Mutant SUMO and protease thereof

Abstract

The invention relates to a mutant SUMO and protease thereof, the mutant SUMO is not cut by eukaryotic cell endogenous SUMO protease, and the expression and solubility of protein can be increased by using an eukaryotic SUMOsm expression system (namely transfected expression of a plasmid containing a target gene in eukaryotic cells) just like a prokaryotic SUMO expression system. Meanwhile, a specific SUMOsm protease is designed, and the specific SUMOsm protease can be used for identifying and cutting a SUMOsm tag. The SUMOsm protease has the same property as the original SUMO protease, only recognizes and specifically cuts the tertiary structure of SUMOsm, and does not cut in the target protein. The unique functional protein production advantage of the SUMO system is expanded to a eukaryotic expression host through the SUMOsm and the specific SUMOsm protease.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering, and in particular to a mutant SUMO and its protease. Background Technology

[0002] SUMO (Small Ubiquitin-like Modifier) ​​is a class of small ubiquitin-like modified proteins found in eukaryotic cells. Unlike ubiquitin, SUMOylation typically does not directly mediate protein degradation, but rather acts as a key post-transcriptional modification, widely involved in core cellular processes such as subcellular localization, stability, transcriptional activity, and protein-protein interactions.

[0003] SUMO itself is a tightly packed globular protein of about 100 amino acids, exhibiting a typical ubiquitin fold structure. This natural characteristic lays the foundation for its application in biotechnology. In the early stages of recombinant protein technology development, researchers often used fusion tags such as glutathione S-transferase (GST), maltose-binding protein (MBP), or thioredoxin (Trx) to promote the soluble expression and purification of target proteins. However, these tags are usually large in size, and their removal depends on non-specific proteases (such as thrombin and factor Xa), which often have low cleavage efficiency, low specificity, and the risk of causing unintended cleavage within the target protein.

[0004] The emergence of the SUMO tag offers an elegant and efficient solution to these challenges. As a fusion tag, it increases the expression levels of many different proteins and offers significant advantages compared to other gene fusion systems, such as: Strong "molecular chaperone" function: This is the most compelling advantage of the SUMO tag. When fused with target proteins that are difficult to express, prone to inclusion bodies, or insoluble, the SUMO tag can significantly improve their solubility and correct folding ratio. Efficient and specific protease cleavage: SUMO can be precisely cleaved by its specific protease (SUMO Protease, such as the catalytic core fragment of yeast Ulp1) after the strictly conserved C-terminal Gly-Gly residue. This protease recognizes the complete three-dimensional structure of the SUMO itself, rather than a simple linear sequence, resulting in extremely high cleavage specificity and virtually no non-specific cleavage within the target protein. Native sequence after cleavage: After cleavage by the SUMO protease, the target protein is released after the last Gly residue. This means that by simply appending the start codon of the target protein immediately after the SUMO tag during cloning, a completely natural N-terminal target protein without any additional amino acid residues can be obtained after cleavage. This is crucial for structural biology, antibody production, and functional studies. The SUMO tag is structurally stable and small in size: compared to large tags like GST and MBP, its small size places less metabolic burden on the host cell and has less impact on the structure and function of the target protein.

[0005] SUMO fusion tags are widely used in prokaryotic expression systems due to these advantages, such as for the expression of conventional proteins, as well as toxic proteins, antimicrobial peptides, and protein dimers.

[0006] However, some recombinant proteins require expression in eukaryotic systems to provide a suitable environment for protein folding and post-translational modification. Unfortunately, eukaryotic cells possess endogenous SUMO proteases that can effectively cleave SUMO fusion tags, limiting the expression and purification of recombinant proteins with fusion tags to prokaryotes. Therefore, it is necessary to express mutant SUMO in eukaryotic cells and to design a protease that can recognize and cleave mutant SUMO tags. Summary of the Invention

[0007] The purpose of this invention is to provide a mutant SUMO tag (SUMOsm) that is not cleaved by endogenous SUMO proteases in eukaryotic cells, and also to provide a specific SUMOsm protease that can recognize and cleave the SUMOsm tag. By using the mutant SUMO (SUMOsm) and the specific SUMOsm protease, the unique functional protein production advantages of the SUMO system are extended to eukaryotic expression hosts.

[0008] To achieve the objectives of this invention, the technical solution is as follows:

[0009] A mutant SUMO, which, relative to the wild-type SUMO, contains at least the following mutation sites: D at position 68 replaced by K, T at position 77 replaced by L, and E at position 94 replaced by R.

[0010] Furthermore, the mutant SUMO, compared to the wild-type SUMO, also includes a 55th position where R is replaced by K.

[0011] The base sequence of the mutant SUMO is shown in SEQ ID NO.1; the amino acid sequence of the mutant SUMO is shown in SEQ ID NO.2.

[0012] Furthermore, the mutant SUMO is linked with 6 His bases to form 6His-SUMOsm, the base sequence of which is shown in SEQ ID NO.3.

[0013] The base sequence of the plasmid pcDNA3.1 / 6His-SUMOsm containing the bases 6His-SUMOsm is shown in SEQ ID NO. 6.

[0014] A mutant SUMO protease, MutUlp1, contains at least the following mutation sites relative to the wild-type ULP (SUMO protease): N at position 197 is replaced by S, and D at position 224 is replaced by E.

[0015] Furthermore, MutUlp1, relative to wild-type ULP (the protease of SUMO), also includes a G-substitution at position 202.

[0016] The base sequence of MutUlp1 is shown in SEQ ID NO.4; the amino acid sequence of MutUlp1 is shown in SEQ ID NO.5.

[0017] The base sequence of a plasmid pET28a-MutUlp1 is shown in SEQ ID NO.7, which contains the base sequence of MutUlp1.

[0018] A method for expressing a recombinant protein, the method comprising the following steps:

[0019] S1. The target gene was cloned into the pcDNA3.1 / 6His-SUMOsm expression vector. The base sequence of the pcDNA3.1 / 6His-SUMOsm expression vector is shown in SEQ ID NO.6.

[0020] S2. The recombinant expression vector is introduced into host cell 1, the recombinant host cell 1 is cultured, the recombinant protein is expressed, and the expressed protein is collected and purified.

[0021] S3. The SUMOsm tag is cleaved by the mutant SUMO protease MutUlp1 to obtain the recombinant protein.

[0022] The host cell 1 was selected from eukaryotic HEK-293T cells.

[0023] In step S2, the introduction process refers to a technique that introduces plasmids into host cells through transformation, transfection, or infection to ultimately achieve the amplification and expression of the construct plasmid within the host cells. This is a commonly used technique in the biological field, and the present invention does not impose any particular limitations on it. Further, the introduction process includes one or more of the following: calcium chloride method, electroporation method, and λ phage infection method.

[0024] In this invention, step 2, purification, refers to a technique that separates the target gene and SUMO tags from the cell culture medium and removes other impurities to obtain high-purity target protein and SUMO tag protein. This is a commonly used technique in the biological field, and this invention does not specifically limit it. In some specific embodiments, specific examples of the purification process include, but are not limited to, one or more of the following: ultrasonic disruption, centrifugation, Ni-NTA column chromatography, and dialysis.

[0025] Furthermore, the method for constructing the pcDNA3.1 / 6His-SUMOsm expression vector includes:

[0026] S21. The gene sequence of wild-type SUMO was mutated to obtain mutant SUMO (SUMOsm). The base sequence of mutant SUMO is shown in SEQ ID NO.1, and the amino acid sequence of mutant SUMO is shown in SEQ ID NO.2.

[0027] S22. Link the six His bases together to form 6His-SUMOsm, the base sequence of which is shown in SEQ ID NO.3; clone 6His-SUMOsm into the pcDNA3.1(+) vector to obtain the pcDNA3.1 / 6His-SUMOsm expression vector plasmid.

[0028] Furthermore, the preparation method of the mutant SUMO protease MutUlp1 includes the following steps:

[0029] S31. The protease gene sequence of wild-type SUMO was mutated to obtain the protease MutUlp1 of mutant SUMO, as shown in SEQ ID NO.4. The amino acid sequence of the mutant SUMO is shown in SEQ ID NO.5. The base sequence of plasmid pET28a-MutUlp1 is shown in SEQ ID NO.7.

[0030] S32. Introduce the recombinant Escherichia coli expression strain BL21 (DE3), culture the recombinant Escherichia coli expression strain BL21 (DE3), express the recombinant protein, collect and purify it to obtain the mutant SUMO protease MutUlp1.

[0031] Compared with existing technologies, the significant advantages of this invention are as follows: By designing a mutant SUMO tag (SUMOsm) that is not cleaved by endogenous SUMO protease in eukaryotic cells, this invention utilizes a eukaryotic SUMOsm expression system (i.e., transfecting and expressing a plasmid containing the target gene in eukaryotic cells) to increase the expression and solubility of the target gene protein, just like the prokaryotic SUMO expression system. Simultaneously, a specific SUMOsm protease is designed that can recognize and cleave the SUMOsm tag. The SUMOsm protease possesses the same properties as the original SUMO protease, recognizing and specifically cleaving only the tertiary structure of SUMOsm without cleaving within the target protein. Through SUMOsm and the specific SUMOsm protease, the unique functional protein production advantages of the SUMO system are extended to eukaryotic expression hosts. Attached Figure Description

[0032] Figure 1 This is the map of the recombinant expression vector pcDNA3.1 / 6His-SUMOsm in Example 1 of this invention.

[0033] Figure 2 This is the map of the recombinant expression plasmid pcDNA3.1 / 6His-SUMOsm-EGFP in Example 2 of the present invention.

[0034] Figure 3 This is an electrophoresis diagram of the expression results of the 6His-SUMOsm-EGFP eukaryotic system in Example 3 of the present invention.

[0035] In the figure, 1 represents the untransfected group; 2 represents the transfected group; and M represents the molecular weight marker.

[0036] Figure 4 This is a diagram showing the purification results of 6His-SUMOsm-EGFP nickel column protein in Example 3 of the present invention.

[0037] In the figure, M is the molecular weight marker; 1 is the cell lysis buffer; 2 is the flow-through buffer; 3 is the elution buffer with 100 mM imidazole; 4 is the elution buffer with 200 mM imidazole; 5 is the elution buffer with 400 mM imidazole; and 6 is the elution buffer with 800 mM imidazole.

[0038] Figure 5 This is a graph showing the results of a small amount of MutUlp1 expression in Embodiment 5 of the present invention.

[0039] In the figure, M is the molecular weight marker; 1 is the lysis supernatant before induction; and 2 is the lysis supernatant after induction.

[0040] Figure 6 This is a diagram showing the purification results of MutUlp1 nickel column protein in Example 5 of the present invention.

[0041] In the figure, M is the molecular weight marker; 1 is the total protein; 2 is the flow-through buffer; 3 is the elution buffer with 50 mM imidazole; 4 is the elution buffer with 100 mM imidazole; 5 is the elution buffer with 200 mM imidazole; 6 is the elution buffer with 400 mM imidazole; 7 is the elution buffer with 800 mM imidazole; and 8 is the elution buffer with 2000 mM imidazole.

[0042] Figure 7 This is an electrophoresis diagram of the EGFP protein after the 6His-SUMOsm tag was removed in Example 6 of the present invention.

[0043] In the figure, M is the molecular weight marker; 1 is HisSUMOsm-EGFP; 2 is HisSUMOsm-EGFP+MutUlp1, with MutUlp1 at 0.5 ng; 3 is HisSUMOsm-EGFP+MutUlp1, with MutUlp1 at 0.25 ng; 4 is HisSUMOsm-EGFP+MutUlp1, with MutUlp1 at 0.125 ng; 5 is HisSUMOsm-EGFP+MutUlp1, with MutUlp1 at 0.0625 ng; and 6 is HisSUMOsm-EGFP+MutUlp1, with MutUlp1 at 0.03125 ng. Detailed Implementation

[0044] The embodiments of the present invention will be described in detail below with reference to the accompanying drawings.

[0045] Example 1: Construction of pcDNA3.1 / 6His-SUMOsm expression vector

[0046] Using the commercially available pET-28a(+)-sumo (NovoPro) plasmid as a template, the wild-type SUMO gene was mutated via directed mutagenesis to obtain a mutant SUMO (denoted as SUMOsm). SUMOsm contains the following mutation sites relative to wild-type SUMO: D at position 68 is replaced by K, T at position 77 is replaced by L, and E at position 94 is replaced by R. The amino acid sequence of SUMOsm is shown in SEQ ID NO.2, and the base sequence is shown in SEQ ID NO.1. Subsequently, using pET-28a(+)-SUMOsm as a template, the 6His-SUMOsm fragment was amplified. The base sequence of 6His-SUMOsm is shown in SEQ ID NO.3. The addition of six His bases facilitates subsequent protein purification. Using pcDNA3.1(+) (SnapGene) as a template, the pcDNA3.1 fragment was amplified. 6His-SUMOsm was then cloned into the pcDNA3.1(+) vector using a seamless ligation method, resulting in the pcDNA3.1 / 6His-SUMOsm expression vector plasmid. The specific procedures are as follows:

[0047] 1. Design upstream and downstream primers containing the corresponding mutation points and use the corresponding primers to amplify the circular pET-28a(+)-sumo plasmid by directed mutagenesis. The components are shown in Table 1.

[0048] Table 1

[0049] Using pET-28a(+)-sumo as a template, the primers were primer1 and primer2.

[0050] Mix well and centrifuge. Place in a PCR instrument with the following cycling parameters: 95°C pre-denaturation: 2 min, 95°C denaturation: 10 s, 58°C annealing: 30 s, 72°C extension: 80 s, cycle = 15, 72°C final extension: 5 min. The reaction system and conditions are based on Inno-Pfu DNA polymerase (InnoZyme DP1006).

[0051] 2. Dpn I digestion

[0052] The PCR product amplified in step 1 is digested with Dpn I endonuclease to remove the plasmid template, leaving newly synthesized double-stranded circular DNA molecules with mutated bases.

[0053] The Dpn I enzyme digestion system is shown in Table 2:

[0054] Table 2

[0055]

[0056] Water bath at 37°C for 2 hours.

[0057] 3. Purification of PCR products

[0058] The PCR product digested with Dpn I in step 2 was purified using the SanPrep column PCR product purification kit (Sangon Biotech NO. B518141). The method was as follows: (1) Add 5 times the volume of Buffer B3 to the PCR reaction solution and mix thoroughly.

[0059] (2) Centrifuge at 8,000 x g for 30 seconds. Discard the liquid in the collection tube.

[0060] (3) Add 500 μl Wash Solution, centrifuge at 9,000 X g for 30 seconds, and discard the liquid in the collection tube.

[0061] (4) Repeat step 3 once.

[0062] (5) Centrifuge the empty adsorption column at 9000 x g for 1 minute.

[0063] (6) Place the adsorption column into a clean 1.5 mL centrifuge tube, add 15-40 μl of Elution Buffer to the center of the adsorption membrane, let it stand at room temperature for 1 minute, and then centrifuge for 1 minute. Save the DNA solution in the tube.

[0064] 4. Transformation and screening of plasmids after positive mutation

[0065] (1) Take 200 μl of competent cell (DH-5α) suspension from the -80℃ freezer and place it on ice to thaw.

[0066] (2) Add the DNA solution (10 μl) obtained in step 3, shake gently, and place on ice for 30 minutes.

[0067] (3) Heat shock in a dry bath at 42℃ for 90s, and then quickly place on ice to cool for 2min.

[0068] (4) Add 200 μl of LB liquid medium (without antibiotics) to the tube, mix well, and culture at 37°C with shaking for 0.5 h to allow the bacteria to return to normal growth and express the antibiotic resistance gene encoded by the plasmid.

[0069] (5) Spread the above bacterial solution on a screening plate containing ampicillin antibiotic, place it face up for half an hour, and after the bacterial solution is completely absorbed by the culture medium, invert the culture dish and incubate at 37°C for 16 hours.

[0070] (6) Pick 5 single clones and put them into 3ml LB (Amp). After shaking overnight at 37°C, extract the plasmid and send it for sequencing.

[0071] The recombinant plasmid samples were sequenced by Sangon Biotech (Shanghai) Co., Ltd. The sequencing results were compared with the template sequence, and a single-mutant recombinant expression vector plasmid pET-28a(+)-sumo1 was obtained through screening. The mutation site was where D was replaced by K at position 68.

[0072] 5. Construction of recombinant expression vector plasmid with double mutation

[0073] Using pET-28a(+)-sumo1 obtained in step 4 as the mutation template, primers 3 and 4 were used. Steps 1-4 were repeated to screen and obtain the double-mutated recombinant expression vector plasmid pET-28a(+)-sumo2, with the mutation sites being L substitution at position 77 and K substitution at position 68.

[0074] 6. Construction of recombinant expression vector plasmid with three mutations

[0075] Using pET-28a(+)-sumo2 obtained in step 5 as the mutation template, primers were primer5 and primer6, respectively. Steps 1-4 were repeated to screen and obtain the recombinant expression vector plasmid pET-28a(+)-SUMOsm with three mutations: E at position 94 was replaced by R, T at position 77 was replaced by L, and D at position 68 was replaced by K.

[0076] 7. Construction of pcDNA3.1 / 6His-SUMOsm expression vector plasmid

[0077] Using the methods described in steps 1-3, pET-28a(+)-SUMOsm was used as a template, with primers 7 and 8. Cycling parameters were: 95°C pre-denaturation: 2 min, 95°C denaturation: 10 s, 60°C annealing: 30 s, 72°C extension: 5 s, cycle = 15, final extension at 72°C for 5 min. A 6His-SUMOsm fragment was obtained.

[0078] Using pcDNA3.1(+) as a template, primers were primer9 and primer10. Cycling parameters were: 95°C pre-denaturation: 2 min, 95°C denaturation: 10 s, 60°C annealing: 30 s, 72°C extension: 60 s, cycle = 15, final extension at 72°C: 5 min. The 6His-SUMOsm fragment was obtained. The pcDNA3.1 fragment was also obtained.

[0079] The two fragments were seamlessly cloned according to the kit instructions (InnoClone Universal One StepCloning Kit).

[0080] The reaction system is shown in Table 3 below:

[0081] Table 3

[0082]

[0083] Mix well, centrifuge, and place in a PCR instrument to react at 37°C for 30 min.

[0084] Using the method in step 4, the pcDNA3.1 / 6His-SUMOsm expression vector plasmid was obtained through screening. The plasmid map is shown below. Figure 1 As shown, the base sequence of pcDNA3.1 / 6His-SUMOsm is shown in SEQ ID NO.6.

[0085] Example 2: Construction of the pcDNA3.1 / 6His-SUMOsm-EGFP recombinant expression vector

[0086] The commercially available pEGFP-C2 (SnapGene) plasmid was seamlessly cloned with the pcDNA3.1 / 6His-SUMOsm plasmid vector obtained in Example 1 to construct the pEF1a-HisSUMO-Mut-GFP plasmid, as follows: Using pEGFP-C2 (SnapGene) as a template, primers 11 and 12 were used to amplify the EGFP fragment. Using pcDNA3.1 / 6His-SUMOsm as a template, primers 13 and 14 were used to amplify the pcDNA3.1 / 6His-SUMOsm fragment. The EGFP fragment was then cloned into the pcDNA3.1 / 6His-SUMOsm vector using a seamless ligation method to obtain the pcDNA3.1 / 6His-SUMOsm-EGFP recombinant expression vector plasmid.

[0087] The EGFP sequence and the pcDNA3.1 / 6His-SUMOsm sequence were amplified using primers, respectively. The reaction system for amplifying the EGFP sequence is shown in Table 4.

[0088] Table 4

[0089]

[0090] Mix well, centrifuge, and place in a PCR instrument. Cycling parameters: 95°C pre-denaturation: 2 min, 95°C denaturation: 10 s, 57°C annealing: 30 s, 72°C extension: 8 s, cycle = 30, 72°C final extension: 5 min.

[0091] The amplified pcDNA3.1 / 6His-SUMOsm sequence is shown in Table 5.

[0092] Table 5

[0093]

[0094] Mix well, centrifuge, and place in a PCR instrument. Cycling parameters: 95°C pre-denaturation: 2 min, 95°C denaturation: 10 s, 57°C annealing: 30 s, 72°C extension: 80 s, cycle = 30, 72°C final extension: 5 min.

[0095] DpnI digestion, PCR product purification

[0096] The EGFP fragment was seamlessly cloned with the pcDNA3.1 / 6His-SUMOsm fragment according to the kit instructions (InnoClone Universal One Step Cloning Kit). The reaction system is shown in Table 6.

[0097] Table 6

[0098]

[0099] Mix well, centrifuge, and place in a PCR instrument to react at 37°C for 30 min.

[0100] Transformation and screening of positive mutant plasmids: The seamless cloning product was transformed into *E. coli* DH-5α, single clones were picked, plasmids were extracted and sequenced, and the recombinant expression plasmid pcDNA3.1 / 6His-SUMOsm-EGFP containing the target gene EGFP was obtained. The plasmid map is shown below. Figure 2 As shown.

[0101] Example 3: Expression and purification of the 6His-SUMOsm-EGFP gene in HEK-293T cells

[0102] The 6His-SUMOsm-EGFP plasmid was expressed in HEK-293T cells through the following steps:

[0103] (1) Seed HEK-293T cells into 6-well plates and add 1.5 ml of complete culture medium to each well. Incubate at 37°C and 5% CO2. Transfect the cells when the confluence reaches 70%.

[0104] (2) Dilute plasmid DNA: Take a sterile 1.5ml centrifuge tube and add 250 μL of Opti-MEM serum-free medium. Add 2ug of 6His-SUMOsm-EGFP plasmid DNA and gently pipette to mix.

[0105] (3) Diluting the transfection reagent: Take another sterile 1.5ml centrifuge tube and add 250 μL of Opti-MEM serum-free culture medium. Add 5uL of liposome nucleic acid transfection reagent and gently pipette to mix.

[0106] (4) Mixing and incubation: Add the diluted DNA directly to the diluted transfection reagent, gently mix with a pipette, and incubate at room temperature for 20 min to form a DNA-liposome complex.

[0107] (5) Carefully aspirate the old complete culture medium from the 6-well plate, gently wash the cells once with preheated PBS, and then aspirate the PBS. Add 2 ml of antibiotic-free complete culture medium to each well, and add 500 μL of the incubated DNA-liposome complex to the well. After adding, gently shake the culture plate back and forth and side to side to mix it evenly.

[0108] (6) Place the cells back into an incubator at 37°C and 5% CO2 and continue culturing.

[0109] (7) Six hours after transfection, the culture medium containing the complex was aspirated and replaced with 2 ml of fresh complete culture medium for continued culture.

[0110] (8) After 48 hours, remove the cell culture plate from the incubator and discard the culture medium.

[0111] (9) Add 1 ml of pre-cooled PBS to each well. Gently shake the culture plate, then completely aspirate the PBS. Repeat this step once.

[0112] (10) Prepare RIPA lysis buffer: Add 10 μL PMSF to each 1 ml RIPA solution to achieve a final PMSF concentration of 1 mM, and mix well. Add 200 μL of lysis buffer to each well of a 6-well plate. Swish the plate several times with a pipette to ensure the lysis buffer and cells are in full contact. The viscous liquid that comes off the pipette is the cell lysis buffer.

[0113] (11) Use a pipette to transfer the viscous cell lysate to a pre-chilled 1.5 ml centrifuge tube. Centrifuge at 12000 g for 8 minutes and collect the supernatant (total protein solution).

[0114] (12) Protein concentration determination: Take a small amount of supernatant and use the BCA method to determine the protein concentration of the sample.

[0115] (13) Preparation of electrophoresis samples: Take 40 μL of protein sample, add 10 μL of 5× protein loading buffer, and mix thoroughly. Add to a metal bath at 100°C for 10 min to allow the protein to denature completely.

[0116] (14) 20 μL of sample was loaded onto a protein gel and subjected to SDS-PAGD electrophoresis at 90 V for 120 minutes. After electrophoresis, the sample was stained with Coomassie Brilliant Blue dye for half an hour, then destained and the protein expression was observed.

[0117] The results are as follows Figure 3 As shown, the pcDNA3.1 / 6His-SUMOsm-EGFP transfected group showed a significant increase in protein expression at the size position of the target gene compared to the untransfected group (HEK-293T cells transfected without the addition of the 6His-SUMOsm-EGFP plasmid), indicating that the newly constructed SUMOsm tag can be stably expressed in the eukaryotic system.

[0118] Scale-up culture of 6His-SUMOsm-EGFP in HEK-293T cells:

[0119] (1) The cells were cultured, transfected, and lysed according to the conditions described in Example 3.

[0120] (2) Connect the nickel affinity column to the purification instrument and slowly pass the protein lysis supernatant through the nickel affinity column at a constant rate under 4 °C conditions.

[0121] (3) Use a low concentration of imidazole buffer (10 mM imidazole) to flow through the nickel affinity column at a rate of 1 mL / min for about 5 column volumes.

[0122] (4) Perform elution in stages using imidazole buffers containing 100, 200, 400 and 800 mM respectively, at a flow rate of 1 mL / min, collect the elution peaks of each stage, and pass each concentration of imidazole buffer through 2 column volumes.

[0123] (5) Collect the protein samples and use SDS-PAGE to detect the molecular weight and purity of the fusion protein.

[0124] The results are as follows Figure 4 As shown, there were no other obvious bands besides the target protein band in lane 6, indicating that high-purity protein can be obtained after nickel column purification following the large-scale expression of 6His-SUMOsm-EGFP.

[0125] Example 4: Construction of the pET28a-MutUlp1 expression vector

[0126] Using the commercially available pET28a-ULP(SUMO) (NovoPro) plasmid as a template, the wild-type ULP(SUMO) gene was mutated via directed mutagenesis to obtain the mutant ULP (MutUlp1). MutUlp1, compared to the wild-type ULP(SUMO), contains the following mutation sites: D at position 224 is replaced by E, and N at position 197 is replaced by S. The amino acid sequence of the mutated MutUlp1 is shown in SEQ ID NO. 5. The specific operational steps are as follows:

[0127] 1. Design upstream and downstream primers containing the corresponding mutation points and use the corresponding primers to amplify the circular pET28a-ULP(SUMO) plasmid by directed mutagenesis. The reaction system is shown in Table 7.

[0128] Table 7

[0129]

[0130] Mix well and centrifuge, then place in a PCR instrument. Cycling parameters: 95°C pre-denaturation: 2 min, 95°C denaturation: 10 s, 60°C annealing: 30 s, 68°C extension: 65 s, cycle = 15, 72°C final extension: 5 min.

[0131] 2. The PCR product was digested with Dpn I, purified, transformed, and screened to obtain a single mutant plasmid with the mutation site being the substitution of N for S at position 197.

[0132] 3. Using the single mutant plasmid obtained in step 2 as a template, primers 17 and 18 were used. Cycling parameters were: 95°C pre-denaturation: 2 min, 95°C denaturation: 10 s, 56°C annealing: 30 s, 68°C extension: 65 s, cycle = 15, 72°C final extension: 5 min. The reaction system is shown in Table 7, except that primers 17 and 18 were replaced. The above process was repeated to obtain the double mutant plasmid (pET28a-MutUlp1), with mutations at positions 224 (D replaced by E) and 197 (N replaced by S). The base sequence of pET28a-MutUlp1 is shown in SEQ ID NO. 7.

[0133] Example 5: Expression and purification of the MutUlp1 gene

[0134] MutUlp1 was expressed in small amounts in the prokaryotic system, and the steps were as follows:

[0135] (1) Transformation: Escherichia coli BL21 (DE3) competent cells were thawed on ice, 1 μl (150 ng) of pET28a-MutUlp1 plasmid obtained in Example 4 was added, the cells were placed on ice for 30 minutes, heat-shocked at 42°C for 90 seconds, 200 μl of LB liquid (without Kan) was added, the cells were shaken at 37°C for 10 minutes, then plated and incubated upside down at 37°C overnight.

[0136] (2) Expression:

[0137] ① Activation: Pick a single colony and place it in 3 ml LB (50 μg / ml Kan), shake overnight at 37°C; ② Secondary activation: Inoculate 1 / 100 of the bacteria in 3 ml LB (50 μg / ml Kan), shake at 37°C for 6-8 hours until OD600 = 0.6; ③ Induction: Set up two groups and induce them according to the following induction conditions: control group without IPTG; experimental group with IPTG added to a final concentration of 0.5 mM. Temperature: 20°C, rotation speed: 200 r / min, induction time: 16 hours. (3) Collect bacterial cells: centrifuge at 4°C, 8000 r / min for 2 minutes and collect the bacterial cell pellet. (4) Lysis: add 250 μl of bacterial lysis buffer to resuspend the cells and vortex to mix thoroughly. Place the centrifuge tube in a -80°C freezer for 5 minutes, take out the centrifuge tube, and thaw in a water bath at room temperature for 5 minutes. Repeat this process 3 times. (5) Preparation of electrophoresis samples: Electrophoresis at 12000×g, centrifuged at 4ºC for 10 min, collected the supernatant, took 40ul of protein sample, added 10ul of 5× protein loading buffer, and mixed thoroughly. Added to a metal bath at 100℃ for 10 min to allow the protein to denature fully. (6) Take 20ul of sample and load it onto a protein gel, and perform SDS-PAGD electrophoresis at 90V for 120 minutes. After electrophoresis, stain with Coomassie brilliant blue dye for half an hour, destain, and observe the protein expression.

[0138] The SDS-PAGE electrophoresis results of MutUlp1 are as follows: Figure 5 As shown, the induction conditions used in the experimental group can significantly improve the soluble expression of the protein.

[0139] The methods for large-scale expression and purification of MutUlp1 are as follows:

[0140] (1) Purification: The recombinant protein expression strain was cultured and the target protein was induced to express by scaling up the culture to 500 ml according to the conditions described in Example 5.

[0141] (2) Centrifuge at 5000g for 10min, discard the culture medium, and collect the bacterial precipitate.

[0142] (3) Add 20 ml Binding Buffer to resuspend the cells and vortex to fully resuspend them. Transfer the cell suspension to a 50 ml centrifuge tube.

[0143] (4) Ultrasonic fragmentation: 10s on, 10s off, total 20min.

[0144] (5) After sonication, centrifuge at 15000×g, 4ºC for 30min, and transfer the supernatant to a new 50 ml centrifuge tube.

[0145] (6) Connect the nickel affinity column to the purification instrument and slowly pass the protein lysis supernatant through the nickel affinity column at a constant rate under 4 °C conditions.

[0146] (7) Use a low concentration of imidazole buffer (10 mM imidazole) to flow through the nickel affinity column at a rate of 5 mL / min for about 5 column volumes.

[0147] (8) Perform staged elution with imidazole buffer containing 50, 100, 200, 400, 800 and 2000 mM respectively, at a flow rate of 5 mL / min, collect the elution peaks of each stage, and pass each concentration of imidazole buffer through 2 column volumes.

[0148] (9) Collect the protein samples and use SDS-PAGE to detect the molecular weight and purity of the fusion protein.

[0149] The results are as follows Figure 6 As shown, MutUlp1 expression can be purified to high purity using a nickel column.

[0150] Example 6: MutUlp1 Activity Assay

[0151] The activity of the MutUlp1 protease was identified using purified 6His-SUMOsm-EGFP protein as a substrate. The reaction system is shown in Table 8.

[0152] Table 8

[0153]

[0154] 6His-SUMOsm-EGFP was mixed with different amounts of MutUlp1 and reacted at 30°C for 1 hour. After the reaction, electrophoresis samples were prepared and analyzed by SDS-PAGE electrophoresis.

[0155] The results are as follows Figure 7 As shown, MutUlp1 can remove the 6His-SUMOsm tag from the 6His-SUMOsm recombinant protein that is stably expressed in a eukaryotic system to obtain the native EGFP structure.

[0156] The sequence listings of all the primers mentioned above are shown in Table 9.

[0157] Table 9

[0158]

[0159] The above description is merely a specific embodiment of the present invention. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple changes or substitutions can be made to the technical solution of the present invention, and these should all be within the protection scope of the present invention.

Claims

1. A mutant SUMO, characterized in that: The mutant SUMO comprises at least the following mutation sites of wild-type SUMO: D at position 68 is replaced by K, T at position 77 is replaced by L, and E at position 94 is replaced by R.

2. The mutant SUMO of claim 1, wherein: The mutant SUMO further comprises R at position 55 is replaced by K.

3. The mutant SUMO of claim 1, wherein: The base sequence of the mutant SUMO is shown in SEQ ID NO. 1; the amino acid sequence of the mutant SUMO is shown in SEQ ID NO.

2.

4. A recombinant expression vector, characterized by: The recombinant expression vector contains the base sequence of the mutant SUMO as claimed in claim 3.

5. A plasmid, characterized by: The base sequence of the plasmid is shown in SEQ ID NO.

6.

6. A mutant SUMO protease MutUlp1, characterized in that: The mutant ULP, i.e., the protease of wild-type SUMO, comprises at least the following mutation sites: N at position 197 is replaced by S, and D at position 224 is replaced by E.

7. The mutant SUMO protease MutUlp1 according to claim 6, characterized in that: The MutUlp1 further comprises G at position 202 is replaced by H.

8. The mutant SUMO protease MutUlp1 according to claim 6, characterized in that: The base sequence of the MutUlp1 is shown in SEQ ID NO. 4; the amino acid sequence of the MutUlp1 is shown in SEQ ID NO.

5.

9. A recombinant expression vector, characterized by: The recombinant expression vector contains the base sequence of the MutUlp1 as claimed in claim 8.

10. A method for expressing a recombinant protein, characterized in that: The method comprises the following steps: S1, cloning a target gene into a pcDNA3.1 / 6His-SUMOsm expression vector, the base sequence of the pcDNA3.1 / 6His-SUMOsm expression vector is shown in SEQ ID NO. 6; S2, introducing the recombinant expression vector into a host cell 1, culturing the recombinant host cell 1, expressing the recombinant protein, collecting and purifying the expressed protein; S3, using the mutant SUMO protease MutUlp1 to cut the SUMOsm tag, to obtain the recombinant protein; The host cell 1 is selected from a eukaryotic HEK-293T cell; The MutUlp1 preparation method comprises the following steps: S31, mutating the wild-type SUMO protease gene sequence to obtain the MutUlp1 base sequence, the mutant SUMO amino acid sequence is shown in SEQ ID NO. 5, and the base sequence of the plasmid pET28a-MutUlp1 is shown in SEQ ID NO. 7; S32, introducing the plasmid pET28a-MutUlp1 into an E. coli expression strain BL21 (DE3), culturing the recombinant E. coli expression strain BL21 (DE3), expressing the recombinant protein, collecting and purifying, to obtain the MutUlp1.

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