Prx5-Trx2 protein compound as well as preparation method and application thereof

Abstract

The invention discloses a Prx5-Trx2 protein compound, a preparation method and application, and relates to the technical field of biology, the method comprises the following steps: mutating cysteine of cysteine residue C47 in Prx5 into serine to obtain a Prx5C47S mutant, and preparing Prx5C47S mutant protein; the method comprises the following steps: mutating cysteine of a cysteine residue C34 in Trx2 into serine, so as to obtain a Trx2C34S mutant; the preparation method comprises the following steps: preparing Trx2C34S mutant protein; the Prx5C47S mutant protein and the Trx2C34S mutant protein are subjected to a disulfide bond cross-linking reaction, and the Prx5-Trx2 protein compound is prepared. According to the invention, a large amount of stable Prx5-Trx2 protein compounds can be prepared, and a basis is provided for research and development of novel anti-tumor drug-resistant drugs targeting the Prx5-Trx2 protein compounds.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and more specifically to a Prx5-Trx2 protein complex, a preparation method and an application thereof. Background Art

[0002] Reactive oxygen is a kind of oxygen-containing substances with high oxidizing ability, mainly including hydrogen peroxide H2O2, organic peroxide ROOH, superoxide anion O 2− , hydroxyl free radical HO·, etc. Both the external environment and normal cell metabolism can induce the production of reactive oxygen species. The large accumulation of reactive oxygen species in cells can induce oxidative stress, causing them to attack the proteins, DNA, phospholipids, etc. of cells and cause cell damage. Abnormal regulation of reactive oxygen species is associated with the occurrence of many diseases such as cancer, neurodegenerative diseases, and diabetes. Therefore, there are a variety of antioxidant proteins in cells to resist the toxic effects of reactive oxygen species on cells, thereby maintaining redox homeostasis.

[0003] Peroxide reductase Prx is a class of antioxidant proteins widely present in cells. It decomposes hydrogen peroxide and organic peroxides through cysteine, which has its own redox activity. The decomposition of peroxide substrate catalyzed by Prx depends on two key active cysteines, namely peroxidatic cysteine, abbreviated as C P and decomposition of cysteine, the English name is resolving cysteine, the English abbreviation is C R . C P It is highly conserved at the evolutionary level and is responsible for the nucleophilic attack on the peroxide bond of the peroxide. P The nearby residues form the conserved peroxide reductase active site motif PXXXS / TXXC. R Responsible for C P The catalytic cycle of Prx is divided into three steps: ① peroxidation. P Nucleophilic attack on peroxide substrates reduces the substrates to the corresponding alcohols. P Oxidized to cysteine ​​in the form of sulfenic acid −SOH; ② Decomposition. C P With C RForming disulfide-linked oxidized Prx; ③ Recycling. Thioredoxin Trx regenerates the inactivated oxidized Prx into catalytically capable reduced Prx through a disulfide exchange reaction. Studies have found that 90% of intracellular peroxides are catalytically decomposed by Prx. Therefore, Prx plays a leading role in resisting the attack of reactive oxygen species. Clinical and animal experiments have shown that defects in peroxide reductase increase the generation of reactive oxygen species, leading to oxidative stress, which is closely related to the occurrence and development of various diseases such as inflammatory diseases, Fanconi anemia, and hemolytic anemia. Studies have found that the protein and mRNA expression levels of Prx are upregulated in most cancer patients, and the overexpression of Prx is associated with the prognosis of cancer patients and resistance to chemotherapy and radiotherapy.

[0004] Peroxisome reductase 5, or Prx5, is the only atypical bicysteine ​​Prx in mammals. Its N- and C-termini have mitochondrial and peroxisome localization signal peptides, respectively, and are widely distributed in mitochondria, peroxisomes, nuclei, and cytoplasm. Prx5 can resist the oxidative damage of reactive oxygen species to cells through its peroxisome function, thus playing a protective role in cell survival. For example, Prx5 alleviates obesity-induced fatty liver by regulating oxidative stress; using an obese mouse model, it was found that Prx5 reduces fat production by regulating reactive oxygen species; Prx5 participates in the formation of vertebrate cilia by regulating mitochondrial reactive oxygen species. Studies have found that the protein expression level of Prx5 is upregulated in most tumor patients, such as lung cancer, gastric cancer, and breast cancer. Its overexpression enhances the occurrence and metastasis of tumors and is associated with poor prognosis.

[0005] Thioredoxin 2, or Trx2, is a highly conserved and widely expressed antioxidant protein that can reduce covalent disulfide bonds in target proteins and plays a key role in regulating cellular redox homeostasis. The notable feature of Trx2 is the presence of two adjacent cysteines in the CXXC motif. These two cysteines are the key amino acid residues for Trx2 to reduce other proteins. Because the C31 residue of Trx2 is exposed to the solvent, C31 is more likely to obtain electrons than C34 to form a thiol anion with nucleophilic attack ability, opening the disulfide bonds of the substrate protein to form thiol groups. After losing electrons, C31 forms a disulfide bond with C34.

[0006] In summary, it is necessary to develop a method for preparing a large amount of stable Prx5-Trx2 protein complex. Summary of the invention

[0007] To solve the above problems, the present invention provides a Prx5-Trx2 protein complex, a preparation method and an application thereof, which can be used to prepare a large amount of stable Prx5-Trx2 protein complex.

[0008] The present invention is achieved through the following technical solutions: A method for preparing a Prx5-Trx2 protein complex comprises the following steps: The cysteine ​​residue C47 in Prx5 is mutated to serine to obtain a Prx5C47S mutant, and a Prx5C47S mutant protein is prepared; the nucleotide sequence of the Prx5C47S mutant is shown in SEQ ID NO.1.

[0009] The cysteine ​​residue C34 in Trx2 is mutated to serine to obtain a Trx2C34S mutant; the Trx2C34S mutant protein is prepared; the nucleotide sequence of the Trx2C34S mutant is shown in SEQ ID NO.3.

[0010] 5,5'-dithiobis(2-nitrobenzoic acid) and Prx5C47S mutant protein were subjected to disulfide bond cross-linking reaction to obtain the product Prx5C47S-TNB.

[0011] The Prx5C47S-TNB and the Trx2C34S mutant protein were subjected to a disulfide cross-linking reaction to obtain a reaction product, thereby obtaining a Prx5-Trx2 protein complex.

[0012] Preferably, the 5,5'-dithiobis(2-nitrobenzoic acid) and the Prx5C47S mutant protein are mixed at a molar ratio of 10-20:1 to perform a disulfide bond cross-linking reaction.

[0013] Preferably, the molar ratio of the Prx5C47S-TNB to the Trx2C34S mutant protein is 1:1.5-1:2.

[0014] Preferably, the reaction product is purified to obtain a purified Prx5-Trx2 protein complex, and the purification steps are as follows: (1) Incubate the reaction product in a water bath at 25°C~30°C for 25 min~30 min.

[0015] (2) The sample is then loaded onto a nickel affinity chromatography column, washed, and the target protein is eluted.

[0016] (3) Further purification using a molecular sieve chromatography column to obtain a purified Prx5-Trx2 protein complex.

[0017] Preferably, buffer H is used for washing, wherein the components of the buffer H are 15 mM to 25 mM tris(hydroxymethyl)aminomethane hydrochloride, 140 mM to 160 mM sodium chloride and 40 mM to 60 mM imidazole.

[0018] Preferably, buffer I is used for elution, wherein the components of the buffer I are 15 mM to 25 mM tris(hydroxymethyl)aminomethane hydrochloride, 140 mM to 160 mM sodium chloride and 240 mM to 260 mM imidazole.

[0019] Preferably, the molecular sieve chromatography column is further purified using buffer E, wherein the components of buffer E are 20 mM tris(hydroxymethyl)aminomethane hydrochloride and 150 mM sodium chloride.

[0020] The Prx5-Trx2 protein complex prepared by the preparation method of the Prx5-Trx2 protein complex is used in analyzing the three-dimensional structure of the Prx5-Trx2 protein complex.

[0021] The Prx5-Trx2 protein complex prepared by the preparation method of the Prx5-Trx2 protein complex is used in the development of new anti-tumor drug resistance drugs targeting the Prx5-Trx2 protein complex target.

[0022] Compared with the prior art, the present invention has the following beneficial effects: (1) The present invention mutates the cysteine ​​of cysteine ​​residue C47 in Prx5 to serine, and mutates the cysteine ​​of cysteine ​​residue C34 in Trx2 to serine, and prepares Trx2C34S mutant protein and Prx5C47S mutant protein respectively; then, the disulfide bond exchange between Prx5 and Trx2 proteins is accelerated by 5,5'-disulfide bis(2-nitrobenzoic acid), i.e., DTNB disulfide bond cross-linking technology, thereby preparing a large number of disulfide bond-linked Prx5-Trx2 protein complexes, providing a basis for studying the structural changes of Prx5 during the catalytic cycle and the development of new anti-tumor resistance drugs targeting the Prx5-Trx2 protein complex.

[0023] (2) The method for purifying and preparing the Prx5-Trx2 protein complex provided by the present invention has the characteristics of simple operation, reliable technology, low cost, and rapid purification. The obtained Prx5-Trx2 protein complex has high purity and can be applied to subsequent protein complex structure analysis and drug discovery. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0025] Figure 1This is a design strategy diagram for forming a disulfide bond between Prx5 and Trx2 based on DTNB cross-linking in the present invention.

[0026] Figure 2 Five groups of reaction result graphs are set for the present invention; among them, 1 is the Trx2C34S+DTT swimming lane; 2 is the Prx5C151S-His-TNB+DTT swimming lane; 3 is the Prx5C47S-His-TNB+DTT swimming lane; 4 is the Trx2C34S+Prx5C151S-His-TNB+DTT swimming lane; 5 is the Trx2C34S+Prx5C47S-His-TNB+DTT swimming lane; M is the Marker swimming lane; 6 is the Trx2C34S+DTT swimming lane; 7 is Prx5C151S-His-TNB; 8 is Prx5C47S-His-TNB; 9 is Trx2C34S+Prx5C151S-His-TNB; 10 is Trx2C34S+Prx5C47S-His-TNB.

[0027] Figure 3 The molecular sieve chromatography column 16 / 600 Superdex75 is used for purification and solution polymerization state analysis of the Prx5C47S-Trx2C34S protein complex of the present invention.

[0028] Figure 3 In the figure, A is the purification diagram of molecular sieve chromatography column 16 / 600 Superdex 75; B is the SDS-PAGE diagram of the corresponding molecular sieve chromatography peak tube, and DTT with a final concentration of 5mM was added before loading; C is a fitting diagram for calculating the molecular weight of the target protein based on the molecular sieve peak volume of the target protein using molecular weight standards; black dots represent molecular weight standards; the peak volume corresponding to the red dots is 56.96ml, representing the Prx5C47S-Trx2C34S complex; the peak volume corresponding to the blue dots is 81.65ml, representing the remaining Trx2C34S after the cross-linking reaction is completed. DETAILED DESCRIPTION

[0029] In order to facilitate the understanding of the present invention, the present invention will be described more comprehensively below, and preferred embodiments of the present invention are provided. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0030] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used in the specification of the present invention herein are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0031] The inventive concept of the present invention is as follows: During the Prx5-catalyzed substrate peroxide decomposition cycle, Trx2 can reduce the inactive oxidized Prx5 to the catalytically competent reduced Prx5 through a disulfide exchange reaction, thereby entering the next round of catalytic cycle. Therefore, capturing the Prx5-Trx2 protein complex in a covalently linked state through a disulfide bond is a key step in the Prx5 catalytic cycle.

[0032] Based on this, the present invention provides a method for preparing a Prx5-Trx2 protein complex, characterized in that it comprises the following steps: The cysteine ​​residue C47 in Prx5 is mutated to serine to obtain a Prx5C47S mutant, and a Prx5C47S mutant protein is prepared; the nucleotide sequence of the Prx5C47S mutant is shown in SEQ ID NO.1.

[0033] The cysteine ​​residue C34 in Trx2 is mutated to serine to obtain a Trx2C34S mutant; the Trx2C34S mutant protein is prepared; the nucleotide sequence of the Trx2C34S mutant is shown in SEQ ID NO.3.

[0034] 5,5'-dithiobis(2-nitrobenzoic acid) was subjected to disulfide bond cross-linking reaction with the Prx5C47S mutant protein to obtain Prx5C47S-TNB.

[0035] The Prx5C47S-TNB and the Trx2C34S mutant protein are subjected to a disulfide cross-linking reaction to obtain a reaction product, thereby obtaining a Prx5-Trx2 protein complex.

[0036] The present invention is the first to use DTNB to carry out disulfide cross-linking reaction with thiol-containing Prx5 and Trx2, and to clarify that C47 and C151 are the corresponding catalytically active cysteine ​​residues C of Prx5. P and C R , identifying cysteine ​​that prefers to form an intermolecular disulfide bond with Trx2, thereby forming an intermediate form of a Prx5-Trx2 complex connected by an intermolecular disulfide bond during the catalytic regeneration of Prx5. The preparation method can be used to prepare a large number of stable Prx5-Trx2 protein complexes, overcoming the defects of the prior art, and the present invention has the characteristics of simple operation, reliable technology, low cost, rapid purification, etc. The obtained Prx5-Trx2 protein complex has a high purity and can be applied to subsequent protein complex structure analysis and drug discovery.

[0037] The technical solution of the present invention will be further described below in conjunction with specific embodiments.

[0038] Example 1: A method for preparing a Prx5-Trx2 protein complex The cysteine ​​of the cysteine ​​residue C47 in Prx5 is mutated to serine to obtain a Prx5C47S mutant; the nucleotide sequence of the Prx5C47S mutant is cloned into a pET28a vector to obtain a Prx5C47S recombinant plasmid; the Prx5C47S recombinant plasmid is transformed into Escherichia coli Rosetta DE3 competent cells for culture, the cells are collected, the cells are resuspended and lysed, and the cells are frozen and centrifuged to obtain a Prx5C47S mutant protein; the nucleotide sequence of the Prx5C47S mutant is shown in SEQ ID NO.1.

[0039] The cysteine ​​of the cysteine ​​residue C34 in Trx2 is mutated to serine to obtain a Trx2C34S mutant; the nucleotide sequence of the Trx2C34S mutant is cloned into a pET28a vector to obtain a Trx2C34S recombinant plasmid; the Trx2C34S recombinant plasmid is transformed into Escherichia coli Rosetta DE3 competent cells for culture, the cells are collected, the cells are resuspended and lysed, and frozen centrifuged to obtain a Trx2C34S mutant protein; the nucleotide sequence of the Trx2C34S mutant is shown in SEQ ID NO.3.

[0040] Using 5,5'-dithiobis(2-nitrobenzoic acid), i.e., DTNB disulfide bond cross-linking technology, 5,5'-dithiobis(2-nitrobenzoic acid) and Prx5C47S mutant protein were subjected to disulfide bond cross-linking reaction at a molar ratio of 10:1 to obtain Prx5C47S-TNB.

[0041] Prx5C47S-TNB and Trx2C34S were subjected to disulfide cross-linking reaction at a molar ratio of 1:1.5 to obtain a reaction product, thereby obtaining a Prx5-Trx2 protein complex.

[0042] Example 2: A method for preparing a Prx5-Trx2 protein complex The cysteine ​​of the cysteine ​​residue C47 in Prx5 is mutated to serine to obtain a Prx5C47S mutant; the nucleotide sequence of the Prx5C47S mutant is cloned into a pET28a vector to obtain a Prx5C47S recombinant plasmid; the Prx5C47S recombinant plasmid is transformed into Escherichia coli Rosetta DE3 competent cells for culture, the cells are collected, the cells are resuspended and lysed, and the cells are frozen and centrifuged to obtain a Prx5C47S mutant protein; the nucleotide sequence of the Prx5C47S mutant is shown in SEQ ID NO.1.

[0043] The cysteine ​​of the cysteine ​​residue C34 in Trx2 is mutated to serine to obtain a Trx2C34S mutant; the nucleotide sequence of the Trx2C34S mutant is cloned into a pET28a vector to obtain a Trx2C34S recombinant plasmid; the Trx2C34S recombinant plasmid is transformed into Escherichia coli Rosetta DE3 competent cells for culture, the cells are collected, the cells are resuspended and lysed, and frozen centrifuged to obtain a Trx2C34S mutant protein; the nucleotide sequence of the Trx2C34S mutant is shown in SEQ ID NO.3.

[0044] 5,5'-dithiobis(2-nitrobenzoic acid), i.e., DTNB disulfide bond cross-linking technology was used to react 5,5'-dithiobis(2-nitrobenzoic acid) with the Prx5C47S mutant protein at a molar ratio of 20:1 to obtain Prx5C47S-TNB.

[0045] Prx5C47S-TNB and Trx2C34S were subjected to disulfide cross-linking reaction at a molar ratio of 1:2 to obtain a reaction product, namely, a Prx5-Trx2 protein complex.

[0046] The Prx5-Trx2 protein complex obtained in Example 1 and Example 2 was incubated in a 30°C water bath for 30 minutes, then loaded onto a nickel affinity chromatography column, washed with buffer H, and eluted with buffer I; a molecular sieve chromatography column Superdex75 with a gel inner diameter of 16 mm and a column height of 600 mm was used, and buffer E was used for elution to further obtain a purified Prx5-Trx2 protein complex. The components of the buffer H are 20 mM tris hydrochloride, 150 mM sodium chloride, and 50 mM imidazole; the components of the buffer I are 20 mM tris hydrochloride, 150 mM sodium chloride, and 250 mM imidazole; the components of the buffer E are 20 mM tris hydrochloride and 150 mM sodium chloride.

[0047] The purification diagram of molecular sieve chromatography column is as follows Figure 3 As shown, a Prx5-Trx2 protein complex with a more symmetrical peak shape and better condition was obtained.

[0048] The protein sample corresponding to the peak tube was taken and DTT was added at a final concentration of 5 mM for SDS-PAGE analysis. The results showed that the Prx5C47S-Trx2C34S protein had a high purity and a molar ratio of 1:1. Figure 3As shown in B. According to the molecular sieve elution peak volume of the complex of 56.96 ml, the molecular mass was calculated to be 59.1 kDa, which is close to the theoretical molecular mass of the Prx5C47S-Trx2C34S heterotetramer complex of 60.6 kDa. Figure 3 As shown in C. This indicates that the present application successfully prepared the Prx5-Trx2 protein complex, and the obtained Prx5-Trx2 protein complex has a high purity, which can be applied to the subsequent structural analysis of the protein complex and drug discovery.

[0049] It should be noted that the components of the buffer H are 15mM~25mM tris hydrochloride, 140mM~160mM sodium chloride and 40mM~60mM imidazole; the components of the buffer I are 15mM~25mM tris hydrochloride, 140mM~160mM sodium chloride and 240mM~260mM imidazole; the components of the buffer E are 15mM~25mM tris hydrochloride and 140mM~160mM sodium chloride. Within the composition range of the above buffers, the Prx5-Trx2 protein complex can be purified.

[0050] The screening and identification experiments of the cysteine ​​required for the formation of disulfide bonds between Prx5 and Trx2 are as follows: 4. Mutation of amino acid residues of Prx5 and Trx2 The Prx5 protein sequence contains three cysteine ​​residues, namely C47, C72 and C151. Among them, C47 and C151 are the corresponding catalytically active cysteine ​​residues C P and C R In order to identify the cysteine ​​required for the disulfide bond formation between Prx5 and Trx2, the present invention designed two Prx5 mutants, namely Prx5C151S and Prx5C47S. Since C72 is a non-surface exposed residue and does not participate in the formation of disulfide bonds, it does not need to be mutated. The Trx2 protein sequence contains two cysteine ​​residues, namely C31 and C34. Among them, C31 is the one that attacks C in Prx. P -C R C34 is a cysteine ​​that forms an intramolecular disulfide bond with C31. Therefore, the present invention designs a Trx2C34S mutant to obtain a stable disulfide bond-connected Prx5-Trx2 complex.

[0051] 5. Construction of Prx5 and Trx2 mutant proteins The coding DNA sequences of mature Prx5 mutants Prx5C151S and Prx5C47S without the mitochondrial targeting sequence were cloned into the pET28a vector as shown in Table 1, and the final expressed recombinant protein sequences are shown in Table 2. In order to facilitate subsequent experiments, the present invention relates to two types of recombinant Prx5 mutant proteins, namely, Prx5 mutant proteins with a histidine tag His-tag at the N-terminus and Prx5 mutant proteins after thrombin cleavage of the His-tag.

[0052] The coding DNA sequence of the mature Trx2 mutant Trx2C34S without the mitochondrial targeting sequence, as shown in Table 1, was cloned into the pET28a vector, so that the N-terminus of the recombinant protein had a shorter His-tag without a restriction site, as shown in Table 2.

[0053] Table 1 DNA coding sequences of Prx5 and Trx2 mutants

[0054] Table 2 Recombinant protein sequences of Prx5 and Trx2 mutants

[0055] Note: The protein sequence of the vector in the recombinant protein is underlined.

[0056] 2. Prokaryotic expression of Prx5 and Trx2 mutant proteins The Prx5 and Trx2 recombinant plasmids were transformed into E. coli Rosetta DE3 competent cells using the 42°C heat shock method, and LB agar plates containing 50µg / ml kanamycin and 34µg / ml chloramphenicol were spread on them and cultured overnight in a 37°C incubator. Single clones were picked and cultured overnight in 5ml LB liquid medium containing kanamycin and chloramphenicol. The next day, 400ml LB liquid medium containing kanamycin and chloramphenicol was inoculated at a ratio of 1:500 and cultured in a 37°C shaker. When the bacterial solution OD 600 When the nm was 0.6-1.0, add isopropyl-β-D-thiogalactoside (IPTG) at a final concentration of 0.2 mM to induce overexpression of the target protein, and continue shaking culture at 37°C for 4 h. Use a low-temperature centrifuge to centrifuge at 4000g for 20 min to collect the bacteria.

[0057] 3. Purification of Prx5 and Trx2 mutant proteins (1) Protein purification of Prx5C47S-His and Prx5C151S-His Resuspend the cells in 50 ml buffer A and lyse the cells by ultrasonic disruption. The ultrasonic power used is 260w, the working time is 4s, the pause time is 10s, and the total time is 30min. Centrifuge at 24446g for 40min using a high-speed refrigerated centrifuge to collect the supernatant containing the target protein. The Prx5 mutant protein has a His-tag at the N-terminus, and the target protein can be purified using nickel affinity chromatography. After the protein is loaded on the nickel affinity chromatography column, wash 10 column volumes with buffer B to remove impurities, and then elute the target protein with buffer C. Use a HiPrep26 / 10 desalting column and exchange the buffer with buffer D to remove imidazole.

[0058] (2) Protein purification of Prx5C47S and Prx5C151S Prx5C47S-His and Prx5C151S-His with His-tag were purified by the above nickel affinity chromatography column to obtain the target protein, and then passed through the HiPrep26 / 10 desalting column, and the buffer was exchanged with buffer E to remove imidazole. The His-tag of the target protein was further removed by thrombin at 18°C ​​overnight. The next day, the molecular sieve chromatography column Superdex75 with a gel inner diameter of 16mm and a column height of 600mm was used to further purify the target protein using buffer E. Analysis found that Prx5 mainly existed in the form of dimers in the solution.

[0059] (3) Protein purification of Trx2C34S The method of ultrasonic disruption to lyse Trx2C34S bacteria is the same as the above method. The N-terminus of Trx2C34S protein has a His-tag without a restriction site, and the target protein can be purified by nickel affinity chromatography. After the protein is loaded on the nickel affinity chromatography column, 10 column volumes are washed with buffer F to remove impurities, and then the target protein is eluted with buffer G. Pass through the HiPrep26 / 10 desalting column and exchange the buffer with buffer D to remove imidazole.

[0060] The Trx2C34S protein used to prepare the Prx5C47S-Trx2C34S complex needs to be further purified using a molecular sieve chromatography column Superdex 75 with a gel inner diameter of 16 mm and a column height of 600 mm, and buffer E is used to obtain the target protein.

[0061] 4. Identification of the required cysteines for disulfide bond formation between Prx5 and Trx2 based on DTNB cross-linking 5,5'-dithiobis(2-nitrobenzoic acid), referred to as DTNB, can react rapidly with compounds containing free sulfhydryl groups and mediate the formation of intermolecular disulfide bonds between two proteins. We used DTNB to perform disulfide cross-linking reactions with sulfhydryl-containing Prx5 and Trx2 to identify which active cysteine ​​in Prx5, C P or C R It prefers to form an intermolecular disulfide bond with Trx2, thereby forming an intermediate form of the Prx5-Trx2 complex connected by an intermolecular disulfide bond during the Prx5 catalytic regeneration process, such as Figure 1 shown.

[0062] C47 and C151 are the catalytically active cysteine ​​residues of Prx5 P and C R. In order to identify which active cysteine ​​in Prx5, C47 or C151 prefers to form an intermolecular disulfide bond with Trx2. In order to quickly purify the protein, the present invention uses two mutants, Prx5C47S-His and Prx5C151S-His, with a His-tag for subsequent experiments. 5mM dithiothreitol, i.e., DTT, was added to the above-purified Prx5C47S-His, Prx5C151S-His and Trx2C34S protein solutions, respectively, and incubated on ice for 30 minutes to reduce the cysteine ​​exposed on the protein surface. Then, the HiPrep26 / 10 desalting column was used to exchange the buffer with buffer D to remove excess DTT. The Prx5C47S-His and Prx5C151S-His protein solutions were mixed with 20-fold excess DTNB, respectively, and incubated in a 25°C water bath for 30 min to generate disulfide-linked Prx5C47S-His-TNB and Prx5C151S-His-TNB. The excess DTNB was removed by exchanging the buffer with buffer D through a HiPrep26 / 10 desalting column, and then five groups of experiments were set up for reaction, namely: Trx2C34S, Prx5C151S-His-TNB, Prx5C47S-His-TNB, Trx2C34S+Prx5C151S-His-TNB, and Trx2C34S+Prx5C47S-His-TNB. Among them, Trx2C34S: Prx5C151S-His-TNB = 1:1 in the Trx2C34S+Prx5C151S-His-TNB group; Trx2C34S: Prx5C47S-His-TNB = 1:1 in the Trx2C34S+Prx5C47S-His-TNB group. They were placed in a 25°C water bath and incubated for 30 minutes for reaction. The final concentration of each protein was 35µM. The five groups of samples were placed in a 25°C water bath and incubated for 30 minutes for reaction, and then 5mM DTT was added and no DTT was added for control, and finally 10 samples were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, i.e. SDS-PAGE.

[0063] The results are as follows Figure 2As shown in the figure, after mixing Prx5C47S-His and Trx2C34S, a disulfide-bonded Prx5C47S-His-Trx2C34S was formed. The theoretical molecular weight of this complex is 32.2 kDa, which is consistent with the band size detected by SDS-PAGE. At the same time, this complex can be reduced by DTT, indicating that Prx5 prefers to use C151 to form an intermolecular disulfide bond with C31 of Trx2. However, after mixing Prx5C151S-His with Trx2C34S, there is no complex band at the corresponding position, indicating that C47 of Prx5 and C31 of Trx2 do not tend to form an intermolecular disulfide bond, as shown in the figure. Figure 2 shown.

[0064] The buffer compositions used above are: Buffer A: 20 mM Tris-HCl, 250 mM NaCl, 14 mM β-mercaptoethanol, and 1.5% glycerol at pH 8.5.

[0065] Buffer B: 20 ​​mM Tris-HCl, 250 mM NaCl, 14 mM β-mercaptoethanol, and 50 mM imidazole, pH 8.5.

[0066] Buffer C: 20 mM Tris-HCl, 250 mM sodium chloride, 14 mM β-mercaptoethanol, and 250 mM imidazole at pH 8.5.

[0067] Buffer D: 20 mM Tris-HCl and 150 mM NaCl at pH 7.5.

[0068] Buffer E: 20 mM Tris-HCl and 150 mM NaCl, pH 8.0.

[0069] Buffer F: 20 mM Tris-HCl, 250 mM NaCl, 14 mM β-mercaptoethanol, and 50 mM imidazole, pH 7.5.

[0070] Buffer G: 20 mM Tris-HCl, 250 mM NaCl, 14 mM β-mercaptoethanol, and 250 mM imidazole at pH 7.5.

[0071] Buffer H: 20 mM Tris-HCl, pH 8.0, 150 mM NaCl, and 50 mM imidazole.

[0072] Buffer I: 20 mM Tris-HCl, pH 8.0, 150 mM NaCl, and 250 mM imidazole.

[0073] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0074] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A method for preparing a Prx5-Trx2 protein complex, characterized in that: The following steps are involved: The cysteine ​​residue C47 in Prx5 is mutated to serine to obtain a Prx5C47S mutant, and a Prx5C47S mutant protein is prepared; the nucleotide sequence of the Prx5C47S mutant is shown in SEQ ID NO.1; The cysteine ​​residue C34 in Trx2 is mutated to serine to obtain a Trx2C34S mutant; a Trx2C34S mutant protein is prepared; the nucleotide sequence of the Trx2C34S mutant is shown in SEQ ID NO.3; 5,5'-dithiobis(2-nitrobenzoic acid) was subjected to disulfide cross-linking reaction with the Prx5C47S mutant protein to obtain Prx5C47S-TNB; The Prx5C47S-TNB and the Trx2C34S mutant protein are subjected to a disulfide cross-linking reaction to obtain a reaction product, thereby obtaining a Prx5-Trx2 protein complex.

2. The method for preparing the Prx5-Trx2 protein complex according to claim 1, characterized in that: The 5,5'-dithiobis(2-nitrobenzoic acid) and the Prx5C47S mutant protein are mixed at a molar ratio of 10-20:1 to perform a disulfide bond cross-linking reaction.

3. The method for preparing the Prx5-Trx2 protein complex according to claim 1, characterized in that: The molar ratio of the Prx5C47S-TNB to the Trx2C34S mutant protein is 1:1.5-1:

2.

4. The method for preparing the Prx5-Trx2 protein complex according to claim 1, characterized in that: After the reaction product is purified, a purified Prx5-Trx2 protein complex is obtained, and the purification is as follows: (1) Incubate the reaction product in a water bath at 25°C to 30°C for 25 min to 30 min; (2) The sample is then loaded onto a nickel affinity chromatography column, washed, and the target protein is eluted; (3) Further purification using a molecular sieve chromatography column to obtain a purified Prx5-Trx2 protein complex.

5. The method for preparing the Prx5-Trx2 protein complex according to claim 4, characterized in that: Buffer H is used for washing, wherein the components of the buffer H are 15 mM to 25 mM tris(hydroxymethyl)aminomethane hydrochloride, 140 mM to 160 mM sodium chloride and 40 mM to 60 mM imidazole.

6. The method for preparing the Prx5-Trx2 protein complex according to claim 4, characterized in that: Buffer I was used for elution, wherein the components of the buffer I were 15 mM to 25 mM tris(hydroxymethyl)aminomethane hydrochloride, 140 mM to 160 mM sodium chloride and 240 mM to 260 mM imidazole.

7. The method for preparing the Prx5-Trx2 protein complex according to claim 4, characterized in that: The molecular sieve chromatography column is further purified using buffer E, wherein the components of buffer E are 15 mM to 25 mM tris(hydroxymethyl)aminomethane hydrochloride and 140 mM to 160 mM sodium chloride.

8. The Prx5-Trx2 protein complex prepared by the method for preparing the Prx5-Trx2 protein complex according to any one of claims 1 to 7.

9. Use of the Prx5-Trx2 protein complex according to claim 8 in analyzing the three-dimensional structure of the Prx5-Trx2 protein complex.

10. Use of the Prx5-Trx2 protein complex according to claim 8 in developing new anti-tumor drug resistance drugs targeting the Prx5-Trx2 protein complex target.

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