imidazole concentrations for gradient elution, and collect the eluents: Detect by SDS-PAGE gel electrophoresis, prepare samples from the collected component solutions respectively, and conduct electrophoresis detection.

[0031] The detection results of BmE1 are as Figure 1 shown.

[0032] c. Dialysis of the recombinant protein

[0033] Use a 7Kda dialysis bag to dialyze the obtained recombinant protein. Replace the recombinant protein solution with PBS at a volume ratio of protein to PBS of 1:50. After aliquoting into small tubes, store at -80 °C.

[0034] d. Use the BCA method to determine the protein concentrations of BmE1 and BmUbiquitin-GST and adjust the sample loading amounts.

[0035] The detection results of BmE1 are as Figure 1 shown. The results show that high-purity BmE1 can be obtained after affinity chromatography and dialysis.

[0036] The expression results of BmUbiquitin are as Figure 2 shown. The results show that high-purity BmUbiquitin can be obtained after affinity chromatography and dialysis.

[0037] Example 4: Small-scale expression of the recombinant protein BmE2

[0038] The ten pET30a-BmE2 bacterial solutions and the empty vector obtained in Example 2 were respectively inoculated into 20 mL of 2×YT liquid medium, cultured at 37°C and 220 rpm until the OD600 reached 0.6 - 0.8; 6 mL was taken and cooled to 16°C on an ice-water mixture, IPTG (working concentration 5 μg / mL) was added, and induced culture was carried out at 16°C and 220 rpm for 20 h; then 6 mL was taken, IPTG was added, and induced culture was carried out at 37°C and 220 rpm for 4 h; centrifuged at 12000×g for 15 min, the supernatant was discarded, and the cells were collected; 1 mL of pH 8.0 Binding Buffer was added to resuspend the cells, and ultrasonic disruption was carried out on an ice-water mixture for 10 min, program settings: power 21%, disruption for 1 s, stop for 3 s; centrifuged at 4°C and 15000×g for 15 min, the supernatant was transferred to a new 1.5 mL centrifuge tube, and the precipitate was resuspended with 1 mL of pH 8.0 Binding Buffer; 16 μL of protein solution was taken from both the supernatant and the precipitate, 4 μL of 5×SDS-PAGE loading buffer was added, and after blowing and sucking to mix evenly, it was boiled at 100°C for 10 min.

[0039] For SDS-PAGE detection, a polyacrylamide gel with a lower gel concentration of 12% was used. The voltage of the upper gel was 90 V, and the voltage of the lower gel was 120 V. Electrophoresis was terminated after the protein Maker was clearly separated; after the gel running was completed, the gel was placed in the coomassie staining solution at 55 rpm for 15 min, the coomassie staining solution was poured out, and decolorizing solution was added for decolorization. The decolorizing solution was changed every once in a while until the background was clean.

[0040] Example 5. Construction and detection of the in vitro E3 ubiquitin ligase activity system

[0041] a. Reconstruction of ubiquitination activity

[0042] Purify His-tagged recombinant silkworm E1, BmUbiquitin-GST from Escherichia coli, and Escherichia coli lysates of silkworm E2 (BmE2W, BmE2T, UbcE2F, BmE2J1, BmE2J2, UbcE2M, BmE2G2, BmE2C, BmE2130, BmE2X1) prepared as described above. The reaction mixture contained 40 mM Tris-HCl (pH 7.5), 5 mM MgCl2, 2 mM ATP, 2 mM DTT (dithiothreitol), 300 μg / ml BmUbiquitin-GST, 2.5 μg BmE1 / ml, 0.2 mg Escherichia coli lysate expressing E2 / mL, and 2.5 mg MBP-RING / ml to be tested. The reaction mixture was incubated at 30°C for 3 h, and immunoblotting was performed with anti-MBP antiserum (Beyotime).

[0043] b. Polyacrylamide gel electrophoresis

[0044] Use a 12% polyacrylamide gel as the lower layer gel, load 50 μg of total protein into each well, apply a voltage of 90 V to the upper layer gel and 120 V to the lower layer gel, and stop electrophoresis after the protein markers are clearly separated.

[0045] c. Membrane transfer

[0046] Immerse the PVDF membrane in methanol for 30 s for activation, wash off the excess methanol with ddH2O, then place the membrane in the membrane transfer equilibration solution and let it stand for 1 min. Add an appropriate amount of water to the tray to soak the gel; sequentially place a sponge pad, the PVDF membrane, and the gel on the positive electrode side of the membrane transfer cassette. After removing the bubbles with a roller, cover another sponge pad on the gel, compact it, and then put it back into the membrane transfer instrument and set the program to start membrane transfer.

[0047] d. Western Blotting

[0048] Block the PVDF membrane with 5% skim milk powder at room temperature for 1 h; block it overnight at 4 °C with the corresponding antibodies (anti-MBP 1:5000, anti-His 1:5000); add TBST and wash the primary antibody on a shaker at 55 rpm, 10 min each time for a total of three times; incubate with the secondary antibody corresponding to the primary antibody, HRP-labeled goat anti-mouse or goat anti-rabbit IgG at room temperature for 1 h; add TBST and wash the primary antibody on a shaker at 55 rpm, 10 min each time for a total of three times; prepare the exposure solution according to A solution:B solution = 1:1 and perform exposure.

[0049] The results are as Figure 3 shown. React the MBP-CG30 protein with the reaction systems of 10 different E2 enzymes respectively. The results show that obvious high-molecular-weight ubiquitination bands (marked with red brackets) appeared in the reaction systems of the tested MBP-CG30 combined with BmE2W, BmE2T, UBE2F, BmE2J1, BmE2C, and BmE2130 after three hours of reaction. However, no ubiquitination signal was detected in the reaction systems of MBP-CG30 combined with other E2 enzymes, which proves that CG30 indeed has the function of an E3 ubiquitin ligase and can match with specific E2 enzymes to occur ubiquitination reactions.

[0050] Detection sensitivity experiment: Set different concentration gradients of the E3 enzyme (MBP-CG30) to detect the sensitivity range of this system. The results are as Figure 4 shown. Set the final concentration of E3 to 0.1 μg / mL, 0.5 μg / mL, 1 μg / mL, 5 μg / mL, 10 μg / mL, 50 μg / mL, and 100 μg / mL. React with BmE2W for three hours, and the range of the final concentration of E3 corresponding to the samples with ubiquitination bands is between 0.5 μg / mL and 100 μg / mL.

[0051] The above-described embodiments are only preferred embodiments given to fully illustrate the present invention, and the protection scope of the present invention is not limited thereto. Equivalent substitutions or transformations made by those skilled in the art on the basis of the present invention are all within the protection scope of the present invention. The protection scope of the present invention shall be subject to the claims.

Claims

1. A kit for detecting the activity of multiple E3 ubiquitin ligases, characterized in that: The kit includes recombinant BmE1, recombinant BmUbiquitin-GST or recombinant BmE2; the amino acid sequence of the recombinant BmE1 is shown as SEQ ID NO.25, the amino acid sequence of the recombinant BmUbiquitin-GST is shown as SEQ ID NO.36, and the recombinant BmE2 is shown as SEQ ID NO.26 to SEQ ID NO.

35.

2. The kit for detecting the activities of multiple E3 ubiquitin ligases according to claim 1, characterized in that: The kit also includes a reaction solution, and the concentrations of the components of the reaction solution are as follows: 40mM pH 7.5 Tris-HCl, 5mM MgCl2, 2mM ATP, and 2mM DTT dithiothreitol.

3. The kit for detecting the activities of multiple E3 ubiquitin ligases according to claim 1, characterized in that: The concentration of recombinant BmE1 was 2.5 μg / mL, the concentration of BmUbiquitin-GST was 300 μg / mL, and the concentration of recombinant BmE2 was 0.2 mg / mL.

4. Use of the kit for detecting multiple E3 ubiquitin ligase activities according to any one of claims 1 to 3 in detecting E3 ubiquitin ligase activity.

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