HRV3C protease mutant and application thereof

Abstract

The invention relates to protease, in particular to an HRV3C virus protease mutant and application thereof. The HRV3C protease variant is characterized in that the amino acid sequence of the HRV3C protease variant is as shown in SEQ ID NO. 4. The HRV3C protease mutant provided by the invention is not easy to aggregate and precipitate, tolerant to imidazole, obviously higher in thermal stability and yield than a wild type, and equivalent to the wild type in activity.

Description

Technical Field

[0001] This invention relates to proteases, and more specifically to an HRV3C protease mutant and its applications. Background Technology

[0002] HRV3C protease, derived from human rhinovirus, belongs to the serine protease class. Its core characteristic lies in its high substrate recognition specificity; it can precisely recognize the octapeptide sequence Leu-Glu-Val-Leu-Phe-Gln-Gly-Pro or the core pentapeptide sequence Leu-Phe-Gln-Gly-Pro, and specifically cleave between the glutamine (Gln) and glycine (Gly) amino acid residues. The optimal cleavage temperature of HRV3C protease is 4℃-30℃, compared to 30℃ for TEV protease, making it more suitable for proteins with poor thermostability. Based on this characteristic, HRV3C protease is widely used in the biotechnology field for the removal of fusion protein tags, such as effectively removing glutathione S-transferase (GST) tags, maltose-binding protein (MBP) tags, small ubiquitin-like modified protein (SUMO) tags, and polyhistidine (His-Tag) tags, providing crucial support for the isolation and purification of target proteins.

[0003] However, the HRV3C protease still faces many unresolved technical challenges. The HRV3C protease is primarily prepared using an E. coli heterologous expression system and purified via polyhistidine tagging and immobilized metal chromatography (IMAC). However, the protease suffers from poor stability; high concentrations of imidazole and protease itself easily lead to protein precipitation, limiting its large-scale production and application. Some literature reports the use of MBP or GST tags for affinity purification, but these purification methods require expensive starch or glutathione resins, significantly increasing production costs without improving yield.

[0004] Therefore, it is necessary to improve the stability of HRV3C protease, enhance its solubility, and increase its yield, thereby reducing costs and facilitating its wider application. Summary of the Invention

[0005] Purpose of the invention: This invention provides an HRV3C protease mutant that is not easily precipitated, is resistant to high concentrations of imidazole, has higher thermal stability and yield, while having activity comparable to the wild type.

[0006] Technical solution: The present invention provides an HRV3C protease mutant, the nucleic acid sequence of which is shown in SEQ ID NO.2 and the amino acid sequence of which is shown in SEQ ID NO.4; Specifically, compared to the wild-type HRV3C protease, it has the following mutations: amino acid 52 is changed from glutamine to glutamic acid, amino acid 67 is changed from asparagine to aspartic acid, amino acid 80 is changed from asparagine to aspartic acid, amino acid 107 is changed from asparagine to aspartic acid, and amino acid 110 is changed from asparagine to aspartic acid.

[0007] The application of the HRV3C protease in the removal of recombinant protein fusion tags.

[0008] The application of the HRV3C protease in detecting protein-protein interactions.

[0009] This invention purifies HRV3C protease using IMAC (immobilized metal chromatography). Compared with the wild type, the mutant is significantly more tolerant to high concentrations of imidazole and exhibits higher thermal stability.

[0010] The key points of this invention are as follows: 1) An amino acid mutation was introduced into wild-type HRV3C. 2) Compared to the wild type, the mutant exhibits improved thermal stability and increased yield. 3) The mutant HRV3C protease can tolerate high concentrations of imidazole. 4) The mutant HRV3C protease can withstand rapid heating at 95°C and still maintain its activity.

[0011] 5) Mutant proteases can be used to remove fusion protein tags.

[0012] Beneficial effects: The HRV3C protease mutant provided by this invention, purified by immobilized metal chromatography (IMAC), can tolerate high concentrations of imidazole without aggregation and precipitation, has high yield, and has significantly higher thermal stability than the wild type. It can withstand rapid heating to 95°C and still maintain its activity. Attached Figure Description

[0013] Figure 1 Plasmid maps of wild-type HRV3C protease (A) and mutant HRV3C protease (B); Figure 2 Molecular sieve diagrams showing the purification of wild-type HRV3C protease and mutant HRV3C protease; Figure 3 Gel images of proteins purified by molecular sieve from wild-type HRV3C protease and mutant HRV3C protease; Figure 4 The stability of wild-type HRV3C protease and mutant HRV3C protease under high concentrations of imidazole; Figure 5Gel images of wild-type HRV3C protease and mutant HRV3C protease after heating at 4℃, 25℃, 35℃, 45℃, 55℃, 65℃, 75℃, 85℃, and 95℃. Figure 6 Comparison of the enzymatic cleavage activities of wild-type HRV3C protease and mutant HRV3C protease at 1, 2, 4, 8, and 24 hours; Figure 7 Michaelis constants for wild-type HRV3C protease and mutant HRV3C protease; Figure 8 This study compares the activities of wild-type HRV3C protease and mutant HRV3C protease after heating at 4℃, 45℃, 55℃, 65℃, 75℃, 85℃, and 95℃. Detailed Implementation

[0014] Table 1 Nucleotide sequence list: .

[0015] Table 2 Amino acid sequence listing: ; Note: HHHHHH represents His-tag.

[0016] Example 1: Construction and expression of wild-type and mutant HRV3C protease expression plasmids: The nucleotide sequence encoding the wild-type HRV3C protease is shown in SEQ ID NO.1, and the amino acid sequence is shown in SEQ ID NO.3. It was cloned into the pCDFDuet1 vector. A site-directed mutagenesis technique was used to introduce a mutation, resulting in a plasmid expressing the mutant HRV3C protease. The nucleotide sequence encoding the mutant HRV3C protease is shown in SEQ ID NO.2, and the amino acid sequence is shown in SEQ ID NO.4; sequencing was performed to ensure the sequence was correct.

[0017] The correctly sequenced plasmid was transformed into BL21(DE3) competent cells using a heat shock method. The transformed cells were then plated on agar plates containing 50 μg / mL streptomycin and incubated overnight at 37°C. Colonies were scraped from the solid medium and resuspended in 10 mL LB broth. This was then inoculated into 500 mL LB broth and cultured at 37°C until an OD600 of 0.6 was achieved. Subsequently, IPTG at a final concentration of 0.4 mM was added to induce the expression of wild-type HRV 3C protease or its mutant, and the cells were cultured at 25°C for 12–15 h.

[0018] Example 2, Purification of wild-type and mutant HRV3C protease: Transfer the overnight culture to a centrifuge flask and centrifuge at 2500g for 20 minutes to collect the bacterial cells. Resuspend the cells in approximately 35 mL of lysis buffer (20mM Tris-Cl, 150mM NaCl), then use an ultrasonic disruptor to disrupt the *E. coli* cells. The instrument parameters are set as follows: sonication on for 2 seconds, off for 3 seconds, for a total duration of 20 minutes. Transfer the disrupted solution to centrifuge tubes and centrifuge at 30,000g for 30 minutes to remove cell debris and undisrupted cells. Collect the supernatant containing soluble proteins.

[0019] The obtained protein supernatant was purified by immobilized metal ion affinity chromatography (IMAC). The protein supernatant was passed twice through a gravity column packed with nickel ion packing material, followed by washing with 20 volumes of buffer containing 20 mM imidazole (20 mM Tris-Cl, 150 mM NaCl, 20 mM imidazole) to remove contaminating proteins. Then, the target protein (i.e., wild-type or mutant HRV3C protease) was eluted with 4-6 volumes of buffer containing 300 mM imidazole (20 mM Tris-Cl, 150 mM NaCl, 300 mM imidazole).

[0020] The eluted protein solutions were transferred to concentration tubes with a molecular weight cutoff of 10 kDa and concentrated to a volume of approximately 1 mL by centrifugation at 2500 g. Further purification was performed using molecular sieve chromatography to remove imidazole and other impurities, such as... Figure 2 As shown; protein components corresponding to the ultraviolet absorption peaks were collected, and protein purity was analyzed by SDS-PAGE, as shown. Figure 3 As shown.

[0021] Example 3, Effects of high concentrations of imidazole on wild-type and mutant HRV3C protease: Add 300 mM imidazole to both wild-type and mutant proteins, and incubate on ice for 10 minutes. Figure 4 As shown, wild-type HRV3C protease exhibited significant precipitation, while no precipitation was observed in the mutant.

[0022] Example 4, Comparison of protease thermal stability: 1.2 mL of HRV3C protease purified by molecular sieve was used to prepare a protein solution with a final concentration of 20 μM. 100 μL of each solution was transferred to nine PCR tubes, centrifuged, and then placed in a PCR instrument. The tubes were heated at 4℃, 25℃, 35℃, 45℃, 55℃, 65℃, 75℃, 85℃, and 95℃ for 10 minutes each. After centrifugation, 10 μL of the supernatant was collected from each tube, and 10 μL of 2× protein loading buffer was added. SDS-PAGE analysis was performed to analyze the amount of residual protein in the supernatant after heating at different temperatures. The results are as follows: Figure 5As shown, after heating to 55°C, the wild-type HRV3C protease showed no protein residue in the supernatant, as evidenced by the absence of corresponding bands on the proteoglycan, with only a very small amount remaining at 95°C. In contrast, the mutant HRV3C protease still showed significant protein residue in the supernatant at 55°C, and the amount of residual protein in the supernatant gradually increased with increasing temperature. This indicates that the mutant can tolerate higher temperatures and has greater thermal stability than the wild-type.

[0023] Example 5, Comparison of protease activities: 1. Removal of protein tags The MBP-tagged mCherry fusion protein (containing the HRV3C protease cleavage site), MBP-HRV3Ccs-mCherry, was used as the substrate. A 400 μM stock solution of MBP-HRV3Ccs-mCherry and 1 μM stock solutions of wild-type and HRV3C protease mutants were prepared. In two PCR tubes, 35 μL of HRV3C protease substrate, 3 μL of lysis buffer (20 mM Tris-Cl, 150 mM NaCl), and 2 μL of either the wild-type or mutant HRV3C protease were added, mixed, and centrifuged. The tubes were immediately placed on ice at 4°C, and samples were collected at 1, 2, 4, 8, and 24 hours. Each sample was centrifuged first, then 5 μL of the reaction solution was taken, diluted with 25 μL of buffer solution (20 mM Tris-Cl, 150 mM NaCl), centrifuged again, and 10 μL of the reaction solution was taken and mixed with 10 μL of 2× protein loading buffer. The mixture was then immediately flash-frozen in liquid nitrogen and stored at -80°C until use. After all samples were collected, the enzyme digestion efficiency of wild-type and mutant strains was analyzed by SDS-PAGE. The results are attached. Figure 6 As shown, both the reduction of substrate and the formation of product can reflect the enzyme digestion efficiency. The mutant HRV3C protease showed significantly better digestion performance than the wild type at all time points.

[0024] 2. Determination of Michaelis constant A stock solution of wild-type and mutant HRV3C protease at a concentration of 1 μM and a substrate concentration of 400 μM were prepared. The concentrations of wild-type and mutant HRV3C protease were kept constant at 50 nM, and the substrate concentrations were 10, 20, 50, 100, 200, and 300 μM, with a total volume of 40 μL. After mixing, the mixture was immediately placed on ice at 4°C for one hour. After the reaction was complete, protein loading buffer was added immediately to terminate the reaction, and SDS-PAGE was performed. Red fluorescence images were obtained by scanning the gel using a gel imaging system after electrophoresis. The experiment was repeated three times. ImageJ was used to process the images, the enzyme digestion rate at each substrate concentration was calculated, and the Michaelis constant was fitted using GraphPad Prism. The results are attached. Figure 7As shown, the Km and Kcat values ​​of the HRV3C protease mutant are higher than those of the wild type. Specifically, for the HRV3C wild type: Km: 189 μM, Kcat: 0.355; for the HRV3C mutant: Km: 291.6 μM, Kcat: 0.467. The Kcat / Km value of the mutant is comparable to that of the wild type.

[0025] 3. Activity assay of wild-type and mutant HRV3C proteases after heating at various temperatures The concentrations of constant wild-type and mutant HRV3C protease were 10 μM. After heating at 4℃, 45℃, 55℃, 65℃, 75℃, 85℃, and 95℃ for 10 minutes each, centrifugation was performed. 12 μL of the supernatant was collected and added to 18 μL of buffer solution (20 mM Tris-Cl, 150 mM NaCl), mixed well, and set aside. In 14 PCR tubes, the constant HRV3C protease substrate concentration was 300 μM. 1 μL of either heated and diluted wild-type or mutant HRV3C protease was added to each tube, resulting in a total volume of 20 μL. The mixture was mixed and incubated at 4℃ on ice for one hour. Immediately after the reaction was complete, 2× protein loading buffer was added to terminate the reaction. Enzyme digestion activity was analyzed by SDS-PAGE. Results are attached. Figure 8 As shown, the wild type showed only minimal activity after heating at 95°C, while the mutant HRV3C protease maintained activity after heating at 55°C, 65°C, 75°C, 85°C, and 95°C.

Claims

1. An HRV3C protease mutant, characterized in that, Compared to the wild-type HRV3C protease, it has the following amino acid mutations: amino acid at position 52 is mutated from glutamine to glutamic acid, amino acid at position 67 is mutated from asparagine to aspartic acid, amino acid at position 80 is mutated from asparagine to aspartic acid, amino acid at position 107 is mutated from asparagine to aspartic acid, and amino acid at position 110 is mutated from asparagine to aspartic acid; the amino acid sequence of the wild-type HRV3C protease is shown in SEQ ID NO.3, and the amino acid sequence of the HRV3C protease mutant is shown in SEQ ID NO.

4.

2. The application of the HRV3C protease according to claim 1 in the removal of recombinant protein fusion tags.

3. The application of the HRV3C protease according to claim 1 in the detection of protein-protein interactions.

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