Expression and purification method of high-purity IRGQ protein

Abstract

The invention discloses an expression and purification method of a high-purity IRGQ protein. According to the method, protein is primarily purified through Ni affinity chromatography, and most of impure protein is removed. And further purification is carried out through anion exchange chromatography, nucleic acid can be removed, and influence on subsequent experiments caused by pollution of the nucleic acid to protein is reduced. Further purification is carried out through size exclusion chromatography, the sample uniformity can be detected, and a basis is provided for subsequent crystallization experiments. The protein polymerization state and the protein interaction condition can be observed through size exclusion chromatography, and a basis is provided for related functional experiment research.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of biotechnology, and relates to the field of protein expression and purification, in particular to an expression and purification method of high-purity IRGQ protein. BACKGROUND

[0002] Generally, the expression and purification of exogenous proteins is achieved by constructing a recombinant plasmid with a specific affinity tag through genetic engineering, and then inducing expression in a suitable host system. Subsequently, the protein crude extract is obtained by cell lysis and centrifugation, and then the target protein is specifically captured by affinity chromatography to achieve preliminary purification. Thereafter, tag removal is often required, and impurities are removed by ion exchange chromatography, hydrophobic interaction chromatography and other intermediate purification steps, followed by size exclusion chromatography for refining and buffer replacement. Finally, high-purity and high-activity target protein samples are obtained through ultrafiltration concentration, quality verification and storage.

[0003] Human IRGQ protein (immunorelated GTPase family Q, alias IRGQ1, FKSG27) is an immunorelated GTPase that plays a key role in cellular autophagy. It is involved in the defense response to intracellular pathogens and plays an important role in host defense mechanisms by regulating cellular processes such as autophagy. Due to its important biological function, IRGQ protein has become a key research object in the fields of host-pathogen interaction, autoimmune diseases and cancer immunity. In order to further carry out structural and functional research, as well as related drug screening and biological agent development, it is crucial to obtain high-purity, high-activity and structurally intact IRGQ protein. IRGQ protein can bind to GABARAPL2 (GL2), which belongs to the ATG8 family of autophagy regulators in mammals and is essential for autophagosome formation, cargo recruitment and autophagosome-lysosome fusion. The interaction between human IRGQ protein and GABARAPL2 protein is a key link in the cellular autophagy mechanism. Studies have shown that the interaction between IRGQ and GABARAPL2 depends on specific domains in the IRGQ protein. For example, the N-terminal domain (residues 1-184) and a motif called LIR1 (residues 185-190) of IRGQ are essential for binding to GABARAPL2.

[0004] It is known that human IRGQ protein can be induced to express in the prokaryotic expression system Escherichia coli by constructing a recombinant plasmid with a GST tag. Subsequently, the protein crude extract is obtained by cell lysis and centrifugation, and then the target protein is specifically captured by GST affinity chromatography to achieve preliminary purification.

[0005] Due to the important biological function of human IRGQ protein, its original expression and purification method cannot meet the sample requirements for related structural and functional research. SUMMARY

[0006] The present application aims at the above-mentioned problems, and provides an expression and purification method of high-purity IRGQ protein.

[0007] In order to achieve the purpose, the technical scheme adopted by the present application is as follows:

[0008] The present application provides an expression and purification method of high-purity IRGQ protein, comprising the following steps:

[0009] I. Construction of expression vector of fusion protein

[0010] The IRGQ gene is connected into the skeleton vector pETDuet-1 to obtain the pETDuet-1-IRGQ recombinant plasmid for expressing the fusion protein 6×His-IRGQ;

[0011] II. Transformation of recombinant plasmid into E. coli and induction of expression

[0012] The pETDuet-1-IRGQ recombinant plasmid containing the coding sequence of the fusion protein is transformed into E. coli to induce the expression of the 6×His-IRGQ fusion protein, and the E. coli bacteria are collected after the induction of expression;

[0013] III. Resuspension and cell disruption

[0014] The E. coli bacteria obtained in step II are resuspended using a resuspension buffer, the bacterial suspension is subjected to bacterial lysis using a low-temperature ultrahigh-pressure cell disrupter, and the cell lysis supernatant is collected after centrifugation of the lysis solution, thereby obtaining a crude protein extract;

[0015] IV. Protein purification

[0016] The cell lysis supernatant obtained in step III is subjected to protein chromatography purification, comprising the following steps:

[0017] S1. Ni column affinity chromatography

[0018] The cell lysis supernatant is loaded onto the Ni column, and is first incubated with the Ni-NTA filler, and the mixed solution after incubation is subjected to washing and elution on the Ni column, and the eluted sample is collected;

[0019] S2. Q column affinity chromatography

[0020] The protein purification system is connected with the anion exchange chromatography Q column, the Q column is equilibrated using anion exchange purification Buffer A and Buffer B, the eluted sample obtained in step S1 is loaded after adding the anion exchange purification Buffer A, and the target protein is eluted, and the sample at the elution peak is collected;

[0021] S3. Size exclusion chromatography

[0022] The sample from the elution peak obtained in step S2 is subjected to size exclusion chromatography; the protein purification system is connected to a size exclusion column, the size exclusion column is equilibrated, the sample from the elution peak obtained in step S2 is balanced and concentrated and then loaded onto the column, and the molecular sieve elution program is executed. The collected sample from the elution peak is the purified IRGQ protein.

[0023] In step one, using the DNA fragment shown in SEQ ID NO.3 as a template, the IRGQ gene fragment was obtained by PCR amplification using the primers shown in SEQ ID NO.1 and SEQ ID NO.2. The amplification product and the pETDuet-1 empty vector were digested with restriction endonucleases BamHI and XhoI, respectively, and then ligated into competent E. coli cells. Positive clones were screened, which are the pETDuet-1-IRGQ recombinant plasmids.

[0024] The amino acid sequence of the IRGQ protein is shown in SEQ ID NO.4.

[0025] The washing buffer used in step S1 consists of: 20 mM Tris-HCl, 20 mM imidazole, 100 mM NaCl, 10% (m / V) glycerol, pH 8.0; the elution buffer used in step S1 consists of: 20 mM Tris-HCl, 500 mM imidazole, 100 mM NaCl, 10% (m / V) glycerol, pH 8.0.

[0026] In step S2, the anion exchange purification buffer A component consists of 20 mM Tris-HCl, 10% (m / V) glycerol, and pH 8.0; the anion exchange purification buffer B component consists of 20 mM Tris-HCl, 1 M NaCl, 10% (m / V) glycerol, and pH 8.0.

[0027] In step S3, the size exclusion column is a Superdex™ 200Increase 10 / 300GL, and the gel filtration chromatography purification buffer consists of 50mM Tris-HCl, 300mM NaCl, and pH 7.8.

[0028] This invention also provides a method for studying the interaction between IRGQ and GL2 proteins. IRGQ and GL2 proteins are mixed and co-incubated, then centrifuged to obtain the supernatant, which is injected into a pre-equilibrated size exclusion column of a protein purification system. A molecular sieve elution program is executed, and the protein polymerization state and protein interaction are observed through size exclusion chromatography. The sample obtained from the elution peak is used for SDS-PAGE analysis to verify the interaction between IRGQ and GL2 proteins.

[0029] The above-mentioned method for studying the interaction between IRGQ and GL2 proteins, wherein the IRGQ protein is obtained by any of the above-mentioned methods.

[0030] The present application has the following advantages compared with the conventional IRGQ purification method:

[0031] (1) Different recombinant plasmid vectors: compared with the conventional IRGQ purification method using a GST-tagged pGEX6P1 vector, the recombinant plasmid used in the present application is a His-tagged pETDuet1 vector. Since the His tag is smaller, it has less impact on protein crystallization experiments, and the crystallization experiment can be directly performed, thereby eliminating the step of removing the tag by enzyme digestion, avoiding protein loss or poor enzyme digestion effect that affects the purity of the protein, and being more conducive to the structural study of IRGQ. At the same time, since the His tag is smaller, it has less impact on the size exclusion chromatography method for detecting protein-protein interaction experiments, and the size exclusion chromatography method can be directly used to detect protein-protein interaction.

[0032] (2) Different purification steps: compared with the conventional IRGQ purification method which only uses GST affinity chromatography for IRGQ purification, the present application combines three different purification methods, namely Ni affinity chromatography-anion exchange chromatography-size exclusion chromatography. The principle of Ni affinity chromatography is that nickel ions have strong affinity with histidine residues that make up the His tag. This affinity allows the target protein with His tag to specifically bind to the nickel ions on the affinity chromatography column filler when it comes into contact with the nickel ions, and proteins without His tag will not bind to the nickel ion column, thereby achieving the purification of the target protein. The principle of ion exchange chromatography is to use the different charge properties between the target protein and other proteins to further separate and purify the target protein, thereby improving the purity. Specifically, proteins will be adsorbed on anion / cation exchange resin, while other components will naturally flow through, and then the solution ion concentration will be changed to compete with the charge of the protein, thereby eluting the proteins adsorbed on the anion / cation exchange resin, achieving the purification of the proteins. The principle of gel filtration chromatography is to separate proteins by their molecular size. By selecting the appropriate pore size of the molecular sieve filler, the target protein and other impurities can be effectively distinguished to further improve the purity of the target protein.

[0033] The method of the present application preliminarily purifies the protein by Ni affinity chromatography to remove most of the impurities. The protein is further purified by anion exchange chromatography, and nucleic acid can be removed to reduce the contamination of the protein to subsequent experiments. The protein is further purified by size exclusion chromatography, and the homogeneity of the sample can be detected to provide a basis for subsequent crystallization experiments. The size exclusion chromatography can also be used to observe the polymerization state of the protein and the protein interaction, which provides a basis for related functional experimental research. BRIEF DESCRIPTION OF DRAWINGS

[0034] Figure 1 Figure 4 is the SDS-PAGE analysis result of the sample obtained in the purification step of Example 1.

[0035] Figure 2 Figure 5 is the UV280 (blue curve) and Cond profile (red curve) of the IRGQ protein obtained by anion exchange chromatography.

[0036] Figure 3 Figure 6 is the size exclusion chromatography result (A) and SDS-PAGE analysis result (B) of the IRGQ protein.

[0037] Figure 4 Figure 7 is the size exclusion chromatography result (A) and SDS-PAGE analysis result (B) of the IRGQ protein co-incubated with GL2 protein.

[0038] Figure 5 Figure 8 is the size exclusion chromatography result (A) and SDS-PAGE analysis result (B) of the pETDuet-1-GL2 protein. DETAILED DESCRIPTION

[0039] The present application will be further described below in conjunction with examples, but the present application is not limited by the examples.

[0040] The experimental methods in the following examples are conventional methods, unless otherwise specified.

[0041] Main reagent sources:

[0042] E. coli expression vector pETDuet-1 vector: The vector with N-terminal 6xHis tag (N-His) was purchased from Hunan Fenghui Biological Company. BL21(DE3) competent cells: purchased from Shanghai Weidi Biological Company. Inducer IPTG, protein Marker, conventional solid reagents, etc. were purchased from Shengong Biological. The glue solution was purchased from Boaolong Biological Company. PMSF protease inhibitor, restriction enzymes, ligase, etc. were purchased from Thermo Company. 50K specification protein concentration tube (Amicon Ultra-15 Centrifugal Filters, Millipore) was purchased from Merck Company. Gold Mix, primer synthesis, plasmid extraction kit were purchased from Gengke Biological Company. Nickel column affinity chromatography filler was purchased from GE Company Ni SepharoseTM 6 Fast Flow.

[0043] Example 1 Protein expression and purification method of the application

[0044] I. Construction of fusion protein expression vector

[0045] Firstly, using SnapGene software, the upstream and downstream primers containing BamHI and XhoI restriction sites were designed and synthesized for the full-length human IRGQ gene, and the IRGQ gene fragment was obtained by PCR amplification. The PCR amplification primers are as follows: upstream primer (SEQ ID NO. 1): 5'-CGGGATCCGATGCCACCGCCGCAAGGTGACG-3', downstream primer (SEQ ID NO. 2): 5'-CCGCTCGAGTTACTGAGCCGGTTCCGGCGGA-3'. The template DNA fragment for PCR amplification was synthesized by a biological company, and the sequence is shown in SEQ ID NO. 3.

[0046] Subsequently, the amplification product and pETDuet-1 empty vector were digested with restriction enzymes BamHI and XhoI, respectively, and gel electrophoresis was performed for recovery. The linearized vector and the target fragment were mixed at a molar ratio of 1:5, and T4 DNA ligase was used for ligation at 22°C for 30 min. The ligation product was transformed into BL21(DE3) competent cells and plated on LB plates containing the corresponding antibiotic and incubated overnight. The next day, a single colony was picked for amplification, and the plasmid was extracted and sent to Gengke Biological Company for sequencing verification. Finally, the correct sequence of the pETDuet-1-IRGQ recombinant plasmid was obtained, which was used for subsequent protein expression and purification experiments.

[0047] The pETDuet-1-IRGQ recombinant plasmid contains a fusion protein "6xHis-IRGQ" with a molecular weight of about 63.5KDa. The IRGQ amino acid sequence is as follows (SEQ ID NO. 4):

[0048] MPPPQGDVTALFLGPPGLGKSALIAALCDKDVETLEAPEGRPDSGVPSLRAAGPGLFLGELSCPPAAPGPWAAEANVLVLVLPGPEGNGEPLAPALGEAALAALARGTPLLAVRNLRPGDSQTAAQARDQTAALLNSAGLGAADLFVLPANCGSSDGCEELERLRAALQSQAEALRRLLPPAQDGFEVLGAAELEAVREAFETGGLEAALSWVRSGLERLGSARLDLAVAGKADVGLVVDMLLGLDPGDPGAAPASVPTAPTPFPAPERPNVVLWTVPLGHTGTATTAAAASHPTHYDALILVTPGAPTEKDWAQVQALLLPDAPLVCVRTDGEGEDPECLGEGKMENPKGESLKNAGGGGLENALSKGREKCSAGSQKAGSGEGPGKAGSEGLQQVVGMKKSGGGDSERAAALSPEDETWEVLEEAPPPVFPLRPGGLPGLCEWLRRALPPAQAGALLLALPPASPSAARTKAAALRAGAWRPALLASLAAAAAPLPGLGWACDVALLRGQLAEWRRGLGLEPTALARRERALGLASGELAARAHFPGPVTRAEVEARLGAWAGEGTAGGAALGALSFLWPAGGAAATGGLGYRAAHGVLLQALDEMRADAEAVLAPPEPAQ.

[0049] II. Transform the successfully constructed recombinant plasmid into E. coli competent cells, and induce expression under specific conditions using IPTG

[0050] Take 1 μL of the recombinant plasmid and transform it into E. coli BL21(DE3) competent cells. After plating and culturing, pick a single colony and perform small-scale culture in 5 mL of LB medium containing 100 μg / mL ampicillin at 37°C and 180 rpm for 12-14 hours. Then, transfer 1:100 to 1 L of LB medium containing the same concentration of antibiotics and incubate at 37°C and 220 rpm until the OD600 is 0.6-0.8. Next, reduce the culture temperature to 16°C and incubate with shaking for 40 min. Add 0.5 mM of the inducer IPTG and induce expression at 16°C and 220 rpm for 16-18 hours.

[0051] III. Collect the bacteria, resuspend, and lyse

[0052] The induced bacterial solution was transferred to a centrifuge bottle, and after balancing, it was centrifuged at 4°C and 4000 rpm for 20 minutes. The supernatant was discarded, and the bacterial precipitate was collected and transferred to a pre-cooled beaker and placed on ice. Then, resuspension buffer was added at a ratio of 10 mL per 1 L of LB medium, and a glass rod was used to stir until the bacterial precipitate was completely resuspended without visible clumps. The resuspension buffer used in this example had the following composition: 50 mM Tris-HCl, 20 mM imidazole, 500 mM NaCl, 10% (m / V) glycerol, pH 8.0.

[0053] Cell lysis: The bacterial resuspension was lysed using a pre-cooled ultrahigh pressure cell crusher (4°C, 1500 bar). Before lysis, the instrument pipeline was cleaned with ultrapure water to replace the stored ethanol and the system was equilibrated with the resuspension buffer. After adding 100 mM PMSF stock solution at a ratio of 100 μL per 10 mL of bacterial resuspension, the bacterial solution was subjected to 5 cycles of lysis, and 20 μL of the lysed bacterial solution was taken as the broken bacteria sample for SDS-PAGE analysis. The lysed bacterial solution was transferred to a centrifuge tube, balanced, and centrifuged at 4°C and 20000 rpm for 1 hour. The cell lysis supernatant was collected, which was the crude protein extract, for subsequent purification. 20 μL of the supernatant and precipitate were taken for SDS-PAGE analysis.

[0054] Four, protein purification

[0055] S1, crude separation stage - Ni column affinity chromatography:

[0056] Washing buffer composition: 20 mM Tris-HCl, 20 mM imidazole, 100 mM NaCl, 10% (m / V) glycerol, pH 8.0.

[0057] Elution buffer composition: 20 mM Tris-HCl, 500 mM imidazole, 100 mM NaCl, 10% (m / V) glycerol, pH 8.0.

[0058] S1.1 Column equilibration: 2 mL of Ni-NTA filler was packed into the column, and the column was equilibrated with ultrapure water (3 column volumes) and resuspension buffer in sequence.

[0059] S1.2 Incubation: 70 mL of cell lysis supernatant was divided into 50 mL centrifuge tubes, and 1 mL of cell lysis supernatant was taken and added to the column containing 2 mL of Ni-NTA filler after equilibration in step S1.1. The resuspension was mixed with the Ni-NTA filler in the column, and the resuspension was divided into the 50 mL centrifuge tubes containing the cell lysis supernatant, and placed on a 4°C roller bed for a total of 1 hour of incubation.

[0060] S1.3 Loading: The mixed solution after 1 hour co-incubation was added to the column in batches until all the mixed solution was naturally flowed through, and 20 μL of the flow-through sample was taken for SDS-PAGE analysis.

[0061] S1.4 Washing: 5 mL of the washing buffer was added to the column, and after resuspension, it was left to stand for 2 min, and then naturally flowed through. This operation was repeated three times. 20 μL of the mixed solution after resuspension after the first time of adding the washing buffer was taken, and was recorded as the column-loading sample for SDS-PAGE analysis. 20 μL of the flow-through sample after the second time of adding the washing buffer was taken, and was recorded as the post-washing loading sample for SDS-PAGE analysis.

[0062] S1.5 Elution: 5 mL of the elution buffer was added to the column, and after resuspension, it was left to stand for 2 min, and then naturally flowed through into a clean beaker. This operation was repeated three times, and a total of 15 mL of the eluate was collected for step S2 after the three times of elution. 20 μL of the mixed solution after resuspension after the first time of adding the elution buffer was taken, and was recorded as the post-washing loading sample for SDS-PAGE analysis. 20 μL of the mixed flow-through sample after the three times of elution was taken, and was recorded as the post-elution loading sample for SDS-PAGE analysis. Ultra-pure water was added to the column, and the resuspended mixture was mixed well. 20 μL of the resuspended solution was taken, and was recorded as the post-elution loading sample for SDS-PAGE analysis.

[0063] S1.6 Column regeneration: After the ultra-pure water in the column was naturally flowed through, 5 mL of a 0.1M EDTA solution was added, and was left to stand on a 4°C roller bed for 7 min, and was naturally flowed through. Then, the resuspended mixture was flowed through three times with the addition of ultra-pure water. Then, 5 mL of a 6M guanidine hydrochloride solution was added, and was left to stand on a 4°C roller bed for 7 min, and was naturally flowed through. Then, the resuspended mixture was flowed through three times with the addition of ultra-pure water. Finally, 10 mL of a 0.1M NiSO4 solution was added, and was left to stand on a 4°C roller bed overnight. The next day, the resuspended mixture was flowed through twice with the addition of ultra-pure water, and an appropriate amount of ultra-pure water was added for storage.

[0064] S2, Moderate purification stage-Q column affinity chromatography:

[0065] Anion exchange purification Buffer A component: 20 mM Tris-HCl, 10% (m / V) glycerol, pH 8.0.

[0066] Anion exchange purification Buffer B component: 20 mM Tris-HCl, 1M NaCl, 10% (m / V) glycerol, pH 8.0.

[0067] S2.1 Column equilibration: The anion exchange column (Q column: HiTrap Q FF 5 mL) was connected to the AKTA pure 25 system, and the column was equilibrated with the anion exchange purification Buffer A and Buffer B. TM Q FF 5 mL) was connected to the AKTA pure 25 system, and the column was equilibrated with the anion exchange purification Buffer A and Buffer B.

[0068] S2.2 Sample loading: Add 35 mL of anion exchange purification Buffer A into 15 mL elution sample collected in step S1.5, and perform desalting dilution. After desalting dilution, use a syringe to inject it into the sample loading device 150 mL Superloop of AKTA pure 25 system for affinity separation.

[0069] S2.3 Target protein elution: By setting a linearly increasing salt ion concentration gradient elution program, gradually increase the salt concentration to competitively neutralize the charge interaction between the protein and the ligand, so as to realize the specific elution of the target protein, and the elution flow rate is 2 mL / min, and the sample at the elution peak is collected for size exclusion chromatography. Respectively take 20 μL flow-through and elution peak samples for SDS-PAGE analysis.

[0070] S3, Fine purification stage-size exclusion chromatography:

[0071] S3.1 Column equilibration: Connect the size exclusion chromatography column (Superdex™ 200 Increase 10 / 300 GL, GE) to the AKTA pure 25 system, and use gel filtration purification Buffer to equilibrate the column. The components of the gel filtration purification Buffer are: 50 mM Tris-HCl, 300 mM NaCl, pH 7.8.

[0072] S3.2 Sample loading: Collect the target protein at the elution peak of the Q column affinity chromatography stage into a 50KDa concentration tube, and after equilibration, centrifuge at 4°C, 4000g to concentrate to less than 1 mL. Then transfer the concentrated sample to a 1.5 mL centrifuge tube, and after equilibration, centrifuge at 4°C, 12000 rpm for 10 min. Use a syringe to inject the supernatant sample through the sample loading device 1 mL Loop into the size exclusion chromatography column.

[0073] S3.3 Target protein elution: Perform molecular sieve elution program, elution flow rate is 0.4 mL / min, and the sample at the elution peak collected is the purified IRGQ protein, and 20 μL of the sample at the elution peak is taken for SDS-PAGE analysis.

[0074] Five, SDS-PAGE analysis

[0075] Take the foregoing sampling of broken bacteria, supernatant, precipitate, flow through, hanging column, washing miscellaneous upper sample, washing miscellaneous lower sample, elution upper sample, elution lower sample, add 10 μL 1x Loading Buffer to each sample, add 5 μL 5x Loading Buffer to the broken bacteria sample, denature at 98°C for 10 min, and then centrifuge instantly. Take 3 μL of pre-stained protein two-color Marker and 5 μL of denatured protein sample, add them to the loading slot, and run at 80V until the separation gel, and then run at 120V until the gel bottom. After taking it out, dye, decolorize, and wash with water overnight.

[0076] The results are shown in Figure 1 The SDS-PAGE results of the broken bacteria sample, supernatant sample, and precipitate sample show that the pETDuet-1-IRGQ protein is successfully expressed, but multiple impurity bands appear because it has not been purified. The hanging column sample has impurity bands because part of the impurity proteins are non-specifically combined with the filler without the washing step. Through the washing step, non-specifically combined impurity proteins can be removed. The pETDuet-1-IRGQ protein purified by Ni column affinity chromatography has a single band. Figure 1 The lanes marked as 5, 24, 27, and 33 correspond to the sample tube numbers collected at the flow through, peak start, peak tip, and peak tail of the Q column affinity chromatography, respectively, to ensure that the protein with higher purity in the elution peak can be collected for the next size exclusion chromatography.

[0077] The pETDuet-1-IRGQ UV280 and Cond spectra obtained through the S2.3 step are shown in Figure 2 According to the collection tube number at the absorption peak corresponding to the blue curve in Figure 2 , combined with the conductance value of the red curve (used to assist in judging the salt concentration at which the protein can be eluted), the sample is collected, that is, the IRGQ elution sample of Q column affinity chromatography is obtained, and then concentrated and centrifuged for size exclusion chromatography.

[0078] Six, sample preservation

[0079] Collect the target protein at the elution peak of the size exclusion chromatography stage into a 50KDa concentration tube, and then centrifuge at 4°C and 4000g to concentrate to an appropriate concentration. Then, perform aliquoting, quick-freeze in liquid nitrogen, and store in a -80°C refrigerator.

[0080] The size exclusion chromatography results of the IRGQ protein are shown in Figure 3A shows the elution volume of IRGQ protein on a size exclusion chromatography column Superdex 200 increase 10 / 300 GL column, and the elution peak positions are 12.32 mL and 14.35 mL, respectively, since the molecular weight of IRGQ is 63.5 kDa, it is judged that IRGQ protein can spontaneously dimerize in a natural state. The SDS-PAGE results of the sample at the elution peak obtained after size exclusion chromatography elution Figure 3 B shows that the IRGQ protein purified by size exclusion chromatography has a single band, indicating that the protein has high purity, Figure 3 In B, lanes 6, 7, 8, 10, 11, and 14 represent Figure 3 In A, the start, peak tip, and peak tail of elution peak 1, and the start, peak tip, and peak tail of elution peak 2 correspond to the number of sample tubes on the collection tray, which is used to ensure that the IRGQ protein with high purity in the elution peak can be collected.

[0081] Example 2 verifies the interaction between IRGQ and GL2 protein in vitro

[0082] The GL2 protein obtained after expression and purification of the pETDuet-1-GL2 recombinant plasmid is obtained by the method of Example 1, and the size exclusion chromatography results of the obtained GL2 protein are as shown in Figure 5 A shows the SDS-PAGE analysis results Figure 5 B shows: the blue curve shows the elution volume of the purified GL2 protein on a Superdex 200 increase 10 / 300 GL column, and the elution peak position is 18.98 mL; the SDS-PAGE results show that the pETDuet-1-GL2 protein (MW: 13.6 kDa) purified by size exclusion chromatography has a single band, indicating that the obtained GL2 protein has high purity.

[0083] The IRGQ protein purified by the method of the application and the previously prepared GL2 protein purified by expression and purification of the pETDuet-1-GL2 recombinant plasmid are mixed in a 1:1 molar ratio into a 1.5 mL centrifuge tube, and are incubated on a 4°C roller bed for a total of 2 hours. Then, after equilibration, centrifugation is performed at 4°C and 12000 rpm for 10 min. The supernatant sample is injected into a previously equilibrated size exclusion chromatography column using a syringe, and a molecular sieve elution program is performed. 20 μL of sample is taken from the elution peak, and 5 μL of 5x Loading Buffer is added for sample preparation and gel running. Finally, according to the SDS-PAGE results, the stable complex of the two is collected into a 10KDa concentration tube, and after equilibration, centrifugation is performed at 4°C and 4000g to concentrate to an appropriate concentration. Subsequently, aliquot and store for subsequent co-crystal experiments.

[0084] The experimental results show that the size exclusion chromatography results of pETDuet-l-IRGQ protein after co-incubation with pETDuet-l-GL2 protein are shown in Figure 4 A, Figure 4 The blue curve in A shows the elution volume of pETDuet-l-IRGQ after co-incubation with pETDuet-l-GL2 on a Superdex 200 increase 10 / 300 GL column, with peak positions at 12.59 mL and 15.16 mL, respectively. Compared with the size exclusion chromatography results of pETDuet-l-GL2 (as shown in Figure 5 ), the peak positions of the size exclusion chromatography of pETDuet-l-IRGQ protein after co-incubation with pETDuet-l-GL2 protein are shifted forward, indicating that pETDuet-l-IRGQ protein and pETDuet-l-GL2 protein have interaction and can form stable complexes. The SDS-PAGE results (as shown in Figure 4 B) show that pETDuet-l-IRGQ has interaction with pETDuet-l-GL2. pETDuet-l-IRGQ MW: 63.5 kDa, pETDuet-l-GL2 MW: 13.6 kDa.

[0085] The conventional IRGQ purification method is as follows: the GST-labeled fusion protein is expressed in E. coli strain BL21 (DE3). The bacteria are cultured in LB medium with the addition of 100 mg / mL ampicillin at 37°C in a shaking incubator (150 rpm) to an OD600 of 0.5-0.6. Protein expression is induced by adding 0.5 mM IPTG, and incubation is performed at 16°C for 16 hours. Ultrasonic lysis is performed in GST lysis buffer (20 mM TrisHCl, pH 7.5, 10 mM EDTA, pH 8.0, 5 mM EGTA, 150 mM NaCl, 0.1% b-mercaptoethanol, 1 mm PMSF). The lysate is centrifuged (10000 rpm) to remove the supernatant, 0.05% Triton X-100 is added, and the lysate is incubated with glutathione Sepharose 4B beads (GE Life Sciences) on a 4°C rotary platform for 1 hour. Washing is performed 5 times in GST washing buffer (20 mM TrisHCl, pH 7.5, 10 mM EDTA, pH 8.0, 150 mM NaCl, 0.5% Triton X-100, 0.1% b-mercaptoethanol, 1 mM PMSF). The immobilized protein is reconstituted in GST storage buffer (20 mM TrisHCl, pH 7.5, 0.1% NaN3, 0.1% b-mercaptoethanol), and dialyzed in (25 mM TrisHCl, pH 7.5, 200 mM NaCl) for 16 hours at a temperature of 4°C.

[0086] Compared with the conventional IRGQ purification method, the method of the present application successfully purifies high-purity and high-yield human IRGQ protein with a His tag by using three different purification methods in combination, and does not need to consider the influence of the His tag when performing related functional experiments, reduces the label enzyme cleavage step, and avoids negative effects such as sample loss.

Claims

1. A method for expressing and purifying high-purity IRGQ protein, characterized in that, Includes the following steps: I. Construction of expression vectors for fusion proteins The IRGQ gene was ligated into the backbone vector pETDuet-1 to obtain the pETDuet-1-IRGQ recombinant plasmid expressing the fusion protein 6×His-IRGQ; II. Transformation of E. coli with recombinant plasmid and induction of expression The pETDuet‐1-IRGQ recombinant plasmid containing the fusion protein coding sequence was transformed into Escherichia coli to induce the expression of 6×His‐IRGQ fusion protein. After induction of expression, E. coli cells were collected. III. Resuspension and Cell Disruption The E. coli cells obtained in step 2 were resuspended using resuspension buffer. The bacterial cells were lysed using a low-temperature ultra-high pressure cell disruptor. After centrifugation, the cell lysate supernatant was collected to obtain the crude protein extract. IV. Protein Purification The cell lysate supernatant obtained in step three was purified by protein chromatography, including the following steps: S1, Ni column affinity chromatography: The cell lysis supernatant was loaded onto a Ni column and incubated with Ni-NTA packing material. The incubated mixture was then washed and eluted on the Ni column, and the eluted sample was collected. S2, Q-column affinity chromatography: The protein purification system is connected to an anion exchange chromatography Q column. The Q column is equilibrated with anion exchange purification Buffer A and Buffer B. Anion exchange purification Buffer A is added to the elution sample obtained in step S1 and the sample is loaded to elute the target protein. The sample at the elution peak is collected. S3, Size Exclusion Chromatography The sample from the elution peak obtained in step S2 is subjected to size exclusion chromatography; the protein purification system is connected to a size exclusion column, the size exclusion column is equilibrated, the sample from the elution peak obtained in step S2 is balanced and concentrated and then loaded onto the column, and the molecular sieve elution program is executed. The collected sample from the elution peak is the purified IRGQ protein.

2. The method according to claim 1, characterized in that: In step one, using the DNA fragment shown in SEQ ID NO.3 as a template, the IRGQ gene fragment was obtained by PCR amplification using the primers shown in SEQ ID NO.1 and SEQ ID NO.

2. The amplification product and the pETDuet-1 empty vector were digested with restriction endonucleases BamHI and XhoI, respectively, and then ligated into competent E. coli cells. Positive clones were screened, which are the pETDuet-1-IRGQ recombinant plasmids.

3. The method according to claim 1, characterized in that: The amino acid sequence of the IRGQ protein is shown in SEQ ID NO.

4.

4. The method according to claim 1, characterized in that: The washing buffer used in step S1 consists of: 20 mM Tris-HCl, 20 mM imidazole, 100 mM NaCl, 10% (m / V) glycerol, pH 8.0; the elution buffer used in step S1 consists of: 20 mM Tris-HCl, 500 mM imidazole, 100 mM NaCl, 10% (m / V) glycerol, pH 8.

0.

5. The method according to claim 1, characterized in that: In step S2, the anion exchange purification buffer A component consists of 20 mM Tris-HCl, 10% (m / V) glycerol, and pH 8.0; the anion exchange purification buffer B component consists of 20 mM Tris-HCl, 1 M NaCl, 10% (m / V) glycerol, and pH 8.

0.

6. The method according to claim 1, characterized in that: In step S3, the size exclusion column is a Superdex™ 200 Increase 10 / 300GL, and the gel filtration chromatography purification buffer consists of 50 mM Tris-HCl, 300 mM NaCl, and pH 7.

8.

7. A method for studying the interaction between IRGQ and GL2 proteins, characterized in that: IRGQ and GL2 proteins were mixed and incubated, then centrifuged to collect the supernatant, which was injected into a pre-equilibrated size exclusion column of a protein purification system. Molecular sieve elution was performed, and the protein polymerization state and protein interaction were observed by size exclusion chromatography. The sample obtained from the elution peak was used for SDS-PAGE analysis to verify the interaction between IRGQ and GL2 proteins.

8. The method according to claim 7, characterized in that: The IRGQ protein is the IRGQ protein obtained by the method according to any one of claims 1 to 6.

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