Ubiquitination detection system and ubiquitination detection method

Abstract

The invention discloses a ubiquitination detection system and a ubiquitination detection method. The ubiquitination detection system comprises a prokaryotic expression vector, and the prokaryotic expression vector comprises a ubiquitin expression cassette, a ubiquitin activating enzyme expression cassette, a ubiquitin binding enzyme expression cassette, a ubiquitin ligase expression cassette and a substrate protein expression cassette. The ubiquitin expression cassette, the ubiquitin binding enzyme expression cassette, the ubiquitin ligase expression cassette and the substrate protein expression cassette respectively express different tag proteins in a fusion manner. According to the technical scheme provided by the invention, the human-derived ubiquitination related gene is introduced into the prokaryotic expression vector in a form of replaceable expression elements, and then corresponding protein expression is induced, so that the whole ubiquitination process is completed in a prokaryote (such as escherichia coli) body, and the ubiquitination reconstruction process is realized.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a ubiquitination detection system and a ubiquitination detection method. Background Art

[0002] Proteins play a vital role as the main executors of cell life activities. They are not only the basis of cell structure, but also catalysts of intracellular chemical reactions and key molecules for energy storage and transport. Proteins play an irreplaceable role in life activities such as cell signaling, material transport, metabolism, and immune defense. There are nearly 20,700 genes encoding proteins in humans, but the actual human proteome includes hundreds of thousands or even millions of proteins with different functions. Post-translational modification of proteins represents an important means of naturally increasing the complexity of the proteome.

[0003] Ubiquitination, as an important post-translational modification of proteins in eukaryotes, is involved in most processes of cellular life activities. Due to the different ways of connecting ubiquitin molecules, ubiquitin can complete different ubiquitination modifications on substrates in a way similar to code programming. Different ubiquitination modifications and types of the same substrate determine the different fates of the substrate, so ubiquitination modification gives it complexity of function.

[0004] Protein ubiquitination modification is an enzymatic reaction process mediated by three enzymes: ubiquitin activating enzyme (E1), ubiquitin conjugating enzyme (E2), and ubiquitin ligase (E3). Under the condition of ATP hydrolysis, the ubiquitin molecule is activated by E1 and covalently linked to the cysteine ​​at the active site of the E1 molecule through its carbon-terminal glycine. The activated ubiquitin is transferred to a cysteine ​​residue of the ubiquitin conjugating enzyme E2 through a thioester bond. At this time, the ubiquitin ligase E3 recognizes and connects to the substrate protein to be degraded, and then binds to E2. Under the action of the ubiquitin ligase E3, ubiquitin is transferred from E2 to the lysine residue of the substrate protein. E3 can bind to both substrate molecules and E2, and plays a "bridge" role in the process of ubiquitin transfer to the substrate. Therefore, E3 ubiquitin ligase plays a vital role in the ubiquitination process and is of great significance to life activities such as maintaining cell homeostasis, regulating the cell cycle, and participating in immune responses.

[0005] The types of ubiquitin modification can be divided into polyubiquitination modification (ubiquitin modifies substrates in the form of ubiquitin chains), single-site monoubiquitination (ubiquitin modifies a single position of a substrate in the form of a single ubiquitin molecule) and multi-site monoubiquitination (ubiquitin modifies multiple positions of a substrate in the form of a single ubiquitin molecule). Among them, polyubiquitination modification is divided into homogeneous polyubiquitin chains and branched polyubiquitin chains according to the shape of the ubiquitin chain, and can be divided into seven types of ubiquitin chains according to the different connection modes between ubiquitins: K6, K11, K27, K29, K33, K48, K63 (the latter ubiquitin is connected to the lysine residue at a specific position on the previous ubiquitin through the carbon-terminal glycine residue) and M1 ubiquitin chain (the latter ubiquitin is connected to the first methionine of the previous ubiquitin through the carbon-terminal glycine residue). Different types of ubiquitin chains determine the different fates of substrates. For example, K48 ubiquitin chains are necessary for substrate degradation by proteasomes, K11 ubiquitin chains regulate the cell cycle, and K27 ubiquitin chains regulate mitochondrial autophagy.

[0006] The human proteome contains 2 E1s, 43 E2s, and more than 1,000 E3s. In the process of substrate ubiquitination modification, E3 plays two main roles: on the one hand, it binds to the substrate, and on the other hand, it binds to a specific E2, thereby facilitating the transfer of ubiquitin from E2 to the substrate. However, how E3 recognizes and pairs with a specific E2 to ensure critical, direct, and precise regulation of specific substrates and life activities remains a hot topic in current research.

[0007] There are two main methods for detecting ubiquitination: one is an in vitro ubiquitination reconstruction experiment, which is based on the in vitro protein purification technology, incubating the purified ubiquitin protein, E1, E2, E3 and substrate protein in vitro, and detecting the ubiquitination level of the substrate protein by immunoblotting. The other is to detect the ubiquitination level of proteins in mammalian cells, which is based on the principle of co-transfecting ubiquitin plasmids, E3 plasmids and substrate proteins with specific expression tags into mammalian cells, enriching the substrate proteins by immunoprecipitation, and then analyzing the ubiquitination of the substrate proteins by immunoblotting. However, both methods have obvious disadvantages, which are mainly manifested in:

[0008] Traditional in vitro ubiquitination reconstruction: 1) Traditional in vitro ubiquitination experiments require protein purification, which is extremely complex and cumbersome, costly and time-consuming. At the same time, some proteins cannot be obtained through in vitro purification. 2) It is extremely dependent on protein activity. If the purified protein has low activity or is inactive, the subsequent immunoblotting experiment will not be able to detect the ubiquitination of the substrate protein, which will produce a huge error. 3) It is extremely cumbersome to determine the type of substrate ubiquitin chain and the specific pairing of E2-E3. Traditional in vitro ubiquitination reconstruction experiments often use universal E2 (which can recognize and bind to all E3 ubiquitin ligases) and wild-type ubiquitin proteins. Only by adding different types of E2 or mutant ubiquitin molecules to the system can the type of ubiquitin chain or the specific pairing of E2-E3 be determined. The workload increases exponentially with the permutations and combinations.

[0009] Ubiquitination detection based on mammalian cells: 1) There are many influencing factors. Since eukaryotic cells have their own ubiquitination system, the possibility of ubiquitination modification of substrate proteins by the endogenous ubiquitination system cannot be ruled out by exogenously transfecting specific E3 and target plasmids into mammalian cells to express substrate proteins. 2) The system is seriously polluted. This method detects immunoprecipitation components, which contain other miscellaneous proteins in addition to the target protein, and there is a lack of direct evidence that the target protein is ubiquitinated. 3) It is impossible to identify specific E2-E3 pairing. Since mammalian cells have their own ubiquitination system, the method of exogenously transferred E3 and substrates still cannot identify specific E2-E3 pairing.

[0010] That is to say, these two methods are currently expensive and extremely cumbersome. Currently, there is no efficient and simple method to identify E2-E3 pairing in vitro. Summary of the invention

[0011] The present invention aims to provide a ubiquitination detection system and a ubiquitination detection method to solve the technical problems of high ubiquitination cost and complicated operation in the prior art.

[0012] In order to achieve the above-mentioned object, according to one aspect of the present invention, a ubiquitination detection system is provided. The ubiquitination detection system comprises a prokaryotic expression vector, the prokaryotic expression vector comprises a ubiquitin expression frame, a ubiquitin activating enzyme expression frame, a ubiquitin conjugating enzyme expression frame, a ubiquitin ligase expression frame and a substrate protein expression frame, and the ubiquitin expression frame, the ubiquitin conjugating enzyme expression frame, the ubiquitin ligase expression frame and the substrate protein expression frame are fused to express different tag proteins respectively.

[0013] Furthermore, the ubiquitin expression cassette, ubiquitin activating enzyme expression cassette, ubiquitin conjugating enzyme expression cassette, ubiquitin ligase expression cassette and substrate protein expression cassette are constructed in the same prokaryotic expression vector and express proteins independently.

[0014] Furthermore, the ubiquitin expression cassette, ubiquitin activating enzyme expression cassette, ubiquitin ligase expression cassette and substrate protein expression cassette are constructed in a first prokaryotic expression vector, the ubiquitin conjugating enzyme expression cassette is constructed in a second prokaryotic expression vector, and the first prokaryotic expression vector and the second prokaryotic expression vector have compatible replication initiation sites and different resistance genes.

[0015] Furthermore, the ubiquitin expression frame is fused to express His tag protein, the ubiquitin activating enzyme expression frame does not express tag protein, the ubiquitin conjugating enzyme expression frame is fused to express HA tag protein, the ubiquitin ligase expression frame is fused to express V5 tag protein, and the substrate protein expression frame is fused to express FLAG tag protein.

[0016] Further, the ubiquitin expression cassette expresses wild-type ubiquitin or a ubiquitin mutant, and the ubiquitin mutant is ΔGG, K6R, K11R, K27R, K29R, K33R, K48R, K63R, K0, K6 only, K11 only, K27 only, K29 only, K33 only, K48 only or K63 only mutant.

[0017] Furthermore, the ubiquitin ligase expression frame and the substrate protein expression frame are set correspondingly.

[0018] Furthermore, the prokaryotic expression vector is used to express the protein in Escherichia coli.

[0019] According to another aspect of the present invention, a method for detecting ubiquitination is provided, which comprises: using any of the above ubiquitination detection systems to perform ubiquitination detection.

[0020] Further, it includes: transferring the prokaryotic expression vector into Escherichia coli; inducing the ubiquitin expression frame, the ubiquitin activating enzyme expression frame, the ubiquitin conjugating enzyme expression frame, the ubiquitin ligase expression frame and the substrate protein expression frame to express proteins; lysing the Escherichia coli, and detecting the ubiquitination level and protein expression level of the lysed protein.

[0021] Furthermore, the ubiquitination level and protein expression level of the cleaved protein were detected by Western blot technology.

[0022] Furthermore, ubiquitin antibodies were incubated to detect the ubiquitination level of the cleaved protein; and tag protein antibodies were incubated to detect the protein expression levels of ubiquitin-binding enzymes, ubiquitin ligases and substrate proteins, respectively.

[0023] Furthermore, the ubiquitination detection method includes the detection of reconstructing the ubiquitination process, and the detection includes: analyzing the protein expression product by Western blot technology, using ubiquitin antibody and substrate tag protein antibody to respectively detect the ubiquitination level and the substrate protein expression level; if the ubiquitination process can be reconstructed, the ubiquitin antibody can detect the ubiquitination level; if the substrate protein is ubiquitinated, the substrate tag protein antibody can also detect the presence of ubiquitination bands on the substrate protein.

[0024] Furthermore, the ubiquitination detection method includes identifying the type of ubiquitin chain, and identifying the type of ubiquitin chain includes: constructing a prokaryotic expression vector containing different ubiquitin mutant expression frames, ubiquitin ligase expression frames and substrate protein expression frames; inducing protein expression and performing protein ubiquitination analysis using Western blot protein blotting technology; and identifying the type of specific ubiquitin ligase and substrate protein ubiquitin chain based on the strength or presence of ubiquitination of the expressed proteins of different ubiquitin mutants.

[0025] Furthermore, the ubiquitination detection method includes identifying the pairing of ubiquitin conjugating enzyme and ubiquitin ligase based on a ubiquitination detection system including a first prokaryotic expression vector and a second prokaryotic expression vector: if the protein product co-expressed by the first prokaryotic expression vector and the second prokaryotic expression vector is ubiquitinated, it indicates that the ubiquitin conjugating enzyme and the ubiquitin ligase can form a pair; if the protein product co-expressed by the first prokaryotic expression vector and the second prokaryotic expression vector is not ubiquitinated, it indicates that the ubiquitin conjugating enzyme and the ubiquitin ligase are not paired.

[0026] By applying the technical solution of the present invention, human ubiquitination-related genes are introduced into prokaryotic expression vectors in the form of replaceable expression elements, and then the corresponding protein expression is induced, so that the entire ubiquitination process is completed in prokaryotes (e.g., Escherichia coli), thereby realizing the ubiquitination reconstruction process. Compared with the traditional in vitro ubiquitination reconstruction process, this method is simple and convenient to operate, does not require purification of specific proteins, has a short time period, and is low in cost. Moreover, prokaryotes themselves do not have ubiquitination systems, so introducing human ubiquitination systems into Escherichia coli can reduce interference. Compared with detection methods based on mammalian cells, it is not interfered by endogenous ubiquitination factors, which increases the credibility and authenticity of the results. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] The drawings constituting a part of the present application are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the drawings:

[0028] Figure 1The principle flow chart of the present application is shown, wherein RBS (ribosomal binding site) is the ribosome binding site, ORI (replication origin) is the replication start site, and ORF (open reading frames) is the open reading frame.

[0029] Figure 2 Western blot analysis of protein expression and ubiquitination levels after simultaneous expression of ubiquitin (ub), UBA1 (E1) and UBE2D1 (E2) is shown.

[0030] Figure 3 A in the middle is Western blot analysis of the ubiquitination level after simultaneous expression of ubiquitin, E1, E2, TRAIP (E3) and its activity-deficient mutants. The wild-type TRAIP can produce ubiquitination, while its catalytic activity-deficient group does not produce ubiquitination. Figure 3 B is a Western blot analysis of the ubiquitination level and RNF220 autoubiquitination level after simultaneous expression of ubiquitin, E1, E2, RNF220 (E3) and its activity-deficient mutants. This technology successfully showed that the wild-type RNF220 has ubiquitination and autoubiquitination activities, while its catalytic activity-deficient group did not produce ubiquitination and autoubiquitination.

[0031] Figure 4 Western blot analysis shows the ubiquitination level of substrates after simultaneous expression of ubiquitin, E1, E2, MARCH8 (E3) and cGAS (substrate). RBS (ribosomal binding site) is the ribosome binding site, ORI (replication origin) is the replication origin, and ORF (open reading frames) is the open reading frame.

[0032] Figure 5 Shown are the types of ubiquitin mutants constructed based on this technology.

[0033] Figure 6 A in the middle is Western blot analysis of the RNF220 autoubiquitination level after expressing different ubiquitin proteins (WT, WT-ΔGG, K0 and K0-ΔGG). Figure 6 Middle B shows the level of RNF220 autoubiquitination after expressing different ubiquitin KxR mutants. Figure 6 Middle C shows the level of RNF220 autoubiquitination after expressing different ubiquitin Kx only mutants.

[0034] Figure 7 The plasmid information map and screening principle in the dual-plasmid system screening method are shown.

[0035] Figure 8 A shows Western blot analysis of the expression of dual plasmids plasmid 1 (TRAIP or RNF220) and plasmid 2 (UBE2D1). Figure 8 Middle B is Western blot analysis of the ubiquitination level after RNF220 was co-expressed with different E2 plasmids as the fixed E3 plasmid. DETAILED DESCRIPTION

[0036] It should be noted that, in the absence of conflict, the embodiments and features in the embodiments of the present application can be combined with each other. The present invention will be described in detail below with reference to the accompanying drawings and in combination with the embodiments.

[0037] It should be noted that the terms "first", "second", etc. in the specification and claims of the present application are used to distinguish similar objects, and are not necessarily used to describe a specific order or sequence. It should be understood that the terms used in this way can be interchangeable where appropriate, so that the embodiments of the present application described herein can be implemented in an order other than those described herein, for example.

[0038] In view of the technical problems that the ubiquitination detection in the prior art described in the background art is high in cost and extremely complicated, the present application proposes the following technical solutions.

[0039] According to a typical embodiment of the present application, a ubiquitination detection system is provided. The ubiquitination detection system includes a prokaryotic expression vector, which includes a ubiquitin expression frame, a ubiquitin activating enzyme expression frame, a ubiquitin conjugating enzyme expression frame, a ubiquitin ligase expression frame and a substrate protein expression frame, and the ubiquitin expression frame, the ubiquitin conjugating enzyme expression frame, the ubiquitin ligase expression frame and the substrate protein expression frame are fused to express different tag proteins. For example, the ubiquitin expression frame is fused to express His tag protein, the ubiquitin activating enzyme expression frame does not express tag protein, the ubiquitin conjugating enzyme expression frame is fused to express HA tag protein, the ubiquitin ligase expression frame is fused to express V5 tag protein, and the substrate protein expression frame is fused to express FLAG tag protein.

[0040] According to a typical embodiment of the present application, the ubiquitination detection using the above ubiquitination system includes: transferring the prokaryotic expression vector into Escherichia coli; inducing the ubiquitin expression frame, the ubiquitin activating enzyme expression frame, the ubiquitin conjugating enzyme expression frame, the ubiquitin ligase expression frame and the substrate protein expression frame to express proteins; lysing the Escherichia coli, and detecting the ubiquitination level and protein expression level of the lysed protein. Typically, the ubiquitination level and protein expression level of the lysed protein are detected by Western blot protein blotting technology.

[0041] By applying the technical solution of the present invention, human ubiquitination-related genes are introduced into prokaryotic expression vectors in the form of replaceable expression elements, and then the corresponding protein expression is induced, so that the entire ubiquitination process is completed in prokaryotes (e.g., Escherichia coli), thereby realizing the ubiquitination reconstruction process. Compared with the traditional in vitro ubiquitination reconstruction process, this method is simple and convenient to operate, does not require purification of specific proteins, has a short time period, and is low in cost. Moreover, prokaryotes themselves do not have ubiquitination systems, so introducing human ubiquitination systems into Escherichia coli can reduce interference. Compared with detection methods based on mammalian cells, it is not interfered by endogenous ubiquitination factors, which increases the credibility and authenticity of the results.

[0042] In a typical embodiment of the present application, the ubiquitin expression cassette, the ubiquitin activating enzyme expression cassette, the ubiquitin conjugating enzyme expression cassette, the ubiquitin ligase expression cassette and the substrate protein expression cassette are constructed in the same prokaryotic expression vector and are independently expressed. In this way, multiple proteins can be expressed in Escherichia coli with only one plasmid, avoiding the need for multiple transformations of multiple proteins and improving the feasibility of the operation.

[0043] In the present application, the ubiquitination level of the cleaved protein can be detected by incubating with ubiquitin antibodies; the protein expression levels of ubiquitin conjugating enzymes, ubiquitin ligases and substrate proteins can be detected by incubating with tag protein antibodies. Specifically, the protein expression level of ubiquitin conjugating enzymes can be detected by incubating with HA tag protein antibodies; the protein expression level of ubiquitin ligases can be detected by incubating with V5 tag protein antibodies; and the protein expression level of substrate proteins can be detected by incubating with FLAG tag protein antibodies.

[0044] According to a typical embodiment of the present application, the ubiquitin expression frame expresses wild-type ubiquitin or a ubiquitin mutant, and the ubiquitin mutant is ΔGG, K6R, K11R, K27R, K29R, K33R, K48R, K63R, K0, K6 only, K11 only, K27only, K29 only, K33 only, K48 only or K63 only mutant, which can be used to identify the ubiquitin chain type. The wild-type ubiquitin DNA sequence is SEQ ID NO.1: atgcagatcttcgtgaagaccctgactggtaagaccatcactctcgaag tggagccgagtgacaccattgagaatgtcaaggcaaagatccaagacaaggaaggcatccctcctgaccagcagaggttgatctttgctgggaa acagctggaagatggacgcaccctgtctgactacaacatccagaaagagtccaccctgcacctggtcctccgtctcagaggtggt; the wild-type ubiquitin amino acid sequence is SEQ ID NO.2: MQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQ QRLIFAGKQLEDGRTLSDYNIQKESTLHLVLRLRGG. The mutants are named according to the naming rules commonly used in the art, for example, the ubiquitin mutant ΔGG is obtained by deleting the last GG on the basis of SEQ ID NO.2; the ubiquitin mutant K6R is obtained by mutating the 6th K on the basis of SEQ ID NO.2 to R; the ubiquitin mutant K0 is obtained by mutating the 6th, 11th, 27th, 29th, 33rd, 48th and 63rd K on the basis of SEQ ID NO.2 to R; and K6 only is obtained by retaining the 6th K on the basis of SEQ ID NO.2 and mutating the 11th, 27th, 29th, 33rd, 48th and 63rd K to R.

[0045] In one embodiment of the present application, the ubiquitin ligase expression frame and the substrate protein expression frame can be set accordingly by a person skilled in the art according to the requirements, for example, if it is planned to study whether a certain E3 ubiquitin ligase A can ubiquitinate a certain substrate B. Under this system, the expression frame position of E3 on the plasmid backbone can be replaced with the DNA sequence of A, so that it can express protein A; correspondingly, the expression frame position of "substrate" on the plasmid backbone is replaced with the DNA sequence of B, so that it can express protein B. Then, if it is planned to study which type of ubiquitin is ubiquitinated by E3 ubiquitin ligase A on substrate protein B, the position of "ubiquitin" on the plasmid backbone can be replaced with different ubiquitin mutant DNA sequences to detect which ubiquitin mutant will affect the ubiquitination of A to B.

[0046] According to a typical embodiment of the present application, the ubiquitin expression cassette, the ubiquitin activating enzyme expression cassette, the ubiquitin ligase expression cassette and the substrate protein expression cassette are constructed in a first prokaryotic expression vector, the ubiquitin conjugating enzyme expression cassette is constructed in a second prokaryotic expression vector, and the first prokaryotic expression vector and the second prokaryotic expression vector have compatible replication initiation sites and different resistance genes, which can be used to identify E2-E3 pairing.

[0047] In a typical embodiment of the present application, the prokaryotic expression vector includes five expression cassettes, which can express proteins independently at the same time. Four of the expression cassettes carry different tags, the first expression cassette is ubiquitin (fused with 6×His tag protein), the second expression cassette is UBA1 (E1, untagged protein), the third expression cassette is E2 (fused with HA tag protein), the fourth expression cassette is E3 (fused with V5 tag protein), and the fifth expression cassette is substrate protein (fused with 3×FLAG tag protein), where E2, E3 and substrate can be replaced as needed.

[0048] In a typical embodiment of the present application, the technical process is as follows. After E3 and substrate are constructed into the above-mentioned vector as needed, the plasmid is transformed into BL21 (DE3) Escherichia coli competent state, and the bacterial solution is coated on an ampicillin-resistant plate. The next day, 5-10 monoclones are picked and added to 6mL of LB medium containing ampicillin resistance, and cultured at 37°C, 200rpm / min shaking table. When the cell density grows to OD600 between 0.6-0.8, 0.5mM IPTG is added to induce protein expression. The protein expression conditions are 28°C, 200rpm / min, and the expression time is 3h. The expressed bacterial solution is centrifuged at 4000rpm / min for 10min, and the bacterial precipitate is collected. The supernatant is removed, 400ul lysis solution is added to the precipitate and resuspended, and the bacterial cells are lysed in an ice water bath by an ultrasonic disruptor. The ultrasonic conditions are 300W, the ultrasonic is turned on for 2s, and the ultrasonic is turned off for 4s, and the total time is 3min. The bacterial solution after ultrasonic disruption was centrifuged at 4°C, 20000g / min for 15min, and the supernatant was taken. The supernatant protein was quantified using the BCA protein concentration determination kit and the protein concentration was adjusted to 2μg / ul. The cleaved protein was analyzed by Western blot protein blotting technology. The ubiquitination level of the cleaved protein was detected by incubation with ubiquitin antibody, the expression level of E2 protein was detected by incubation with HA tag antibody, the expression level of E3 protein was detected by incubation with V5 tag antibody, and the expression level of substrate protein was detected by incubation with FLAG tag antibody.

[0049] The ubiquitination detection system of the present application can be mainly applied to:

[0050] 1. Reconstruct the ubiquitination process.

[0051] For example: clone E3 and substrate genes onto a plasmid vector that already contains ubiquitin, E1, and E2 genes to obtain a new plasmid, transform the plasmid into BL21 (DE3) Escherichia coli competent cells for protein expression, analyze the protein expression product by Western blot, use ubiquitin antibody and FLAG antibody to detect the ubiquitination level and substrate protein level, respectively. If the ubiquitination process can be reconstructed, the ubiquitination antibody can detect the ubiquitination level. If the substrate protein is ubiquitinated, the FLAG antibody can also detect the presence of ubiquitination bands on the substrate protein.

[0052] 2. Identify the ubiquitin chain type.

[0053] For example, the wild-type ubiquitin on the first expression frame on the plasmid vector is replaced with different ubiquitin mutants, including K6R, K11R, K27R, K29R, K33R, K48R, K63R with single lysine site mutations and K6, K11, K27, K29, K33, K48, K63 with only single lysine site retention. Specific E3 and substrate proteins are constructed using plasmid vectors with different ubiquitin mutants, protein expression is induced, and protein ubiquitination analysis is performed using Western blot technology. The type of ubiquitin chain of specific E3 and substrate proteins can be identified based on the strength of protein ubiquitination expressed by different ubiquitin mutant vectors or the presence or absence of ubiquitination.

[0054] 3. Identification of E2-E3 pairing based on a dual-plasmid system.

[0055] For example, the principle of this method is to clone E2 separately into another inducible prokaryotic expression plasmid vector, which is chloramphenicol resistant and the vector's ORI (origin of replication) is p15A. The remaining genes involved in the ubiquitination process, including ubiquitin, E1, E3 and substrate protein, are maintained on the original plasmid vector, the original plasmid vector resistance is ampicillin resistance, and the vector's ORI is pBR322. In order to ensure that both different plasmids can be expressed in Escherichia coli, the two plasmids must have compatible ORIs and different resistance genes to facilitate subsequent screening. The plasmid containing the E2 gene is co-transformed with the plasmid containing the specific E3 and substrate protein into BL21 (DE3) Escherichia coli, and the bacteria are cultured in LB medium containing chloramphenicol and ampicillin, and protein expression and ubiquitination analysis are performed. If the protein product co-expressed by the two plasmids is ubiquitinated, it indicates that the E2 and E3 can form an E2-E3 pairing. If the co-expressed protein product is not ubiquitinated, it indicates that the E2 and E3 are not paired.

[0056] Method for identifying E2-E3 pairing by dual-plasmid system: When using dual-plasmid system to screen E2, two plasmids can be co-transformed into E. coli at the same time, or the plasmid containing E3 can be transformed into E. coli to prepare specific competent cells, and then different E2 vectors can be transformed into specific competent cells respectively.

[0057] The beneficial effects of the present invention will be further described below in conjunction with embodiments.

[0058] Example 1

[0059] Reconstructing the ubiquitination process

[0060] Based on the principle of reconstructing ubiquitination in this application, e.g. Figure 1 As shown, a main prokaryotic expression plasmid skeleton used in this embodiment contains five "expression frames" (ORF1, ORF2, ORF3, ORF4, ORF5), that is, up to five different proteins can be expressed simultaneously. The first expression frame (ORF1) is used to express "ubiquitin" and has a 6×His tag; the second expression frame (ORF2) is used to express the E1 gene without a tag; the third expression frame (ORF3) is used to express the E2 gene with an HA tag; the fourth expression frame (ORF4) is used to express the E3 gene with a V5 tag; the fifth expression frame (ORF5) is used to express the "substrate" gene with a 3×FLAG tag. Take 10 ng of the expression plasmid and transform it into BL21 Escherichia coli competent cells, spread on a plate for overnight culture, pick a single clone the next day, perform protein induction expression, ultrasonically disrupt the bacteria to harvest the protein, and perform protein ubiquitination analysis by immunoblotting. The specific steps are as follows:

[0061] 1. Plasmid construction. The DNA sequence expressing ubiquitin was amplified by PCR, and the ubiquitin gene was cloned into the first expression frame of the pBER322 prokaryotic expression plasmid skeleton through the BamHI and AgeI restriction sites by restriction ligation to obtain a plasmid that only expresses ubiquitin protein. On this basis, UBA1 (E1) was cloned into the plasmid vector through AgeI and SacII, UBE2D1 (E2) was cloned into the plasmid vector through PacI and AscI, TRAIP (E3), RNF220 (E3) and MARCH8 (E3) were cloned into the plasmid vector through AsiSI and MluI, and cGAS (substrate) was cloned into the plasmid vector through NheI and KpnI. Finally, a plasmid vector expressing only ubiquitin, a plasmid vector expressing ubiquitin and E1, a plasmid vector expressing ubiquitin, E1 and E2, a plasmid vector expressing ubiquitin, E1, E2 and E3, and a plasmid vector expressing ubiquitin, E1, E2, E3 and substrate were obtained.

[0062] 2. Transform the plasmid into BL21 competent cells. The plasmids transformed in Example 1 include: ubiquitin-UBA1; ubiquitin-UBA1-UBE2D1; ubiquitin-UBA1-UBE2D1-RNF220 (including wild type and mutants); ubiquitin-UBA1-UBE2D1-TRAIP (including wild type and mutants); ubiquitin-UBA1-UBE2D1-MARCH8; ubiquitin-UBA1-UBE2D1-MARCH8-cGAS. Take 10 ng of plasmid and add it to 30 μL BL21 competent cells, place on ice for 30 min, heat shock at 42 ° C for 45 s, place on ice for 5 min, then add 500 μL of non-resistant LB liquid culture medium, and culture at 37 ° C, 220 rpm / min for 1 hour. Take 100 μL of bacterial solution, spread it on ampicillin-resistant LB solid agar plate, and culture it at 37 ° C overnight.

[0063] 3. Pick a single clone and inoculate it into 5 mL of LB liquid culture medium containing ampicillin antibiotics, culture at 37°C, 220 rpm / min, and culture until OD600 is about 0.6. Add 0.5 mM isopropylthiogalactoside (IPTG) to the culture medium to induce protein expression. After continuing to culture for 4 hours, centrifuge at 4000 rpm / min for 10 minutes to collect the bacteria.

[0064] 4. Ultrasonic disruption of bacteria and collection of proteins. Resuspend the centrifuged bacteria in 500 μL lysis buffer (50 mM Tris-HCl, 2 mM EDTA, 100 mM NaCl, 0.5% Triton X-100, pH 8.5-9.0, add phenylmethylsulfonyl fluoride (PMSF) before use). Ultrasonic disruption of the resuspended bacteria, ultrasonic conditions are 30% power (300 W), ultrasound on 2s, off 2s, total duration of 3min, the whole process is carried out in an ice water bath. The ultrasonic bacterial lysate is centrifuged at 20,000 g for 15 min to remove the precipitate and retain the supernatant. Add the obtained supernatant to the protein loading buffer and boil at 95°C for 10 min. Follow up with immunoblotting analysis.

[0065] 5. Western blot analysis of protein ubiquitination levels. The protein samples prepared in the previous step were separated by electrophoresis using a 12.5% ​​polyacrylamide gel. After electrophoresis, the proteins on the gel were transferred to a nitrocellulose membrane at a constant voltage of 100 V for 100 min.

[0066] 6. Blocking and antibody incubation. Use 5% skim milk powder to block the nitrocellulose membrane at room temperature for 1 hour. After blocking, incubate with the primary antibody. The primary antibody includes ubiquitin primary antibody, HA tag primary antibody, V5 tag primary antibody and FLAG tag primary antibody. The concentration of the primary antibody is referred to the corresponding product manual. Incubate the primary antibody at 4°C overnight. After the primary antibody is blocked, use TBST buffer to wash the nitrocellulose membrane 5 times, each time for 6 minutes. After washing, incubate with the secondary antibody (the secondary antibody used in this example is a horseradish peroxidase-labeled secondary antibody) at room temperature for 1 hour. After the secondary antibody incubation, use TBST buffer to wash 5 times, each time for 6 minutes.

[0067] 7. Development: Use developer and developer to expose, develop and analyze the nitrocellulose membrane.

[0068] Results: Figure 2 As shown, in the IPTG-induced group ( Figure 2 , Lanes 2 and 4), both can express ubiquitin protein, the expression target band is about 10KDa, and the vector containing the E2 gene can express the corresponding E2 protein, the expression target band is about 17KDa. Without the addition of IPTG, the bacteria transformed with the two prokaryotic expression vectors hardly expressed the target protein.

[0069] On the basis of ubiquitin, E1, and E2, TRAIP (E3) ubiquitin ligase is added to the vector to detect whether ubiquitination reaction can occur. Figure 3 As shown in A, after the insertion of TRAIP, the ubiquitinated band can be clearly detected by the ubiquitin antibody ( Figure 3 A in the middle, lane 4). The RING domain is one of the catalytically active domains of E3 ubiquitin ligases. Mutation or deletion of the core amino acids of this domain will lead to the loss of ubiquitin ligase activity, thus failing to achieve the ubiquitination process. In this system, both the RING domain deletion group (ΔRING) and the mutant group (C7A) lost the ubiquitination band ( Figure 3 A, lanes 6 and 8), demonstrating the reliability of this method. At the same time, this method was also used to reconstruct other E3 and substrate ubiquitination processes including RNF220 ( Figure 3 Middle B) and MARCH8 ( Figure 4 ), similar to TRAIP, ubiquitinated bands were also detected in wild-type RNF220 under this method ( Figure 3 B, lane 4), while no ubiquitination band was detected when the RING domain was deleted or mutated ( Figure 3B, lanes 6 and 8). RNF220, as an E3 ubiquitin ligase, has self-ubiquitination activity, that is, it can use itself as a substrate to ubiquitinate itself. We can see the self-ubiquitination of RNF220 through the V5 tag antibody, while RNF220 self-ubiquitination does not exist in the RNF220 activity-deficient mutant. Cyclic guanosine monophosphate-adenylate synthetase (cGAS) is a substrate of MARCH8 and can be ubiquitinated by it. This method also reconstructs the ubiquitination process of MARCH8 and cGAS, such as Figure 4 The FLAG tag antibody shown shows that the substrate protein cGAS is ubiquitinated. These experiments show that this method can be used to reconstruct the ubiquitination process.

[0070] Example 2

[0071] Identify ubiquitin chain types

[0072] In addition to being able to reconstruct the ubiquitination process, this application also has the function of identifying different types of ubiquitin chains. The principle of achieving this function is as follows Figure 5 As shown, on the basis of the prokaryotic expression vector, the wild-type ubiquitin DNA sequence is replaced with different ubiquitin mutant DNA sequences, so that different ubiquitin mutant proteins can be expressed. By analyzing the changes in the ubiquitination strength of the target protein when expressing different ubiquitin mutants by immunoblotting, it can be determined which type of ubiquitin mutant the ubiquitination of the target protein depends on.

[0073] The specific steps are as follows:

[0074] 1. Construction of ubiquitin mutant plasmids. Based on the ubiquitin (wild type)-UBA1-UBE2D1-RNF220 expression plasmid, ubiquitin (wild type) was replaced with different types of ubiquitin mutants, including ΔGG, K6R, K11R, K27R, K29R, K33R, K48R, K63R; K0, K6 only, K11 only, K27 only, K29 only, K33 only, K48 only, K63only, to obtain different ubiquitin mutant plasmids.

[0075] 2. The remaining steps are the same as in Example 1.

[0076] Taking RNF220 as an example, different types of ubiquitin mutants were constructed, and protein expression and ubiquitination analysis were performed. When the ubiquitin mutant was ΔGG, ubiquitination modification could not be completed because ubiquitin could not be activated ( Figure 6In the figure A, lanes 2 and 4). There is no ubiquitination modification in lanes 2 and 4 of the ubiquitin antibody incubation group, and there is no self-ubiquitination modification of RNF220 in lanes 2 and 4 of the V5 tag antibody incubation group. The ubiquitin K0 mutant can prevent the generation of polyubiquitin chains, and the self-ubiquitination band of RNF220 can still be detected under the conditions of the ubiquitin K0 mutant, indicating that there are multi-site monoubiquitination modifications in the self-ubiquitination modification of RNF220. Analysis of the self-ubiquitination type of RNF220 under different ubiquitin mutant conditions shows that in the ubiquitin single-site mutant (KxR), the mutations of K6R, K27R, K48R and K63R significantly weakened the multi-polymer form of RNF220 self-ubiquitination, while the mutations of K11R, K29R and K33R did not affect the multi-polymer form of RNF220 self-ubiquitination ( Figure 6 B, lanes 4, 6, and 7), these results indicate that the types of RNF220 polyubiquitin chains include K6, K27, K48, and K63. In the mutant that retains only a single lysine (Kxonly), since the polyubiquitinated forms of RNF220 include K6, K27, K48, and K63, if only these single-site lysine residues are retained unchanged, RNF220 self-ubiquitination can still form polyubiquitinated forms. On the contrary, if only the remaining single-site lysine residues are retained unchanged, the polyubiquitinated forms of RNF220 are impaired ( Figure 6 Middle C). The comprehensive experimental results showed that this method identified the polyubiquitin chain types of RNF220 autoubiquitination, including K6, K27, K48 and K63.

[0077] Example 3

[0078] Identify E2-E3 pairs

[0079] The third application of this application is to identify E2-E3 pairings. The principle of the application is as follows Figure 7 As shown, one plasmid is further split into two plasmids, the E2 gene is cloned into the p15A prokaryotic expression vector alone, and the other ubiquitination elements are retained in the pBER322 expression vector. The two plasmids are co-transformed into E. coli cells to induce protein expression. If the E2-E3 matches, the ubiquitination process can be completed and ubiquitinated bands are produced, otherwise no ubiquitinated bands can be produced. By co-transforming different E2 plasmids with fixed E3 plasmids, E2 that can match a specific E3 can be screened.

[0080] The specific steps are as follows:

[0081] 1. Single E2 plasmid cloning. Different E2 genes (UBE2D1, UBE2D2, UBE2D3, UBE2D4, UBE2E1, UBE2E2, UBE2E3, UBE2Q1 and UBE2Q2 in this example) were cloned into a p15A plasmid vector with chloramphenicol resistance to obtain a plasmid expressing only a single E2 gene.

[0082] 2. Plasmid cloning lacking E2 gene. Similar to the cloning process in Example 1, ubiquitin, UBA1 and RNF220 (or TRAIP) were constructed into pBER322 plasmid vectors, respectively, and the vector resistance was ampicillin, and plasmids "ubiquitin-UBA1-RNF220" and "ubiquitin-UBA1-TRAIP" were obtained, which did not express E2 protein.

[0083] 3. Double plasmid co-transformation of BL21 competent cells. Take 10ng of "ubiquitin-UBA1-RNF220" (or "ubiquitin-UBA1-TRAIP") and 10ng of different p15A-E2 plasmids and add them to 30μL BL21 competent cells, place on ice for 30min, heat shock at 42℃ for 45s, place on ice for 5min, then add 500μL of resistance-free LB medium, and culture at 37℃, 220rpm / min for 1h. Take 100μL of bacterial solution, spread it on ampicillin and chloramphenicol double resistance LB solid agar plate, and culture it at 37℃ overnight.

[0084] 4. Pick a single clone and inoculate it into 5 mL of LB liquid culture medium containing ampicillin and chloramphenicol double antibiotics. Culture at 37°C, 220 rpm / min until OD600 is about 0.6. Add 0.5 mM isopropylthiogalactoside (IPTG) to the culture medium to induce protein expression. After continuing to culture for 4 hours, centrifuge at 4000 rpm / min for 10 minutes to collect the bacterial liquid.

[0085] 5. The following steps are the same as in Example 1.

[0086] The experimental results are as follows Figure 8 As shown in Figure A, TRAIP and RNF220, as two different E3s, successfully achieved ubiquitination with UBE2D1 through this strategy. Using this method, RNF220 was used as a fixed E3 plasmid to screen different E2 plasmids. The screening results of some E2 plasmids are shown in Figure 2. Figure 8 As shown in B, the results show that the UBE2D family and the UBE2E family can match RNF220 to complete the ubiquitination modification process.

[0087] Related gene DNA sequence:

[0088] Ubiquitin-ΔGG (SEQ ID NO: 3):

[0089] ATGCAGATCTTCGTGAAGACCCTGACTGGTAAGACCATCACTCTCGAAGTGGAGCCGAGTGACACCATTGAGAATGTCAAGGCAAAGATCCAAGACAAGGAAGGCATCCCTCCTGACCAGCAGAGGTTGATCTTTGCTGGGAAACAGCTGGAAGATGGACGCACCCTGTCTGACTACAACATCCAGAAAGAGTCCACCCTGCACCTGGTCCTCCGTCTCAGA

[0090] Ubiquitin-K6R (SEQ ID NO: 4):

[0091] ATGCAGATCTTCGTGAGGACCCTGACTGGTAAGACCATCACTCTCGAAGTGGAGCCGAGTGACACCATTGAGAATGTCAAGGCAAAGATCCAAGACAAGGAAGGCATCCCTCCTGACCAGCAGAGGTTGATCTTTGCTGGGAAACAGCTGGAAGATGGACGCACCCTGTCTGACTACAACATCCAGAAAGAGTCCACCCTGCACCTGGTCCTCCGTCTCAGAGGTGGT

[0092] Ubiquitin-K11R (SEQ ID NO: 5):

[0093] ATGCAGATCTTCGTGAAGACCCTGACTGGTAGGACCATCACTCTCGAAGTGGAGCCGAGTGACACCATTGAGAATGTCAAGGCAAAGATCCAAGACAAGGAAGGCATCCCTCCTGACCAGCAGAGGTTGATCTTTGCTGGGAAACAGCTGGAAGATGGACGCACCCTGTCTGACTACAACATCCAGAAAGAGTCCACCCTGCACCTGGTCCTCCGTCTCAGAGGTGGT

[0094] Ubiquitin-K27R (SEQ ID NO: 6):

[0095] ATGCAGATCTTCGTGAAGACCCTGACTGGTAAGACCATCACTCTCGAAGTGGAGCCGAGTGACACCATTGAGAATGTCAGGGCAAAGATCCAAGACAAGGAAGGCATCCCTCCTGACCAGCAGAGGTTGATCTTTGCTGGGAAACAGCTGGAAGATGGACGCACCCTGTCTGACTACAACATCCAGAAAGAGTCCACCCTGCACCTGGTCCTCCGTCTCAGAGGTGGT

[0096] Ubiquitin-K29R (SEQ ID NO: 7):

[0097] ATGCAGATCTTCGTGAAGACCCTGACTGGTAAGACCATCACTCTCGAAGTGGAGCCGAGTGACACCATTGAGAATGTCAAGGCAAGGATCCAAGACAAGGAAGGCATCCCTCCTGACCAGCAGAGGTTGATCTTTGCTGGGAAACAGCTGGAAGATGGACGCACCCTGTCTGACTACAACATCCAGAAAGAGTCCACCCTGCACCTGGTCCTCCGTCTCAGAGGTGGT

[0098] Ubiquitin-K33R (SEQ ID NO: 8):

[0099] ATGCAGATCTTCGTGAAGACCCTGACTGGTAAGACCATCACTCTCGAAGTGGAGCCGAGTGACACCATTGAGAATGTCAAGGCAAAGATCCAAGACAGGGAAGGCATCCCTCCTGACCAGCAGAGGTTGATCTTTGCTGGGAAACAGCTGGAAGATGGACGCACCCTGTCTGACTACAACATCCAGAAAGAGTCCACCCTGCACCTGGTCCTCCGTCTCAGAGGTGGT

[0100] Ubiquitin-K48R (SEQ ID NO: 9):

[0101] ATGCAGATCTTCGTCAAGACGTTAACCGGTAAAACCATAACTCTAGAAGTTGAACCATCCGATACCATCGAAAACGTTAAGGCTAAAATTCAAGACAAGGAAGGCATTCCACCTGATCAACAAAGATTGATCTTTGCCGGTAGGCAGCTCGAGGACGGTAGAACGCTGTCTGATTACAACATTCAGAAGGAGTCCACCCTGCACCTGGTCCTCCGTCTCAGAGGTGGT

[0102] Ubiquitin-K63R (SEQ ID NO: 10):

[0103] ATGCAGATCTTCGTGAAGACCCTGACTGGTAAGACCATCACTCTCGAAGTGGAGCCGAGTGACACCATTGAGAATGTCAAGGCAAAGATCCAAGACAAGGAAGGCATCCCTCCTGACCAGCAGAGGTTGATCTTTGCTGGGAAACAGCTGGAAGATGGACGCACCCTGTCTGACTACAACATCCAGAGAGAGTCCACCCTGCACCTGGTCCTCCGTCTCAGAGGTGGT

[0104] Ubiquitin-K0 (SEQ ID NO: 11):

[0105] ATGCAGATCTTCGTCAGAACGTTAACCGGTAGAACCATAACTCTAGAAGTTGAACCATCCGATACCATCGAAAACGTTAGAGCTAGAATTCAAGACAGAGAAGGCATTCCACCTGATCAACAAAGATTGATCTTTGCCGGTAGACAGCTCGAGGACGGTAGAACGCTGTCTGATTACAACATTCAGAGAGAGTCGACCTTACATCTTGTCTTAAGACTAAGAGGTGGT

[0106] Ubiquitin-K6 only (SEQ ID NO: 12):

[0107] ATGCAGATCTTCGTCAAAACGTTAACCGGTAGAACCATAACTCTAGAAGTTGAACCATCCGATACCATCGAAAACGTTAGAGCTAGAATTCAAGACAGAGAAGGCATTCCACCTGATCAACAAAGATTGATCTTTGCCGGTAGACAGCTCGAGGACGGTAGAACGCTGTCTGATTACAACATTCAGAGAGAGTCGACCTTACATCTTGTCTTAAGACTAAGAGGTGGT

[0108] Ubiquitin-K11 only (SEQ ID NO: 13):

[0109] ATGCAGATCTTCGTCAGAACGTTAACCGGTAAAACCATAACTCTAGAAGTTGAACCATCCGATACCATCGAAAACGTTAGAGCTAGAATTCAAGACAGAGAAGGCATTCCACCTGATCAACAAAGATTGATCTTTGCCGGTAGACAGCTCGAGGACGGTAGAACGCTGTCTGATTACAACATTCAGAGAGAGTCGACCTTACATCTTGTCTTAAGACTAAGAGGTGGT

[0110] Ubiquitin-K27 only (SEQ ID NO: 14):

[0111] ATGCAGATCTTCGTCAGAACGTTAACCGGTAGAACCATAACTCTAGAAGTTGAACCATCCGATACCATCGAAAACGTTAAAGCTAGAATTCAAGACAGAGAAGGCATTCCACCTGATCAACAAAGATTGATCTTTGCCGGTAGACAGCTCGAGGACGGTAGAACGCTGTCTGATTACAACATTCAGAGAGAGTCGACCTTACATCTTGTCTTAAGACTAAGAGGTGGT

[0112] Ubiquitin-K29 only (SEQ ID NO: 15):

[0113] ATGCAGATCTTCGTCAGAACGTTAACCGGTAGAACCATAACTCTAGAAGTTGAACCATCCGATACCATCGAAAACGTTAGAGCTAAAATTCAAGACAGAGAAGGCATTCCACCTGATCAACAAAGATTGATCTTTGCCGGTAGACAGCTCGAGGACGGTAGAACGCTGTCTGATTACAACATTCAGAGAGAGTCGACCTTACATCTTGTCTTAAGACTAAGAGGTGGT

[0114] Ubiquitin-K33 only (SEQ ID NO: 16):

[0115] ATGCAGATCTTCGTCAGAACGTTAACCGGTAGAACCATAACTCTAGAAGTTGAACCATCCGATACCATCGAAAACGTTAGAGCTAGAATTCAAGACAAAGAAGGCATTCCACCTGATCAACAAAGATTGATCTTTGCCGGTAGACAGCTCGAGGACGGTAGAACGCTGTCTGATTACAACATTCAGAGAGAGTCGACCTTACATCTTGTCTTAAGACTAAGAGGTGGT

[0116] Ubiquitin-K48 only (SEQ ID NO: 17):

[0117] ATGCAGATCTTCGTCAGAACGTTAACCGGTAGAACCATAACTCTAGAAGTTGAACCATCCGATACCATCGAAAACGTTAGAGCTAGAATTCAAGACAGAGAAGGCATTCCACCTGATCAACAAAGATTGATCTTTGCCGGTAAGCAGCTCGAGGACGGTAGAACGCTGTCTGATTACAACATTCAGAGAGAGTCCACCCTGCACCTGGTCCTCCGTCTCAGAGGTGGT

[0118] Ubiquitin-K63 only (SEQ ID NO: 18):

[0119] ATGCAGATCTTCGTCAGAACGTTAACCGGTAGAACCATAACTCTAGAAGTTGAACCATCCGATACCATCGAAAACGTTAGAGCTAGAATTCAAGACAGAGAAGGCATTCCACCTGATCAACAAAGATTGATCTTTGCCGGTAGACAGCTCGAGGACGGTAGAACGCTGTCTGATTACAACATTCAGAAGGAGTCCACCCTGCACCTGGTCCTCCGTCTCAGAGGTGGT

[0120] UBA1(SEQ ID NO:19):

[0121]

[0122] UBE2D1(SEQ ID NO:20):

[0123] ATGGCGCTGAAGAGGATTCAGAAAGAATTGAGTGATCTACAGCGCGATCCACCTGCTCACTGTTCAGCTGGACCTGTGGGAGATGACTTGTTCCACTGGCAAGCCACTATTATGGGGCCTCCTGATAGCGCATATCAAGGTGGAGTCTTCTTTCTCACTGTACATTTTCCGACAGATTATCCTTTTAAACCACCAAAGATTGCTTTCACAACAAAAATTTACCATCCAAACATAAACAGTAATGGAAGTATTTGTCTCGATATTCTGAGGTCACAATGGTCACCAGCTCTGACTGTATCAAAAGTTTTATTGTCCATATGTTCTCTACTTTGTGATCCTAATCCAGATGACCCCTTAGTACCAGATATTGCACAAATCTATAAATCAGACAAAGAAAAATACAACAGACATGCAAGAGAATGGACTCAGAAATATGCAATG

[0124] RNF220(SEQ ID NO:21):

[0125]

[0126] RNF220-ΔRING(SEQ ID NO:22):

[0127]

[0128] RNF220-C514A(SEQ ID NO:23):

[0129]

[0130] TRAIP(SEQ ID NO:24):

[0131]

[0132] TRAIP-ΔRING(SEQ ID NO:25):

[0133]

[0134] TRAIP-C7A(SEQ ID NO:26):

[0135]

[0136] MARCH8(SEQ ID NO:27):

[0137] ATGAGCATGCCACTGCATCAGATCTCTGCCATTCCATCCCAGGATGCCATCTCTGCTAGAGTCTACAGAAGTAAGACCAAAGAAAAGGAGAGGGAAGAACAGAATGAGAAGACTTTGGGACATTTCATGAGTCATTCAAGCAACATTTCTAAGGCTGGGAGTCCTCCGTCAGCATCAGCTCCGGCTCCGGTGTCCTCCTTCTCTCGCACTTCTATCACGCCATCCAGCCAGGACATCTGCAGGATCTGCCACTGTGAAGGAGATGATGAGAGCCCCCTGATCACCCCCTGCCACTGCACAGGAAGCCTCCACTTCGTGCACCAGGCCTGCCTGCAGCAGTGGATCAAGAGCTCCGACACGCGCTGCTGCGAGCTCTGCAAGTATGAGTTCATCATGGAGACCAAGCTGAAGCCACTGAGAAAATGGGAGAAGTTGCAGATGACGTCCAGCGAGCGCAGGAAGATCATGTGCTCAGTGACATTCCACGTCATTGCCATCACATGTGTGGTCTGGTCCTTGTATGTGCTCATTGACCGTACTGCTGAGGAGATCAAGCAGGGGCAGGCAACAGGAATCCTAGAATGGCCCTTTTGGACTAAATTGGTGGTTGTGGCCATCGGCTTCACCGGAGGACTTCTTTTTATGTATGTTCAGTGTAAAGTGTATGTGCAATTGTGGAAGAGACTCAAGGCCTATAATAGAGTGATCTATGTTCAAAACTGTCCAGAAACAAGCAAAAAGAATATTTTTGAAAAATCTCCACTAACAGAGCCCAACTTTGAAAATAAACATGGATATGGAATCTGTCATTCCGACACAAACTCTTCTTGTTGCACAGAGCCTGAAGACACTGGAGCAGAAATCATTCACGTC

[0138] cGAS(SEQ ID NO:28):

[0139]

[0140] UBE2D2(SEQ ID NO:29):

[0141] ATGGCTCTGAAGAGAATCCACAAGGAATTGAATGATCTGGCACGGGACCCTCCAGCACAGTGTTCAGCAGGTCCTGTTGGAGATGATATGTTCCATTGGCAAGCTACAATAATGGGGCCAAATGACAGTCCCTATCAGGGTGGAGTATTTTTCTTGACAATTCATTTCCCAACAGATTACCCCTTCAAACCACCTAAGGTTGCATTTACAACAAGAATTTATCATCCAAATATTAACAGTAATGGCAGCATTTGTCTTGATATTCTACGATCACAGTGGTCTCCAGCACTAACTATTTCAAAAGTACTCTTGTCCATCTGTTCTCTGTTGTGTGATCCCAATCCAGATGATCCTTTAGTGCCTGAGATTGCTCGGATCTACAAAACAGATAGAGAAAAGTACAACAGAATAGCTCGGGAATGGACTCAGAAGTATGCGATG

[0142] UBE2D3(SEQ ID NO:30):

[0143] ATGGCGCTGAAACGGATTAATAAGGAACTTAGTGATTTGGCCCGTGACCCTCCAGCACAATGTTCTGCAGGTCCAGTTGGGGATGATATGTTTCATTGGCAAGCCACAATTATGGGACCTAATGACAGCCCATATCAAGGCGGTGTATTCTTTTTGACAATTCATTTTCCTACAGACTACCCCTTCAAACCACCTAAGGTTGCATTTACAACAAGAATTTATCATCCAAATATTAACAGTAATGGCAGCATTTGTCTCGATATTCTAAGATCACAGTGGTCGCCTGCTTTAACAATTTCTAAAGTTCTTTTATCCATTTGTTCACTGCTATGTGATCCAAACCCAGATGACCCCCTAGTGCCAGAGATTGCACGGATCTATAAAACAGACAGAGATAAGTACAACAGAATATCTCGGGAATGGACTCAGAAGTATGCCATG

[0144] UBE2D4(SEQ ID NO:31):

[0145] ATGGCGCTAAAGCGGATCCAGAAGGAATTAACCGACTTGCAGAGGGATCCTCCTGCCCAGTGTTCTGCAGGACCTGTCGGTGATGACTTGTTCCACTGGCAGGCCACCATCATGGGCCCGAATGACAGTCCTTACCAAGGAGGTGTTTTCTTCCTGACCATCCACTTTCCTACAGATTACCCGTTCAAGCCCCCAAAGGTTGCTTTCACAACCAAAATTTATCACCCTAATATCAACAGCAATGGCAGCATCTGCCTTGATATCCTGCGGTCTCAGTGGTCTCCAGCGTTGACTGTGTCAAAAGTTCTCTTGTCCATCTGCTCGCTGCTCTGCGACCCCAACCCCGATGACCCCCTGGTGCCAGAGATAGCACACACCTACAAGGCCGACAGAGAGAAGTACAACAGACTAGCAAGAGAGTGGACACAAAAATATGCTATG

[0146] UBE2E1(SEQ ID NO:32):

[0147] ATGTCGGATGACGATTCGAGGGCCAGCACCAGCTCCTCCTCATCTTCGTCTTCCAACCAGCAAACCGAGAAAGAAACAAACACCCCCAAGAAGAAGGAGAGTAAAGTCAGCATGAGCAAAAACTCCAAACTCCTCTCCACCAGCGCCAAGAGAATTCAGAAGGAGCTGGCGGACATCACTTTAGACCCTCCACCTAATTGCAGTGCTGGTCCCAAAGGCGATAACATCTATGAATGGAGATCAACCATTCTAGGGCCTCCAGGATCCGTGTATGAGGGTGGTGTATTCTTTCTCGATATCACTTTTACACCAGAATATCCCTTCAAGCCTCCAAAGGTTACATTTCGGACAAGAATCTATCATTGTAATATTAACAGTCAAGGTGTTATTTGCTTGGACATATTGAAAGATAATTGGAGTCCAGCACTAACCATTTCTAAAGTCCTCCTTTCTATCTGCTCACTTCTTACAGACTGTAATCCTGCCGACCCCTTGGTGGGAAGTATTGCCACTCAGTATATGACCAACAGAGCAGAACATGACAGAATGGCCAGACAGTGGACCAAGAGATACGCTACA

[0148] UBE2E2(SEQ ID NO:33):

[0149] ATGTCCACTGAGGCACAAAGAGTTGATGACAGTCCAAGCACTAGTGGAGGAAGTTCCGATGGAGATCAACGTGAAAGTGTTCAGCAAGAACCAGAAAGAGAACAAGTTCAGCCCAAGAAAGGAGGGAAAAATATCCAGCAAAACCGCT GCTAAATTGTCAACTAGTGCTAAAAGAATTCAGAAGGAACTTGCAGAAATCACATTGGACCCTCCTCCCAACTGTAGTGCTGGACCCAAAGGAGACAACATTTATGAATGGAGGTCAACTATATTGGGACCCCCAGGATCTGTCTATGAAG GAGGGGTGTTCTTTCTTGACATTACCTTTTCACCAGACTATCCGTTTAAACCCCCTAAGGTTACCTTCCGAACAAGAATCTATCACTGTAATATTAACAGCCAAGGTGTGATCTGTCTGGACATCTTAAAGGACAACTGGAGTCCGGCTTT AACTATTTCTAAAGTTCTCCTCTCCATCTGCTCACTTCTTACAGATTGCAACCCTGCTGACCCTCTGGTGGGCAGCATCGCCACACAGTACATGACCAACAGAGCAGAGCATGACCGGATGGCCAGACAGTGGACCAAGCGGTACGCCACA

[0150] UBE2E3(SEQ ID NO:34):

[0151] ATGTCCAGTGATAGGCAAAGGTCCGATGATGAGAGCCCCAGCACCAGCAGTGGCAGTTCAGATGCGGACCAGCGAGACCCAGCCGCTCCAGAGCCTGAAGAACAAGAGGAAAGAAAACCTTCTGCCACCCAGCAGAAGAAAAACACCAAACTCTCTAGCAAAACCACTGCTAAGTTATCCACTAGTGCTAAAAGAATTCAGAAGGAGCTAGCTGAAATAACCCTTGATCCTCCTCCTAATTGCAGTGCTGGGCCTAAAGGAGATAACATTTATGAATGGAGATCAACTATACTTGGTCCACCGGGTTCTGTATATGAAGGTGGTGTGTTTTTTCTGGATATCACATTTTCATCAGATTATCCATTTAAGCCACCAAAGGTTACTTTCCGCACCAGAATCTATCACTGCAACATCAACAGTCAGGGAGTCATCTGTCTGGACATCCTTAAAGACAACTGGAGTCCCGCTTTGACTATTTCAAAGGTTTTGCTGTCTATTTGTTCCCTTTTGACAGACTGCAACCCTGCGGATCCTCTGGTTGGAAGCATAGCCACTCAGTATTTGACCAACAGAGCAGAACACGACAGGATAGCCAGACAGTGGACCAAGAGATACGCAACA

[0152] UBE2Q1(SEQ ID NO:35):

[0153]

[0154] UBE2Q2(SEQ ID NO:36):

[0155]

[0156] From the above description, it can be seen that the above embodiments of the present invention achieve the following technical effects:

[0157] 1) By introducing human ubiquitination-related genes into the prokaryotic organism Escherichia coli in the form of replaceable expression elements, IPTG induces the expression of the corresponding protein, so that the entire ubiquitination process is completed in Escherichia coli, thereby realizing the ubiquitination reconstruction process. Compared with the traditional in vitro ubiquitination reconstruction process, this method is simple and convenient to operate, does not require purification of specific proteins, has a short time period and low cost.

[0158] 2) Prokaryotes do not have a ubiquitination system themselves, so introducing a human ubiquitination system into Escherichia coli can reduce interference. Compared with mammalian cell-based detection methods, it is not affected by endogenous ubiquitination factors, increasing the credibility and authenticity of the results.

[0159] 3) Since the expression elements of this system are replaceable, different expression elements can be replaced to achieve different functions. For example, the expression element of ubiquitin can be replaced with different ubiquitin mutants to determine the type of substrate ubiquitin chain.

[0160] 4) Based on the ubiquitination detection single plasmid system, the upgraded dual plasmid system has the function of screening E2-E3 pairings, that is, under the conditions of given E3 and substrate, specific E2-E3 pairings can be identified by replacing different E2s.

[0161] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A ubiquitination detection system, characterized in that: It comprises a prokaryotic expression vector, which comprises a ubiquitin expression frame, a ubiquitin activating enzyme expression frame, a ubiquitin conjugating enzyme expression frame, a ubiquitin ligase expression frame and a substrate protein expression frame, wherein the ubiquitin expression frame, the ubiquitin conjugating enzyme expression frame, the ubiquitin ligase expression frame and the substrate protein expression frame are fused to express different tag proteins respectively.

2. The ubiquitination detection system according to claim 1, characterized in that The ubiquitin expression frame, the ubiquitin activating enzyme expression frame, the ubiquitin conjugating enzyme expression frame, the ubiquitin ligase expression frame and the substrate protein expression frame are constructed in the same prokaryotic expression vector and perform protein expression independently.

3. The ubiquitination detection system according to claim 1, characterized in that The ubiquitin expression cassette, the ubiquitin activating enzyme expression cassette, the ubiquitin ligase expression cassette and the substrate protein expression cassette are constructed in a first prokaryotic expression vector, the ubiquitin conjugating enzyme expression cassette is constructed in a second prokaryotic expression vector, and the first prokaryotic expression vector and the second prokaryotic expression vector have compatible replication initiation sites and different resistance genes.

4. The ubiquitination detection system according to any one of claims 1 to 3, characterized in that: The ubiquitin expression frame is fused to express His tag protein, the ubiquitin activating enzyme expression frame does not express tag protein, the ubiquitin conjugating enzyme expression frame is fused to express HA tag protein, the ubiquitin ligase expression frame is fused to express V5 tag protein, and the substrate protein expression frame is fused to express FLAG tag protein.

5. The ubiquitination detection system according to any one of claims 1 to 3, characterized in that: The ubiquitin expression box expresses wild-type ubiquitin or a ubiquitin mutant, and the ubiquitin mutant is ΔGG, K6R, K11R, K27R, K29R, K33R, K48R, K63R, K0, K6 only, K11 only, K27 only, K29 only, K33 only, K48 only or K63only mutant.

6. The ubiquitination detection system according to any one of claims 1 to 3, characterized in that: The ubiquitin ligase expression frame and the substrate protein expression frame are arranged correspondingly.

7. The ubiquitination detection system according to any one of claims 1 to 3, characterized in that: The prokaryotic expression vector is used to express protein in Escherichia coli.

8. A ubiquitination detection method, characterized in that: include: Ubiquitination detection is performed using the ubiquitination detection system according to any one of claims 1 to 7.

9. The ubiquitination detection method according to claim 8, characterized in that include: Transforming the prokaryotic expression vector into Escherichia coli; Inducing the ubiquitin expression frame, the ubiquitin activating enzyme expression frame, the ubiquitin conjugating enzyme expression frame, the ubiquitin ligase expression frame and the substrate protein expression frame to express proteins; The E. coli is lysed, and the ubiquitination level and protein expression level of the lysed protein are detected.

10. The ubiquitination detection method according to claim 9, characterized in that: The ubiquitination level and protein expression level of the cleaved protein were detected by Western blot.

11. The ubiquitination detection method according to claim 10, characterized in that: The ubiquitin antibody is incubated to detect the ubiquitination level of the cleaved protein; and the tag protein antibody is incubated to detect the protein expression levels of the ubiquitin conjugating enzyme, the ubiquitin ligase and the substrate protein respectively.

12. The ubiquitination detection method according to claim 11, characterized in that: The ubiquitination detection method includes the detection of reconstructing the ubiquitination process, and the detection includes: The protein expression products are analyzed by Western blot technology, and the ubiquitination level and the substrate protein expression level are detected respectively using ubiquitin antibodies and substrate tag protein antibodies. If the ubiquitination process can be reconstructed, the ubiquitination level can be detected by the ubiquitination antibody. If the substrate protein is ubiquitinated, the substrate tag protein antibody can also detect the presence of ubiquitination bands on the substrate protein.

13. The ubiquitination detection method according to claim 11, characterized in that: The ubiquitination detection method includes identifying the type of ubiquitin chain, and the identifying the type of ubiquitin chain includes: constructing a prokaryotic expression vector containing different ubiquitin mutant expression frames, ubiquitin ligase expression frames and substrate protein expression frames; Induce protein expression and use Western blot to analyze protein ubiquitination; The specific ubiquitin ligase and the type of ubiquitin chain of the substrate protein can be identified based on the strength or absence of ubiquitination of the expressed proteins of different ubiquitin mutants.

14. The ubiquitination detection method according to claim 11, characterized in that: The ubiquitination detection method comprises identifying the pairing of ubiquitin conjugating enzyme and ubiquitin ligase based on a ubiquitination detection system comprising a first prokaryotic expression vector and a second prokaryotic expression vector: If the protein product co-expressed by the first prokaryotic expression vector and the second prokaryotic expression vector is ubiquitinated, it indicates that the ubiquitin conjugating enzyme and the ubiquitin ligase can form a pair; If the protein product co-expressed by the first prokaryotic expression vector and the second prokaryotic expression vector is not ubiquitinated, it indicates that the ubiquitin conjugating enzyme and the ubiquitin ligase are not paired.

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