A mutant of tobacco etch virus tev protein, and methods of making and using the same

Abstract

The application provides a mutant of tobacco etch virus TEV protein and a preparation method and application thereof, and belongs to the technical field of biotechnology.The application provides a mutant of tobacco etch virus TEV protein, wherein at least one of the following mutations is made on the basis of the TEV protein: (1) the 220th amino acid is mutated into methionine, (2) the 222nd-242nd amino acids are deleted, (3) the 153rd amino acid is mutated into aspartic acid, and (4) the 173rd amino acid is mutated into alanine; the amino acid sequence of the TEV protein is shown as SEQ ID NO:1.The mutant provided by the application has higher enzymatic activity, high enzymatic reaction rate, and high yield compared with the wild type and commercial TEV, and is more suitable for large-scale production and industrial use.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a mutant of the tobacco etch virus TEV protein, its preparation method, and its application. Background Technology

[0002] Tobacco etch virus protease (TEV protease) is a cysteine ​​protease derived from tobacco etch virus. Due to its high substrate specificity, strong cleavage efficiency, and compatibility with multiple expression systems including prokaryotes and eukaryotes, it has become the preferred tool enzyme for tag cleavage of recombinant fusion proteins, and is widely used in bioengineering, biomedicine, and other fields. This protease exhibits highly specific substrate recognition characteristics, specifically cleaving only specific amino acid sequences. This effectively avoids damage to the target protein caused by non-specific cleavage, maximizing the preservation of the target protein's native structure and biological activity. It plays an irreplaceable role in key steps such as recombinant protein purification and pharmaceutical protein preparation.

[0003] Wild-type TEV proteases have significant limitations in practical applications. Their enzyme activity and reaction rate are limited, and their soluble expression levels are low, making them prone to inclusion body formation. This results in insufficient yield and activity levels for enzyme preparations, severely restricting their large-scale industrial application and promotion. Current technologies for mutant modification of TEV proteases mostly focus on single-performance optimization, generally improving only one of the enzyme's activity or increasing its soluble expression level. They fail to simultaneously achieve a synergistic improvement in both enzyme yield and activity, thus failing to effectively address the application bottlenecks of wild-type TEV proteases. Summary of the Invention

[0004] In view of this, the present invention provides a mutant of tobacco etch virus TEV protein, which has the characteristics of high enzyme activity and high yield.

[0005] To achieve the above objectives, the present invention provides the following technical solution: The present invention provides a mutant of tobacco etch virus TEV protein, which is based on the TEV protein by at least one of the following mutations: (1) 220 position is mutated to methionine, (2) 222-242 positions are deleted, (3) 153 position is mutated to aspartic acid and (4) 173 position is mutated to alanine; The amino acid sequence of the TEV protein is shown in SEQ ID NO:1.

[0006] Preferably, the amino acid sequence of the mutant is any one of those shown in SEQ ID NO:2 to SEQ ID NO:5.

[0007] The present invention provides a gene encoding the mutant.

[0008] The present invention provides a derivative product containing the gene, comprising a recombinant vector and / or recombinant bacteria containing the gene.

[0009] This invention provides a method for preparing the mutant, comprising the following steps: The mutant was recombinantly expressed in a host strain, and the recombinant protein was isolated to obtain the mutant.

[0010] This invention provides the application of the mutant, or the mutant prepared by the derivative or the preparation method, in the cleavage of the fusion tag and / or the target protein.

[0011] Preferably, the target protein includes a fluorescent substrate protein, which includes a fluorescent substrate protein for detecting the activity of tobacco etch virus TEV protease.

[0012] Preferably, the amino acid sequence of the fluorescent substrate protein is shown in SEQ ID NO:10.

[0013] Preferably, the nucleotide sequence of the fluorescent substrate protein is shown in SEQ ID NO:11.

[0014] Compared with the prior art, the present invention has the following advantages: This invention provides a mutant of tobacco etch virus (TEV) protein, which is mutated by at least one of the following mutations: (1) methionine at position 220, (2) deletion at positions 222-242, (3) aspartic acid at position 153, and (4) alanine at position 173; the amino acid sequence of the TEV protein is shown in SEQ ID NO:1. Compared with wild-type and commercially available TEV, the mutant provided by this invention has higher enzyme activity, higher enzyme reaction rate, and higher yield, making it more suitable for large-scale production and industrial use.

[0015] This invention provides the application of the mutant, or a mutant prepared from the derivative or the preparation method, in the cleavage of a fusion tag and / or a target protein. Preferably, the target protein comprises a fluorescent substrate protein, the amino acid sequence of which is shown in SEQ ID NO:10. In this invention, the fluorescent substrate protein is composed of two monomeric fluorescent proteins linked together by a TEV-specific cleavage site as a linker. The fluorescent substrate protein exhibits strong fluorescence when uncleaved, and the fluorescence decays after cleavage as the two monomeric fluorescent proteins separate. This fluorescent substrate protein exhibits high sensitivity and rapid reaction speed when used to detect TEV protease activity, and can be used for the detection of TEV protease activity. Attached Figure Description

[0016] Figure 1The figure shows the results of the induction expression identification of recombinant protease TEV-Δ222, where M is the protein molecular weight standard (Marker), 1 is the total bacterial protein before IPTG induction, 2 is the total bacterial protein after IPTG induction, 3 is the bacterial lysis supernatant after induction, and 4 is the bacterial lysis precipitate after induction. Figure 2 The image shows the Ni affinity chromatography purification and identification results of recombinant protease TEV-Δ222, where M: protein molecular weight standard (Marker), 1: Ni affinity chromatography loading solution, 2: Ni affinity chromatography flow-through solution, 3: elution fraction with 100 mM imidazole concentration, and 4: elution fraction with 500 mM imidazole concentration. Figure 3 The image shows the results of fluorescent substrate expression, where M is the protein molecular weight standard (Marker), 1 is the total bacterial protein before IPTG induction; 2 is the total bacterial protein after IPTG induction; 3 is the supernatant of bacterial lysis after induction; and 4 is the bacterial lysis precipitate after induction. Figure 4 The image shows the purification results of the dual-fluorescence substrate, where M is the protein molecular weight standard (Marker), 1 is the Ni affinity chromatography loading solution, 2 is the Ni affinity chromatography flow-through solution, 3 is the Ni affinity chromatography 100 mM imidazole eluent, 4 is the Ni affinity chromatography 250 mM imidazole elution fraction, and 5 is the Ni affinity chromatography 500 mM imidazole elution fraction. Figure 5 A comparison of response curves for different TEV mutants; Figure 6 A graph showing the percentage of substrate consumed after 1 hour of reaction at 30℃ for different substrate-enzyme ratios. Figure 7 The graph shows the enzyme reaction rate results under the same conditions as the recombinant TEV protease. Figure 8 This is a schematic diagram of the TEV structure and mutation sites. Detailed Implementation

[0017] The present invention provides a mutant of tobacco etch virus TEV protein, characterized in that at least one of the following mutations is performed on the TEV protein: (1) 220 position is mutated to methionine, (2) positions 222-242 are deleted, (3) position 153 is mutated to aspartic acid and (4) position 173 is mutated to alanine; the amino acid sequence of the TEV protein is shown in SEQ ID NO:1 and the nucleotide sequence is shown in SEQ ID NO:9.

[0018] In this invention, when positions 222-242 are deleted, the amino acid sequence of the mutant is as shown in SEQ ID NO:2. When position 220 is mutated to methionine and positions 222-242 are deleted, the amino acid sequence of the mutant is as shown in SEQ ID NO:3. When position 153 is mutated to aspartic acid, the amino acid sequence of the mutant is as shown in SEQ ID NO:4. When position 173 is mutated to alanine, the amino acid sequence of the mutant is as shown in SEQ ID NO:5. In this invention, the N-terminus of the mutant can be further supplemented with an MGS sequence and a 6×His tag. Adding the MGS sequence and tag facilitates purification to obtain a high-purity protease tool, and also facilitates removal and separation of the target product after enzyme digestion. A schematic diagram of the TEV structure and mutation sites is shown below. Figure 8 .

[0019] This invention modifies the amino acid sequence of existing TEV enzymes to obtain mutants. Compared with the wild type, the mutants provided by this invention exhibit significantly improved enzyme activity, reaction rate, and yield.

[0020] This invention provides a gene encoding the mutant. The invention does not specifically limit the gene encoding the mutant; it can be an artificial sequence codon-optimized for expression in, for example, *E. coli*. The invention does not limit the specific method of codon optimization; conventional optimization techniques known in the art can be used.

[0021] The present invention provides a derivative product of the gene, comprising a recombinant vector and / or recombinant bacteria containing the gene.

[0022] This invention provides a recombinant expression vector containing the aforementioned gene. The backbone vector of the recombinant expression vector preferably includes pET28a. The multiple cloning sites of the backbone vector are Ncol and Xhol.

[0023] This invention provides a recombinant bacterium containing the gene or the recombinant expression vector. The host bacterium of the recombinant bacterium preferably includes *Escherichia coli*, and more preferably... E. coli BL21(DE3).

[0024] This invention provides a method for preparing the mutant, comprising the following steps: recombinantly expressing the gene in a host strain, isolating the recombinant protein, and obtaining the mutant. The recombinant expression is preferably performed by culturing the recombinant bacteria. The recombinant expression process preferably includes induction culture with the addition of an inducer; the inducer preferably includes isopropyl thiogalactopyranoside. The induction culture temperature is 16-19°C, more preferably 18°C. The induction culture time is preferably 16-24 h, more preferably 17-18 h. After induction culture, the recombinant protein is isolated. The method for isolating the recombinant protein preferably includes separating the supernatant from the lysed bacterial cells. The recombinant protein is preferably purified and enriched. The purification method preferably includes cobalt ion affinity chromatography purification. The enrichment method preferably retains ultrafiltration membrane concentration. The mutant is obtained after purification and enrichment.

[0025] In the embodiments of this invention, the enzyme activity of different mutants was tested. The results showed that compared with the wild type, the enzyme activity, reaction rate and yield were significantly improved.

[0026] The present invention provides the application of the mutant or the mutant prepared by the recombinant expression vector, the recombinant bacteria or the preparation method in the cleavage of the fusion tag and / or the target protein.

[0027] TEV protease, due to its cleavage activity and specificity, is one of the preferred proteases for removing fusion tags after fusion protein expression. This invention, through modification of the TEV protein, further enhances enzyme activity and reaction rate, making it suitable for cleaving fusion tags and / or target proteins. The target protein preferably includes a fluorescent substrate protein, which preferably includes a fluorescent substrate protein suitable for detecting the activity of tobacco etch virus TEV protease. The target protein preferably includes a fluorescent substrate protein, and the amino acid sequence of the fluorescent substrate protein is preferably as shown in SEQ ID NO:10. The nucleotide sequence of the fluorescent substrate protein is preferably as shown in SEQ ID NO:11. The fluorescent substrate protein (mTFP1-TEVc-mRuby3) is composed of two monomeric fluorescent proteins linked together by a TEV-specific cleavage site as a linker. This fluorescent substrate protein exhibits strong fluorescence when uncleaved; after cleavage, the two fluorescent protein monomers separate, and the fluorescence decays. This fluorescent substrate protein demonstrates high sensitivity and rapid reaction rate when used to detect TEV protease activity. Therefore, it can be used to detect TEV protease activity. When detecting the activity of tobacco etched virus TEV protease, the preferred mass ratio of the fluorescent substrate protein to the tobacco etched virus TEV protease is (10~60):1, more preferably (20~50):1. This ratio facilitates accurate and rapid detection of TEV protease activity.

[0028] To further illustrate the present invention, the solutions provided by the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0029] Example 1: Expression and purification of recombinant TEV enzyme in Escherichia coli 1. Design methods for mutation sites The wild-type TEV protein is Sbi(WD) (Beijing Yiqiao Shenzhou Technology Co., Ltd., catalog number 10603-V07E). The amino acid sequence of the TEV protein is shown in SEQ ID NO:1 (GESLFKGPRDYNPISSTICHLTNESDGHTTSLYGIGFGPFIITNKHLFRRNNGTLLVQSLHGVFKVKNTTTLQQHLIDGRDMIIIRMPKDFPPFPQKLKFREPQREERICLVTTNFQTKSMSSMVSDTSCTFPSSDGIFWKHWIQTKDGQCGSPLVSTRDGFIVGIHSASNFTNTNNYFTSVPKNFMELLTNQEAQQWVSGWRLNADSVLWGGHKVFMNKPEEPFQPVKEATQLMNELVYSQ).The nucleotide sequence is shown in SEQ ID NO:9(). Based on the TEV protein, the enzyme active site, cap region, and C-terminal region were optimized. Mutations at key catalytic sites in the active site improved the enzyme's catalytic efficiency and substrate affinity; optimization of the cap region enhanced the substrate affinity to the active site; the C-terminal residue ELVYSQ has a similar site to the substrate recognition sequence and the site ENLYFQ^G, suggesting self-cleavage to close the active site. Mutations in the C-terminal region improved enzyme activity; the mutation site design scheme is shown in Table 1.

[0030] Table 1. Mutation site design scheme for mutants

[0031] The amino acid sequence of TEV-Δ219C is shown in SEQ ID NO:6 (GESLFKGPRDYNPISSTICHLTNESDGHTTSLYGIGFGPFIITNKHLFRRNNGTLLVQSLHGVFKVKNTTTLQQHLIDGRDMIIIRMPKDFPPFPQKLKFREPQREERICLVTTNFQTKSMSSMVSDTSCTFPSSDGIFWKHWIQTKDGQCGSPLVSTRDGFIVGIHSASNFTNTNNYFTSVPKNFMELLTNQEAQQWVSGWRLNADSVLWGGHKVFM).

[0032] The amino acid sequence of TEV-Δ222C is shown in SEQ ID NO:2 (GESLFKGPRDYNPISSTICHLTNESDGHTTSLYGIGFGPFIITNKHLFRRNNGTLLVQSLHGVFKVKNTTTLQQHLIDGRDMIIIRMPKDFPPFPQKLKFREPQREERICLVTTNFQTKSMSSMVSDTSCTFPSSDGIFWKHWIQTKDGQCGSPLVSTRDGFIVGIHSASNFTNTNNYFTSVPKNFMELLTNQEAQQWVSGWRLNADSVLWGGHKVFMNKP).

[0033] The amino acid sequence of TEV-Δ222C / K220M is shown in SEQ ID NO:3 (GESLFKGPRDYNPISSTICHLTNESDGHTTSLYGIGFGPFIITNKHLFRRNNGTLLVQSLHGVFKVKNTTTLQQHLIDGRDMIIIRMPKDFPPFPQKLKFREPQREERICLVTTNFQTKSMSSMVSDTSCTFPSSDGIFWKHWIQTKDGQCGSPLVSTRDGFIVGIHSASNFTNTNNYFTSVPKNFMELLTNQEAQQWVSGWRLNADSVLWGGHKVFMNMP).

[0034] The amino acid sequence of TEV-S153N is as SEQ ID NO:4(GESLFKGPRDYNPISSTICHLTNESDGHTTSLYGIGFGPFIITNKHLFRRNNGTLLVQSLHGVFKVKNTTTLQQHLIDGRDMIIIRMPKDFPPFPQKLKFREPQREERICLVTTNFQTKS MSSMVSDTSCTFPSSDGIFWKHWIQTKDGQCGNPLVSTRDGFIVGIHSASNFTNTNNYFTSVPKNFMELLTNQEAQQWVSGWRLNADSVLWGGHKVFMNKPEEPFQPVKEATQLMNELVYSQ).

[0035] The amino acid sequence of TEV-T173A is as SEQ ID NO:5(GESLFKGPRDYNPISSTICHLTNESDGHTTSLYGIGFGPFIITNKHLFRRNNGTLLVQSLHGVFKVKNTTTLQQHLIDGRDMIIIRMPKDFPPFPQKLKFREPQREERICLVTTNFQTKS MSSMVSDTSCTFPSSDGIFWKHWIQTKDGQCGSPLVSTRDGFIVGIHSASNFANTNNYFTSVPKNFMELLTNQEAQQWVSGWRLNADSVLWGGHKVFMNKPEEPFQPVKEATQLMNELVYSQ).

[0036] 2. Preparation of TEV-Δ222 mutant (1) Obtaining the expression strain of TEV-Δ222 mutant An MGS sequence and a 6×His tag were added to the N-terminus of the amino acid sequence of the TEV-Δ222 mutant. The gene of the TEV-Δ222 mutant was synthesized based on the amino acid sequence and inserted into the Ncol and Xhol sites of the pET28a vector to obtain the TEV-Δ222 mutant plasmid.

[0037] The TEV-Δ222 mutant plasmid contains all the regulatory elements and kanamycin resistance gene required for expression in the host bacterium BL21(DE3). The TEV-Δ222 mutant plasmid was transformed into competent Escherichia coli BL21(DE3) (Tiangen Biotech) using standard methods. The transformation product was screened on LB agar plates containing kanamycin, and single colonies were verified by PCR and sequencing to obtain recombinant bacteria expressing the TEV-Δ222 mutant.

[0038] (2) Induced expression of TEV-Δ222 mutant Single colonies of recombinant bacteria expressing the TEV-Δ222 mutant were picked and inoculated into MDG medium, and incubated at 250 r·min. -1 Incubate overnight at 37 ℃. Take 10 ml of the activated bacterial culture and inoculate it into 1 L of TB medium containing 0.1% kanamycin. Dispense the solution into 1 L Erlenmeyer flasks (200 ml / flask), place them on a shaker, and incubate at 37 ℃ with shaking at 275 rpm until OD reaches 100%. 600 When the value is 1, add isopropyl thiogalactopyranoside (IPTG) to a final concentration of 1 mM, adjust the shaker setting to 18℃, and culture with shaking for 18 h to obtain a culture containing the TEV-Δ222 mutant. Centrifuge the culture at 8℃ and 8000 rpm for 20 min, discard the supernatant, and retain the bacterial cells for cell lysis.

[0039] Cell lysis: The lysis buffer was NiA buffer containing 0.1 mM MTCEP. Experimental procedure: The wet weight of the bacterial cells was resuspended in the lysis buffer at a material-to-liquid ratio of 1:20 (w / w); then placed in an ice-water bath and lysed using an ultrasonic sonicator. Ultrasonic parameters: power 40%, sonication for 3 s, interval 3 s, total sonication time 15 min. After lysis, the samples were centrifuged at 8000 rpm for 45 min at 4 ℃. After centrifugation, the bacterial cell fragments were discarded, and the supernatant was collected for later use.

[0040] Figure 1 The figure shows the results of the induction and expression identification of the TEV-Δ222 mutant recombinant TEV protease at different stages. The results indicate that the TEV-Δ222 mutant was successfully induced and expressed, mainly in the bacterial cell lysis supernatant. Most of the different recombinant TEV enzymes in this invention showed intracellular soluble expression, while a few were expressed in precipitate. During the preparation process, only the soluble products were extracted.

[0041] (3) Purification of TEV-Δ222 mutant The supernatant from bacterial cell lysis was clarified and filtered through a 0.45 μm pore size filter membrane. The filtrate was then purified as the TEV-Δ222 mutant using Ni-IDA affinity chromatography. The packing ratio for the purification media was 1 g of wet bacterial cells to 1 mL of chromatography packing material. The equilibration buffer was NiA buffer containing a final concentration of 0.1 mM TCEP; the elution buffer was NiB buffer containing a final concentration of 0.1 mM TCEP. Purification procedure: First, equilibrate the Ni-IDA column with equilibration buffer, then load the filtrate onto the column. After loading, perform gradient elution and target protein elution sequentially: First, rinse the column with equilibration buffer to remove non-specifically bound proteins, with a wash volume of 10 column volumes (10 CV); then rinse the column with 20% elution buffer to further remove weakly bound proteins, with a wash volume of 10 CV; finally, elute the target protein with 100% elution buffer, with an elution volume of 10 CV, and collect the eluent. Figure 2 The image shows the Ni affinity chromatography purification and identification results of the recombinant protease TEV-Δ222. In the image, M represents the protein molecular weight standard (Marker), 1 is the Ni affinity chromatography loading buffer, 2 is the Ni affinity chromatography flow-through buffer, 3 is the elution fraction with a concentration of 100 mM imidazole, and 4 is the elution fraction with a concentration of 500 mM imidazole. The results indicate that the final collected elution fraction was enriched with the TEVΔ222 mutant.

[0042] The collected elution fractions were desalted using a G25 column to 2×TEV buffer (100 mM Tris, 2 mM EDTA, 10 mM DTT, pH 7.5). Glycerol was added after desalting until the final concentration reached 50% (V / V), yielding the final TEVΔ222 stock solution. The stock solution formulation was: 50 mM Tris, 1 mM EDTA, 5 mM DTT, 50% glycerol, pH 7.5. The TEVΔ222 stock solution was used for subsequent enzyme activity assays. It is important to maintain low temperature (4℃~8℃) throughout the entire preparation process. Immediately after Ni column elution, the 500 mM imidazole elution fraction was desalted and stored at -80℃.

[0043] Example 2 Preparation of TEV-Δ222 / K220M mutant An MGS sequence and a 6×His tag were added to the N-terminus of the amino acid sequence of the TEV-Δ222 / K220M mutant. The gene was synthesized according to the amino acid sequence, and TEV-Δ222 / K220M was prepared according to the method for preparing the TEV-Δ222 mutant.

[0044] Example 3: Preparation of the TEV-T173A mutant An MGS sequence and a 6×His tag were added to the N-terminus of the amino acid sequence of the TEV-T173A mutant. The gene was synthesized according to the amino acid sequence, and the TEV-T173A mutant was prepared by following the method for preparing the TEV-Δ222 mutant.

[0045] Example 4: Preparation of the TEV-S153N mutant The amino acid sequence of the TEV-S153N mutant was modified by adding an MGS sequence and a 6×His tag to the N-terminus. The gene was synthesized based on the amino acid sequence and the TEV-S153N mutant was prepared according to the method for preparing the TEV-Δ222 mutant.

[0046] Example 5: Preparation of the TEV-Δ219 mutant An MGS sequence and a 6×His tag were added to the N-terminus of the amino acid sequence of the TEV-Δ219 mutant. The gene was synthesized according to the amino acid sequence, and the TEV-Δ219 mutant was prepared by following the method for preparing the TEV-Δ222 mutant.

[0047] Comparative Example 1 1. Preparation of wild-type control TEV protease: The MGS sequence and 6×His tag were added to the N-terminus of the TEV amino acid sequence in Example 1. The gene was synthesized according to the amino acid sequence and the TEV protease was prepared according to the method for preparing the TEV-Δ222 mutant in Example 1.

[0048] 2. Preparation of commercially available high-activity mutant controls TEV-uTEV3 and TEV-eTEV mutants: Based on the amino acid sequences of the TEV-uTEV3 and TEV-eTEV mutants, the TEV-uTEV3 mutant (GESLFKGPRDYNPISSTICHLTNESDGHTTSLYGIGFGPFIITNKHLFRRNNGTLLVQSLHGVFKVKNTTTLQQHLIDGRDMIIIRMPKDFPPFPQKLKFREPQREERICLVTTNFQTKSMSSMVSDTSCTFPSSDGIFWKHWIQTKDGQCGSPLVSTRDGFIVGIHSASNFTNTNNYFTSVPKNFMELLTNQEAQQWVSGWRLNADSVLWGGHKVFMVKPEEPFQPVKEATQLMNELVYSQ, SEQ ID) was prepared according to the method for preparing the TEV-Δ222 mutant in Example 1. NO:7) and the TEV-eTEV mutant (GEILFKGPRDYNPISSTICHLTNESDGHTTTLYGIGFGPFIITNKHLFRRNNGTLLVQSLHGVFKVKNTTTLQQHLIDGRDMIIIRMPKDFPPFPQKLKFREPQREERICLVTTNFQTKSMSSMVSDTSCTFPSSDGIFWKHWIQTKDGQCGSPLVSTRDGFIVGIHSASNFANTNNYFTSVPKNFMELLTNQEAQQWVSGWRLNADSVLWGGHKVFMRKPEEPFQPVKEVTQLMNELVYSQ, SEQ ID NO:8). Mutation sites for the wild-type control and the highly active mutant are shown in Table 2.

[0049] Table 2 Mutation sites of wild-type control and highly active mutants

[0050] Example 6: Method for Detecting Recombinant TEV Protease Activity 1. Preparation method of fluorescent substrate

[0051] The gene encoding the fluorescent substrate protein was inserted into the Ncol and Xhol sites of the expression vector pET28a to obtain the fluorescent substrate recombinant expression vector pET28a-mTFP1-TEVc-mRUby3-6his. This expression vector contains all the regulatory elements required for expression in the host bacterium BL21(DE3) and the kanamycin resistance gene. The vector was transformed into competent Escherichia coli BL21(DE3) (Tiangen Biotech) using standard methods. The transformation products were screened on LB agar plates containing kanamycin, and single colonies were verified by PCR and sequencing to obtain recombinant bacteria expressing the fluorescent substrate.

[0052] The methods for inducing expression with fluorescent substrate and cell lysis were the same as those for inducing expression with TEV-Δ222 mutant in Example 1, except that the cell lysis buffer was a NiA buffer without TCEP. Cell lysis supernatant was obtained after cell lysis. Figure 3 The image shows the identification results of recombinant fluorescent substrate-induced expression at different stages. The results indicate that the fluorescent substrate-induced expression was successful. The recombinant dual-fluorescent substrate exhibited intracellular expression. A small portion of the expression product was soluble, while the majority remained in the precipitate. During the preparation process, only the soluble product was extracted.

[0053] Purification of fluorescent substrate: The supernatant from bacterial cell lysis was clarified and filtered through a 0.45 μm pore size filter membrane. The filtrate was then purified as the TEV-Δ222 mutant using Ni-IDA affinity chromatography. The packing ratio for the purification packing material was 1 g of wet bacterial cells to 1 mL of chromatography packing material. The equilibration buffer was NiA buffer; the elution buffer was NiB buffer. Purification procedure: First, equilibrate the Ni-IDA column with equilibration buffer, then load the filtrate onto the column. After loading, perform gradient elution and target protein elution sequentially: First, rinse the column with equilibration buffer to remove non-specifically bound proteins, with a wash volume of 10 column volumes (10⁻⁶ CV); then, rinse the column with 20% elution buffer to further remove weakly bound proteins, with a wash volume of 10⁻⁶ CV; finally, elute the target protein with 50% elution buffer, with an elution volume of 10⁻⁶ CV, and collect the eluent. Figure 4 The figure shows the Ni affinity chromatography purification and identification results of the fluorescent substrate. 1 is the original Ni affinity chromatography loading solution, 2 is the Ni affinity chromatography flow-through solution, 3 is the Ni affinity chromatography 100 mM imidazole eluent, 4 is the Ni affinity chromatography 250 mM imidazole eluent, and 5 is the Ni affinity chromatography 500 mM imidazole eluent. The results indicate that the fluorescent substrate was enriched in the final collected eluent.

[0054] The collected elution fractions were desalted and replaced with a G25 desalting column, and the buffer system was replaced with protease terminus stock solution to obtain the fluorescent substrate. After concentration measurement, the substrate was frozen and stored at -80°C. The protease terminus stock solution formulation was: 50 mM Tris, pH 7.5.

[0055] 2. Detection of TEV mutant protease activity The proportion of substrate consumed was determined by reacting at 30℃ for 1 h with different substrate-enzyme ratios, and the enzyme reaction rate under the same conditions relative to TEV protease was calculated.

[0056] The reaction system buffer was 10× (50mM Tris, 1mM EDTA, 5mM DTT, pH 7.5). Preparation of reaction system with substrate to enzyme mass ratio of 20:1: 10 μl of 1 mg / ml fluorescent substrate, 0.5 μg of protease, 10 μl of reaction system buffer, and ultrapure water were added to make the system volume 100 μl; Preparation of a reaction system with a substrate to enzyme mass ratio of 50:1: 10 μl of 1 mg / ml fluorescent substrate, 0.2 μg of protease, 10 μl of reaction system buffer, and ultrapure water were added to make a total volume of 100 μl; The protease was the TEV protein and TEV mutant prepared in Examples 1-4 and Comparative Example 1, and the concentration of the protease added was 0.5 mg / ml.

[0057] Microplate reader operation procedure: Place the reaction system into the microplate reader, excite at a wavelength of 435nm, and detect the fluorescence intensity at a wavelength of 605nm. Detect the fluorescence intensity every 30 seconds and collect the data.

[0058] Data processing: The overall data is a logarithmic decay curve ( Figure 5 To determine the initial reaction rate, the first 10 data points were selected for linear fitting. The slope (negative value) k when the R value was above 0.99 was calculated. The initial rate v was calculated using formula I, and the enzyme activity was determined by this rate.

[0059] v = -k / (30 × c) Formula I; Where c is the molar concentration.

[0060] Table 3 and Figure 6 Results of substrate consumption ratios for different substrate-enzyme ratios after 1 h of reaction at 30℃.

[0061] Table 3. Substrate consumption ratio at 30℃ for one hour under different enzyme / substrate ratios.

[0062] The results showed that the substrate consumption of the TEV-Δ222 mutant, TEV-T173A mutant, TEV-S153N mutant, and TEV-Δ222 / K220M mutant was significantly higher than that of the wild-type control, with the TEV-Δ222 mutant having the highest substrate consumption ratio.

[0063] Using the maximum response rate (Vmax) of wild-type TEV as a reference, with a value of 1, the relative activities of other mutants were obtained, as shown in Table 4. Figure 7 This is a graph showing the enzyme reaction rate results under the same conditions as the recombinant TEV protease.

[0064] Table 4. Enzyme reaction rate results

[0065] The results showed that the enzyme reaction rates of the TEV-Δ222 mutant, TEV-T173A mutant, TEV-S153N mutant, and TEV-Δ222 / K220M mutant were all significantly higher than those of the wild-type control, with the TEV-Δ222 mutant exhibiting the highest enzyme reaction rate.

[0066] 3. Results of TEV mutant protease yield The TEV mutant protease content in each liter of the culture containing the TEV-Δ222 mutant prepared in Example 1 was measured to obtain the TEV-Δ222 mutant yield. The TEV mutant protease content was quantitatively detected by UV, and the detection method is as follows.

[0067] The TEV-Δ222 mutant stock solution prepared in Example 1 was quantified by UV spectroscopy. The extinction coefficient of the corresponding mutant was calculated based on the amino acid sequence. The absorbance value at UV280 was detected. The protein concentration c was calculated according to Formula II, and then the protein content was obtained.

[0068] c = A / ε (Formula II) Where A is the absorbance (OD value) at UV280; ε is the extinction coefficient (the absorbance value A of the sample at UV280 when the protein concentration is 1 mg / ml); and c is the protein concentration in the solution (mg / mL).

[0069] The content of other TEV mutant proteases was detected using the same method, and the yields of other TEV mutants were obtained. The results are shown in Table 5.

[0070] Table 5 Yields of different TEV mutants

[0071] The yield results indicate that multiplying the yield of each mutant by its specific activity for the same fermentation volume can show the total viability per unit fermentation volume.

[0072] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. Other embodiments can be obtained based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A mutant of the tobacco etch virus TEV protein, characterized in that, Based on the TEV protein, perform at least one of the following mutations: (1) mutate position 220 to methionine, (2) delete positions 222-242, (3) mutate position 153 to aspartic acid, and (4) mutate position 173 to alanine; The amino acid sequence of the TEV protein is shown in SEQ ID NO:

1.

2. The mutant according to claim 1 or 2, characterized in that, The amino acid sequence of the mutant is any one of those shown in SEQ ID NO:2 to SEQ ID NO:

5.

3. A gene encoding the mutant of claim 1 or 2.

4. A derivative product containing the gene of claim 3, characterized in that, A recombinant vector and / or recombinant bacteria containing the gene of claim 4.

5. The derivative product according to claim 4, characterized in that, The backbone vector of the recombinant expression vector preferably includes pET28a; the host bacterium of the recombinant bacteria preferably includes Escherichia coli.

6. A method for preparing the mutant according to claim 1, characterized in that, Includes the following steps: The mutant described in claim 2 was recombinantly expressed in a host strain, and the recombinant protein was isolated to obtain the mutant.

7. The use of the mutant of claim 1 or 2, or the mutant prepared by the derivative of claim 4 or 5, or the preparation method of claim 6, in the cleavage of the fusion tag and / or the target protein.

8. The application according to claim 7, characterized in that, The target protein includes a fluorescent substrate protein, which includes a fluorescent substrate protein used to detect the activity of tobacco etch virus TEV protease.

9. The application according to claim 8, characterized in that, The amino acid sequence of the fluorescent substrate protein is shown in SEQ ID NO:

10.

10. The application according to claim 8, characterized in that, The nucleotide sequence of the fluorescent substrate protein is shown in SEQ ID NO:11.

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