TEV protease variant and application thereof

Abstract

The invention relates to TEV protease, in particular to a TEV protease variant and application thereof. The invention provides a TEV protease mutant, the nucleotide sequence of the TEV protease mutant is shown as SEQ.ID.2, and the amino acid sequence of the TEV protease mutant is shown as SEQ.ID.4. According to the invention, a gene of a TEV mutant is integrated into a pET28b vector and is transformed into a BL21 competent cell for expression, and protein is purified through IMAC (immobilized metal chromatography) and SEC (molecular exclusion chromatography). The TEV mutant shows higher expression quantity which is increased by 6 times compared with wild type TEV, meanwhile, the thermal stability and the activity of the TEV mutant are also improved, and part of protease is still remained after the TEV mutant is quickly heated at 95 DEG C, and the activity is still kept.

Description

Technical Field

[0001] This invention relates to TEV proteases, and more particularly to a TEV protease variant and its applications. Background Technology

[0002] TEV protease is one of the most widely used tool proteases. Derived from tobacco etching virus, it is a 27 kDa cysteine ​​protease with extremely high sequence specificity, precisely recognizing substrate sequences composed of seven amino acid residues (ENLYFQG). It is widely used in research fields such as recombinant protein tag excision and protein-protein interaction detection. Although researchers have developed various mutants to improve its expression level and enzyme activity, such as the S219V mutant which not only completely eliminates autocleavage but also significantly improves stability compared to the wild type, this mutant is still prone to precipitation at high concentrations, indicating that its stability needs further improvement. The carboxyl terminus of this variant is prone to self-degradation (Parks, 1995), and the heterologous expression level is also low. This limits the large-scale application and promotion of this protease. Summary of the Invention

[0003] Purpose of the invention: This invention provides a recombinant TEV protease mutant with higher thermal stability, activity and expression level.

[0004] Technical solution: The present invention provides a TEV protease mutant, the nucleic acid sequence of which is shown in SEQ.ID.2 and the amino acid sequence of which is shown in SEQ.ID.4.

[0005] Application of the TEV protease mutant in the preparation of excision tool enzymes for recombinant protein tags.

[0006] Application of the TEV protease mutant in the preparation of reagents for detecting protein interactions.

[0007] Specifically as follows: All modifications in this invention are based on Super TEV, such as sequence SEQ.ID.1, which we consider to be wild type; Compared with wild-type TEV protease, this invention has a truncation of amino acids 1-10 at the N-terminus and contains the following mutations: The 12th amino acid is mutated from asparagine (N) to aspartic acid (D). The 52nd amino acid is mutated from asparagine (N) to aspartic acid (D). The 73rd amino acid is mutated from glutamine (Q) to glutamic acid (E). The 104th amino acid is mutated from glutamine (Q) to glutamic acid (E). The 105th amino acid is mutated from arginine (R) to lysine (K). The 115th position is mutated from asparagine (N) to aspartic acid (D). The 117th amino acid is mutated from glutamine (Q) to glutamic acid (E). The 145th position is mutated from glutamine (Q) to glutamic acid (E). The 185th amino acid is mutated from asparagine (N) to aspartic acid (D). The 193rd amino acid is mutated from glutamine (Q) to glutamic acid (E). The 197th amino acid is mutated from glutamine (Q) to glutamic acid (E). The 203rd position is mutated from arginine (R) to lysine (K).

[0008] The specific comparison is as follows: .

[0009] Table 1. Nucleotide sequence of TEV protease: .

[0010] Table 2 shows the amino acid sequence of the TEV protease: .

[0011] Note: HHHHHH represents 6x His-tag.

[0012] The key features of this invention are as follows: 1) Design of TEV protease truncation and mutation.

[0013] 2) Higher expression levels, thermal stability, and enzyme activity.

[0014] 3) It retains its activity even after rapid heating to 95℃.

[0015] Beneficial Effects: This invention integrates the TEV mutant gene into the pET28b vector and transforms it into BL21 competent cells for expression. The protein is then purified using IMAC (immobilized metal chromatography) and SEC (size exclusion chromatography). The TEV mutant exhibits significantly higher expression levels, up to 6 times that of wild-type TEV (here, wild-type refers to the highly active SuperTEV variant), while also demonstrating improved thermostability (by approximately 10°C) and activity. Data shows that even under rapid heating at 95°C, some protease remains, and this remaining protease retains its activity. Attached Figure Description

[0016] Figure 1 Plasmid maps of wild-type TEV (A) and mutant TEV (B); Figure 2Molecular sieve chromatography images of wild-type TEV (A) and mutant TEV (B); Figure 3 Gel images of purified wild-type TEV (A) and mutant TEV (B); Figure 4 Thermostatus gel images of wild-type TEV (A) and mutant TEV (B); Figure 5 The activity comparison between wild-type TEV (A) and mutant TEV (B) was performed from 1 to 24 hours. The left figure shows the results of wild-type TEV after enzyme digestion at 1, 2, 4, 8, and 24 hours, and the right figure shows the results of mutant TEV after digestion at 1, 2, 4, 8, and 24 hours. Figure 6 Michaelis-Menten curves for wild-type TEV and mutant TEV; Figure 7 The enzyme digestion activities of wild-type TEV and mutant at 37, 45, and 55 °C are shown. A is a gel image, and B is a fluorescence image. Figure 8 The activity of wild-type and mutant TEV was compared after two hours of digestion at 4℃ (containing DTT), 30℃ (containing DTT), and 30℃ (without DTT). Figure 9 The enzyme digestion activities of wild-type and mutant TEV were compared after heating at 30℃, 45℃, 55℃ and 95℃. Detailed Implementation

[0017] Example 1, plasmid construction: Using molecular cloning technology, wild-type and mutant TEV proteases were synthesized from the whole genome. Seamless cloning was employed, and the target gene was ligated into the pET28a vector. The resulting cells were transformed into DH5α competent cells via heat shock and plated on plates containing 50 ng / μL kanamycin resistance. After overnight culture, single colonies were picked and cultured in 4 mL LB broth, and plasmids were extracted and sequenced. The plasmid structure is shown below. Figure 1 As shown.

[0018] Example 2, Expression of TEV protease: The correctly sequenced plasmid was transformed into BL21(DE3) competent cells using the heat shock method, plated on kanamycin-resistant plates, and cultured overnight. The colonies on the plate were scraped off with 10 mL LB and transferred to 500 mL LB. The kanamycin concentration was increased to 50 ng / uL, and the cells were cultured at 37°C until OD600 = 0.6. Then, 200 uL of 1M IPTG was added, and the culture temperature was adjusted to 25°C and cultured for 12-15 h.

[0019] Result: As Figure 2 As shown, both wild-type and mutant TEV proteases were successfully expressed.

[0020] Example 3, TEV protein purification: Transfer the overnight cultured bacterial culture to a 1L centrifuge cup and centrifuge at 4000 rpm for 20 min using a floor-standing centrifuge. Discard the supernatant and resuspend the cells in 35 mL of Tris-NaCl. Disrupt the cells using an ultrasonic homogenizer with a program of 2 seconds on, 3 seconds off, for a total of 20 min. Transfer the disrupted solution to centrifuge tubes and centrifuge at 16000 rpm for 30 min using an ultracentrifuge. Retain the supernatant, discard the precipitate, and keep the protein supernatant. Equilibrate the nickel gravity column (NTA-Ni) with 4-6 column volumes of water and Tris-NaCl. Pass the protein supernatant through the gravity column twice. Wash away contaminating proteins with 10 column volumes of Tris-NaCl containing 20 mM imidazole, and then elute the target protein with 4-6 column volumes of Tris-NaCl containing 300 mM imidazole. The obtained protein eluent was concentrated to less than 1 mL using a 10 kDa (Sigma) concentrator tube, centrifuged at 15,000 rpm for 30 min to remove the precipitate, and then centrifuged at 15,000 rpm for 10 min to remove air bubbles. The protein sample was further purified by AKTA chromatography system. The A280 peak and the protein solutions from the two tubes before and after the AKTA were collected, and the purity was analyzed by SDS-PAGE.

[0021] Example 4, Comparison of wild-type and mutant TEV proteases: 1. Comparison of protein expression levels Following Examples 1-3, wild-type and mutant TEV proteases were expressed. When purifying the proteins using IMAC, only one-quarter of the protein supernatant was used for purification. Finally, the protein expression level was analyzed by size exclusion chromatography and SDS-PAGE.

[0022] Result: As Figure 3 As shown, the expression level of mutant TEV protease was significantly higher than that of wild-type.

[0023] 2. TEV thermal stability test Purified TEV protease was collected, and its protein concentration was measured using NanoDrop. 1.2 mL of a 10 μM protein solution was prepared, and 100 μL was transferred to a PCR tube. The tube was centrifuged and placed in a PCR instrument with temperature gradients of 4, 25, 35, 45, 55, 65, 75, 85, and 95 °C. After heating at each temperature gradient for 10 minutes, the tube was centrifuged, and 10 μL of the supernatant was collected. 10 μL of 2x protein loading buffer was added, and SDS-PAGE was used to analyze the residual protein amount after heating at each temperature, reflecting its thermal stability.

[0024] The results are as follows Figure 4As shown, wild-type TEV protease precipitates most of the protein after heating at 45°C, and no protein residue is observed under subsequent temperature conditions. In contrast, mutant TEV protease shows almost no denaturation or precipitation at 45°C, and significant protein residue is observed after heating at 95°C.

[0025] 3. TEV activity within 1-24 hours Prepare a 400 μM stock solution of TEV protease substrate (MBP-TEVcs-mCherry); 1 μM stock solutions of wild-type and mutant TEV protease; and a 160 mM stock solution of DTT (freshly prepared). In a PCR tube, add 35 μL of TEV protease substrate, 2 μL of TEV protease, 0.5 μL of DTT, and 2.5 μL of Tris-NaCl buffer. Centrifuge and immediately place in a 30°C water bath. Samples are taken at 1, 2, 4, 8, and 24 hours. For each sample, centrifuge first, then take 5 μL of the reaction solution, dilute with 25 μL of Tris-NaCl buffer, centrifuge, and take 10 μL of the diluted sample. Add 10 μL of 2x protein loading buffer to terminate the reaction, mix well, and immediately freeze in liquid nitrogen at -80°C. After all samples have been collected, analyze the enzyme digestion efficiency of wild-type and mutant TEV protease using SDS-PAGE.

[0026] Results: See attached. Figure 5 As shown, by reducing the substrate and increasing the product generation reaction cleavage rate, the mutant TEV protease showed significantly higher cleavage efficiency than the wild type at all time points.

[0027] 4. Testing the Michaelis constant Prepare stock solutions of wild-type and mutant TEV protease at 1 μM, stock solutions of TEV protease substrate (MBP-TEVcs-mCherry) at 400 μM, and stock solutions of DTT at 40 mM (freshly prepared). The concentrations of wild-type and mutant TEV protease were kept constant at 50 nM, and the DTT concentration at 2 mM. Substrate concentrations were 10, 20, 50, 100, 200, and 300 μM, with a total volume of 40 μL. After mixing, the mixture was immediately incubated at 30°C for one hour, and three parallel experiments were performed. After the reaction was complete, protein loading buffer was added immediately to terminate the reaction. The digestion rate was analyzed by SDS-PAGE. The resulting gel images were scanned using a gel imaging system to obtain fluorescence images. ImageJ was then used to calculate the protein bands as the digestion yield of the protease at each substrate concentration, which was then converted into the digestion rate. Finally, the Michaelis constant was fitted using GraphPad Prism.

[0028] Results: See attached. Figure 6As shown, the Km and Kcat values ​​of the mutant TEV protease were significantly higher than those of the wild type. Specifically, for the wild type TEV: Km: 215.9 uM Kcat: 0.138; for the mutant TEV: Km: 331.6 uM Kcat: 0.248.

[0029] 5. Activity test of TEV protease at 37, 45, and 55℃ The concentrations of constant wild-type and mutant TEV protease were 50 nM, the concentration of enzyme digestion substrate (MBP-TEVcs-mCherry) was 350 μM, the concentration of DTT (freshly prepared) was 2 mM, and the total volume was 40 μL. The mixture was thoroughly mixed, and a blank control was set up. The reaction was carried out at 37, 45, and 55 °C for two hours. After the reaction was completed, the mixture was centrifuged, the supernatant was collected, and protein loading buffer was added. The enzyme digestion efficiency was analyzed by SDS-PAGE.

[0030] Results: See attached. Figure 7 As shown, the cleavage activity of the mutant TEV protease at 37 and 45 ℃ was significantly higher than that of the wild type.

[0031] 6. Activity of TEV protease at 4℃ (containing DTT), 30℃ (containing DTT), and 30℃ (without DTT). The concentrations of constant wild-type and mutant TEV protease were 50 nM, the concentration of enzyme digestion substrate (MBP-TEVcs-mCherry) was 350 μM, the concentration of DTT (freshly prepared) was 2 mM, and the total volume was 40 μL. After centrifugation and mixing, a blank control was also set up. The reaction was carried out at 4℃ and 30℃ for 2 hours. At the same time, a digestion sample without DTT was prepared at 30℃. After the reaction was completed, protein loading buffer was added immediately to terminate the reaction, and the enzyme digestion efficiency was analyzed by SDS-PAGE.

[0032] Results: See attached. Figure 8 As shown, the mutant TEV protease has slightly higher activity than the wild-type TEV protease at 4℃, and at 30℃, the mutant protease has significantly higher activity than the wild-type protease in both the presence and absence of DTT.

[0033] 7. Activity of TEV protease after rapid heating at high temperature The concentrations of wild-type and mutant TEV protease were kept constant at 10 μM. After heating at 4, 30, 45, 55, and 95 °C for 10 minutes, the mixture was centrifuged, and 12 μL of the supernatant was collected. 18 μL of Tris-NaCl buffer was added, and the mixture was mixed thoroughly. The substrate concentration of TEV protease was kept constant at 300 μM, and the concentration of DTT (freshly prepared) was 2 mM. 1 μL of the heated TEV protease diluted sample was added, bringing the total volume to 20 μL. The mixture was centrifuged and mixed thoroughly, and the reaction was carried out at 30 °C for 1 hour. Immediately after the reaction was complete, protein loading buffer was added to terminate the reaction. Enzyme digestion activity was analyzed by SDS-PAGE.

[0034] Results: See attached. Figure 9 As shown, the wild type has almost no activity after heating at 95°C, while the mutant TEV protease still retains some enzymatic activity after heating at 95°C.

Claims

1. A TEV protease mutant, characterized in that, Its nucleic acid sequence is shown in SEQ.ID.2, and its amino acid sequence is shown in SEQ.ID.

4.

2. The application of the TEV protease mutant according to claim 1 in recombinant protein tag excision.

3. The application of the TEV protease mutant according to claim 1 in the preparation of reagents for detecting protein interactions.

Images