A 3c protease mutant, its preparation method and application

Abstract

This invention discloses a 3C protease mutant, its preparation method, and its applications, relating to the field of bioengineering technology. The 3C protease mutant is obtained by modifying the amino acid sequence of the wild-type 3C protease. The 3C protease mutants are 3C protease-M1, 3C protease-M2, or 3C protease-M3, with amino acid sequences shown in SEQ ID NO. 1-3, respectively. Compared to the wild-type 3C protease, the three 3C protease mutants exhibit higher enzyme activity. The activities of 3C protease-M1, 3C protease-M2, and 3C protease-M3 are increased by 3.37, 3.57, and 1.73 times, respectively, compared to the wild-type. In addition, the three 3C protease mutants also have higher thermal stability. Compared with wild-type 3C protease, the thermal stability of 3C protease-M1 increased by 21.69℃, 3C protease-M2 increased by 24.69℃, and 3C protease-M3 increased by 18.86℃, which has a wider range of application conditions and is more suitable for large-scale production and industrial use.

Description

Technical Field

[0001] This invention relates to the field of bioengineering technology, specifically to a 3C protease mutant, its preparation method, and its application. Background Technology

[0002] To eliminate the influence of purification tags on the physical structure, physiological activity, and immunogenicity of the target protein, the purified fusion protein needs to have its purification tag removed. A common method is to use proteases to cleave the fusion tag. Moreover, proteases have high cleavage specificity, and there are many proteases to choose from, making proteases the preferred method for cleaving fusion proteins.

[0003] 3C protease is a cysteine ​​protease derived from human rhinovirus (HRV). It exhibits strong site specificity, specifically recognizing the "LEVLFQGP" sequence in target proteins and cleaving it between the Q and G amino acid residues. HRV 3C protease is characterized by high enzymatic activity, maintaining high activity even at 4°C. It is currently commonly used to remove GST, His, or other protein tags from fusion proteins, making it a frequently used tool enzyme for cleaving affinity tags on fusion proteins.

[0004] Currently, the thermostability and activity of wild-type 3C proteases need to be improved, as they are unstable at high temperatures, limiting their application. To broaden the application conditions of 3C proteases and make them more suitable for large-scale production and industrial applications, this invention proposes a 3C protease mutant, its preparation method, and its application. Summary of the Invention

[0005] The purpose of this invention is to further improve the thermostability and activity of 3C protease by providing a 3C protease mutant, its preparation method, and its application.

[0006] The present invention achieves the above objectives through the following technical solutions:

[0007] As a first aspect of the present invention, a 3C protease mutant is provided, which is obtained by modifying the amino acid sequence of the wild-type 3C protease. The 3C protease mutant is 3Cprotease-M1, 3C protease-M2 or 3C protease-M3, and the amino acid sequences of 3C protease-M1, 3C protease-M2 or 3C protease-M3 are shown in SEQ ID NO.1-3, respectively.

[0008] As a second aspect of the invention, a polynucleotide is also provided, said polynucleotide encoding the 3C protease mutant as described in claim 1.

[0009] As a further optimization of the present invention, the polynucleotide sequences encoding 3C protease-M1, 3C protease-M2 or 3C protease-M3 are shown in SEQ ID NO.7-9, respectively.

[0010] As a third aspect of the present invention, a recombinant plasmid is also provided, wherein the recombinant plasmid is an expression vector containing any of the polynucleotide sequences described above and capable of correspondingly translating and expressing any of the 3C protease mutants described above, wherein the expression vector is pCDFDuet-1.

[0011] As a fourth aspect of the invention, an expression system for a 3C protease mutant is also provided, comprising BL21(DE3) cells containing any of the recombinant plasmids described above or whose genomes integrate any of the polynucleotides described above.

[0012] As a fifth aspect of the present invention, a method for preparing a 3C protease mutant as described in any of the above-described steps is also provided, comprising the following steps:

[0013] (1) Gene sequences that can encode the amino acid sequences of 3C protease-M1, 3C protease-M2 or 3C protease-M3 are synthesized, and the gene sequences are then constructed on expression vectors to obtain recombinant plasmids.

[0014] (2) The recombinant plasmid obtained in step (1) is expressed using the Escherichia coli prokaryotic expression system to obtain the expression product. The 3C protease mutant can be obtained by purifying the expression product.

[0015] As a further optimization of the present invention, in order to facilitate the purification of the expression product, a Strep II tag is added to the N-terminus of the amino acid sequence of 3C protease-M1, 3C protease-M2 or 3C protease-M3, and the expression product is purified by Strep-Tactin XT affinity chromatography column.

[0016] As a sixth aspect of the present invention, the application of the 3C protease mutant as described in any of the above-described methods as a proteolytic enzyme in protein purification is also provided.

[0017] The present invention has the following beneficial effects:

[0018] This invention designs and modifies the amino acid sequence of the wild-type 3C protease to obtain three 3C protease mutants: 3C protease-M1, 3C protease-M2, and 3C protease-M3. Compared to the wild-type 3C protease, these mutants exhibit high activity and thermostability, specifically in the following aspects:

[0019] Regarding the thermostability of the proteases, the Tm value of 3C protease-M1 was 66.68℃, that of 3C protease-M2 was 69.68℃, the Tm value of 3C protease-M3 was 63.85℃, and the Tm value of wild-type 3C protease was 44.99℃. Compared with wild-type 3C protease, the thermostability of 3C protease-M1 increased by 21.69℃, that of 3C protease-M2 increased by 24.69℃, and that of 3C protease-M3 increased by 18.86℃. These three 3C protease mutants exhibited higher stability than the wild-type 3C protease.

[0020] Regarding protease activity, the activity of 3C protease-M1 was measured to be 27.48 × 10⁻⁶. -4 (RFU / s / nM), the activity of 3Cprot ease-M2 was determined to be 29.07 × 10⁻⁶. -4 (RFU / s / nM), the activity of 3C protease-M3 was determined to be 14.12 × 10⁻⁶. -4 (RFU / s / nM), the activity of wild-type 3C protease WT was 8.15 × 10⁻⁶. -4 (RFU / s / nM). The activities of 3C protease-M1, 3C protease-M2, and 3C protease-M3 were increased by 3.37, 3.57, and 1.73 times, respectively, compared with the wild type. The activities of the three 3C protease mutants, 3C protease-M1, 3C protease-M2, and 3C protease-M3, were significantly improved compared with the wild-type 3C protease.

[0021] In summary, compared with wild-type 3C protease, the high activity and high thermal stability of the three 3C protease mutants 3C protease-M1, 3C protease-M2, and 3C protease-M3 overcome the problem of enzyme instability at high temperatures that limits their application, providing better application scenarios for their storage and industrial production, making them more suitable for large-scale industrial use. Attached Figure Description

[0022] Figure 1 Results of low-level expression detection of five 3C protease mutants and wild-type 3C protease WT;

[0023] Figure 2 The results show the thermostability of five 3C protease mutants and the wild-type 3C protease WT.

[0024] Figure 3 The results of activity identification and detection of five 3C protease mutants and wild-type 3C protease WT;

[0025] Figure 4 The results show the affinity purification of the 3C protease mutant 3C protease-M2.

[0026] Figure 5 Results of enzyme digestion application for the 3C protease mutant 3C protease-M2. Detailed Implementation

[0027] The present application will now be described in further detail with reference to the accompanying drawings. It should be noted that the following specific embodiments are only used to further illustrate the present application and should not be construed as limiting the scope of protection of the present application. Those skilled in the art can make some non-essential improvements and adjustments to the present application based on the above application content.

[0028] 1. Materials and Reagents

[0029] Unless otherwise specified, all methods used in this invention are conventional methods known to those skilled in the art. Where specific conditions are not specified, they shall be performed according to conventional conditions or conditions recommended by the manufacturer. Where the manufacturers of reagents or instruments are not specified, they are all conventional products that can be purchased commercially.

[0030] 2. Method

[0031] 2.1 Construction of recombinant 3C protease plasmid

[0032] The recombinant 3C proteases provided by this invention are the wild-type 3C protease WT and its five mutant 3C proteases. The gene sequences of the wild-type 3C protease WT and its five mutant 3C proteases were obtained through gene synthesis. The five mutant 3C proteases are 3C protease-M1, 3C protease-M2, 3C protease-M3, 3C protease-M4, and 3C protease-M5. The amino acid sequences of the five mutant 3C proteases are shown in SEQ ID NO. 1-5, and the amino acid sequence of the wild-type 3C protease WT is shown in SEQ ID NO. 6. The gene sequences corresponding to 3C protease-M1, 3C protease-M2, and 3C protease-M3 are shown in SEQ ID NO. 7-9, respectively.

[0033] The genes synthesized from the wild-type 3C protease WT and its five 3C protease mutants were constructed into the expression vector pCDFDuet-1 (GenScript, pCDFDuet(TM)-1). To facilitate protein purification, a Strep II tag was added to the N-terminus of the sequences shown in SEQ ID NO. 1-6, the sequence of which is shown in SEQ ID NO. 10. All recombinant 3C protease plasmids were sequenced and verified to be completely identical to the target sequences.

[0034] 2.2 Small-scale expression test of recombinant 3C protease

[0035] Using standard molecular biology techniques, the constructed recombinant 3C protease plasmid was transformed into BL21(DE3) *E. coli* competent cells in a clean bench and cultured overnight at 37°C. Single colonies from the overnight culture were picked and transferred to 5 ml of LB broth and incubated at 37°C until the bacterial culture showed an OD value. 600 When the pH is 0.6-0.8, a small amount of bacterial culture is fixed with loading buffer, and a small amount of bacterial culture is added to glycerol and frozen to -80℃. The remaining bacterial culture is added to 0.5mM IPTG and induced at 15℃ for 16 hours. The bacterial cells are then collected and the induced bacterial culture is analyzed by SDS-PAGE.

[0036] See results Figure 1 Compared to the wild-type 3C protease WT, the target bands of the five 3C protease mutants—3C protease-M1, 3C protease-M2, 3C protease-M3, 3C protease-M4, and 3C protease-M5—were clearly visible and significantly expressed in BL21(DE3) Escherichia coli. Small-scale expression tests were collected and eluted samples were used for subsequent thermostability and activity assays.

[0037] 2.3 Thermal stability test of recombinant 3C protease

[0038] The thermal stability of recombinant 3C protease was tested using protein thermal shift (ThermoFluor). This technique utilizes the structural characteristics of proteins. Proteins possess hydrophobic regions hidden internally; as temperature rises, these regions open up, exposing the hydrophobic areas. The fluorescent dye SYPRO Orange binds to these regions, stimulating fluorescence. Changes in fluorescence intensity form a melting curve, and the temperature corresponding to the maximum derivative of the melting curve is the melting point (Tm). The more stable the protein, the higher the measured Tm value.

[0039] The specific procedure for determining the thermal stability of 3C proteases is as follows:

[0040] Add 3C protease to each well of a 96-well PCR plate, and then add 10×SYPRO Orange fluorescent dye (Thermoscientic, S6650) to the corresponding wells. Place the 96-well PCR plate in a qPCR instrument, set the instrument parameters, and increase the temperature from 25℃ to 99℃ at a gradient of 1℃ per minute. Calculate the protein melting curve.

[0041] The results are as follows Figure 2 As shown, the average Tm value of 3C protease-M1 after two tests was 66.68℃, the average Tm value of 3C protease-M2 after two tests was 69.68℃, the average Tm value of 3C protease-M3 after two tests was 63.85℃, the average Tm value of 3C protease-M4 after two tests was 66.06℃, the average Tm value of 3C protease-M5 after two tests was 64.58℃, and the average Tm value of wild-type 3C protease WT after two tests was 44.99℃.

[0042] Compared to the wild-type 3C protease WT, the thermostability values ​​of 3C protease-M1 increased by 21.69℃, 3C protease-M2 by 24.69℃, 3C protease-M3 by 18.86℃, 3C protease-M4 by 21.07℃, and 3C protease-M5 by 19.59℃. These results indicate that all five 3C protease mutants exhibit higher stability than the wild-type 3C protease WT.

[0043] 2.4 Activity test of recombinant 3C protease

[0044] The activity of the 3C protease was detected using fluorescence resonance energy transfer (FRET), specifically, in the presence of two different fluorescent groups, the emission spectrum of one fluorescent group (donor) overlaps to some extent with the absorption spectrum of the other group (acceptor), and the distance between the two fluorescent groups is less than [missing value]. Fluorescent energy is transferred from the donor to the acceptor, resulting in a significantly lower fluorescence intensity in the donor compared to its own (fluorescence quenching). FAM and TAMRA fluorescent groups are attached to both ends of the polypeptide sequence containing the specific recognition site. When the polypeptide is cleaved, the two fluorescent groups separate, releasing a strong fluorescent signal. The higher the activity of the 3C protease, the more fluorescent groups are released, and the greater the fluorescence intensity. Enzyme activity is expressed as the fluorescence intensity absorbed per nanomolar of protein per second.

[0045] The specific procedures for determining 3C protease activity are as follows:

[0046] The peptide substrate 5'-FAM-GLEVLFQGPENLYFQGSGK (TAMRA) was prepared as a 1 mM stock solution and aliquoted for use. The 3C protease activity assay buffer consisted of 50 mM Tris-HCl (pH 8.0), 500 mM NaCl, 1 mM DTT, 1 mM EDTA, and 5% glycerol. The peptide concentration was diluted to 100 nM with the buffer, and the 3C protease was serially diluted 2-fold from 1 μM to a total of 12 concentrations. 30 μL of the substrate was transferred to a 384-well plate with two replicates. 30 μL of the 3C protease to be tested was transferred to the corresponding well, immediately centrifuged, and vortexed to mix. The fluorescence signal values ​​generated by the reaction were collected using a TECAN F200 microplate reader. Data analysis was performed using GraphPad Prism9 software to obtain the enzyme activity parameters of the test protease. Figure 3 The activity parameters of 3C protease were obtained by analyzing the Graph PadPrism9 software.

[0047] Figure 3 The results showed that the activity of 3C protease-M1 was 27.48 × 10⁻⁶. -4 (RFU / s / nM), the activity of 3C protease-M2 was determined to be 29.07 × 10⁻⁶. -4 (RFU / s / nM), the activity of 3C protease-M3 was determined to be 14.12 × 10⁻⁶. -4 (RFU / s / nM), the activity of 3C protease-M4 was determined to be 6.59 × 10⁻⁶. -4 (RFU / s / nM), the activity of 3C protease-M5 was determined to be 5.58 × 10⁻⁶. -4(RFU / s / nM), the activity of wild-type 3C protease WT was 8.15 × 10⁻⁶. -4 (RFU / s / nM). The activities of 3C protease-M1, 3C protease-M2, and 3C protease-M3 compared to wild-type were increased by 3.37, 3.57, and 1.73 times, respectively, while the activities of 3C protease-M4 and 3C protease-M5 compared to wild-type 3C protease were decreased. The results indicate that the three 3C protease mutants, 3C protease-M1, 3C protease-M2, and 3C protease-M3, have higher activities than wild-type 3C protease WT, and their enzyme activities are significantly improved compared to wild-type 3C protease.

[0048] The results of comprehensive thermal stability and activity tests show that the three modified 3C protease mutants, 3Cprotease-M1, 3C protease-M2 and 3C protease-M3, have higher thermal stability and higher enzyme activity than the wild-type 3C protease WT. They overcome the problem of enzyme instability at high temperatures that limits its application and provide application scenarios for its storage and better application in industrial production.

[0049] The following section uses 3C protease-M2 as an example to demonstrate how to scale up and purify this 3C protease mutant for further application.

[0050] 2.5 Large-scale expression and purification of recombinant 3C protease

[0051] 2.5.1 Expression of recombinant 3C protease

[0052] The strain expressing 3C protease-M2 in small quantities was inoculated into 50 ml of LB broth and cultured overnight at 37°C. The overnight cultured bacteria were then inoculated into 1 L of LB broth at a ratio of 1:100 and cultured at 37°C until the bacterial culture reached OD500. 600 When the bacterial growth rate is 0.6-0.8, add 0.5mM IPTG and incubate overnight at 15℃. Collect the bacterial cells by centrifugation at 5000rpm.

[0053] 2.5.2 Purification of recombinant 3C protease

[0054] The collected bacterial cells were weighed and added to a lysis buffer solution (50 mM Tris-HCl (pH 7.5), 500 mM NaCl, 5% glycerol, 1 mM β-ME) at a 1:10 ratio. The cells were homogenized using a high-pressure homogenizer, and the supernatant was collected by centrifugation at 16000 rpm. Because the recombinant 3C protease was tagged with Strep II, a Strep-Tactin XT affinity chromatography column was used for protein enrichment and purification. The specific procedure was as follows: first, the Strep-Tactin XT affinity chromatography column was washed and equilibrated with lysis buffer for 10 column volumes. Then, the lysis supernatant was loaded onto the Strep-Tactin XT affinity chromatography column, and the column was washed with buffer for another 5 column volumes to remove unbound proteins. Finally, elution was performed with a solution containing 75 mM biotin. The eluted proteins were collected and analyzed by SDS-PAGE. The experimental results are shown in [Figure number missing]. Figure 4 The purified 3C protease mutant 3C protease-M2 has high purity and high yield.

[0055] 2.6 Enzyme digestion verification application and stability test

[0056] To test the practical application effect of the modified 3C protease, the recombinant protein POI-3C-eGFP was used as the enzyme digestion target. The sequence of the recombinant protein POI-3C-eGFP is shown in SEQ ID NO.11. In the sequence shown in SEQ ID NO.11, positions 1-375 represent the target protein POI. The C-terminus of the target protein POI is linked to the tag sequence "LEVLFQGP" recognized by the 3C protease. "LEVLFQGP" is located at positions 376-383, and positions 384-620 represent the eGFP protein. The 3C protease can cleave this recombinant protein from the 3C protease cleavage site into two fragments: POI and eGFP. The molecular weight of the POI protein is approximately 40 kDa, and the molecular weight of the eGFP protein is approximately 28 kDa.

[0057] In this experiment, the reaction buffer consisted of 50 mM Tris-HCl (pH 8.0), 500 mM NaCl, and 5% glycerol. The enzymatic digestion reaction was conducted at 4°C. The 3C protease was incubated with the recombinant protein at mass ratios of 1:40, 1:60, and 1:80. Samples were collected one hour after digestion and analyzed by SDS-PAGE to assess the digestion efficiency of the 3C protein.

[0058] See results Figure 5At 4°C, after 1 hour of enzyme digestion, the recombinant POI-3C-eGFP protein was almost completely digested with different enzyme ratios, indicating that the 3C protease-M2 has a high digestion efficiency in practical applications and can be used in a wide range of scenarios.

[0059] 3. Conclusion

[0060] The above description shows that the three recombinant 3C protease mutants 3C protease-M1, 3C protease-M2 and 3C protease-M3 provided by the present invention have higher enzyme activity and better protein stability, have broader application conditions and stronger practical application value, and are more suitable for large-scale production and industrial use.

[0061] The embodiments described above are merely examples of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention.

Claims

1. A 3C protease mutant, characterized in that, The 3C protease mutant was obtained by modifying the amino acid sequence of the wild-type 3C protease. The 3C protease mutant is 3C protease-M1, 3C protease-M2 or 3C protease-M3, and the amino acid sequences of 3C protease-M1, 3C protease-M2 or 3C protease-M3 are shown in SEQ ID NO.1-3, respectively.

2. A polynucleotide, characterized in that, The polynucleotide encodes the 3C protease mutant as described in claim 1.

3. A polynucleotide according to claim 2, characterized in that, The polynucleotide sequences encoding 3C protease-M1, 3C protease-M2, or 3C protease-M3 are shown in SEQ ID NO.7-9, respectively.

4. A recombinant plasmid, characterized in that, The recombinant plasmid is an expression vector containing the polynucleotide sequence as described in any one of claims 2-3 and capable of correspondingly translating and expressing the 3C protease mutant as described in claim 1.

5. The recombinant plasmid according to claim 4, characterized in that, The expression vector is pCDFDuet-1.

6. An expression system for a 3C protease mutant, characterized in that, The BL21(DE3) cells contain the recombinant plasmid as described in any one of claims 4-5 or whose genome integrates the polynucleotide as described in any one of claims 2-3.

7. A method for preparing the 3C protease mutant as described in claim 1, characterized in that, Includes the following steps: (1) Gene sequences that can encode the amino acid sequences of 3C protease-M1, 3C protease-M2 or 3C protease-M3 are synthesized, and the gene sequences are then constructed on expression vectors to obtain recombinant plasmids. (2) The recombinant plasmid obtained in step (1) is expressed using the Escherichia coli prokaryotic expression system to obtain the expression product. The 3C protease mutant can be obtained by purifying the expression product.

8. The method for preparing the 3C protease mutant according to claim 7, characterized in that, To facilitate the purification of the expression products, a Strep II tag was added to the N-terminus of the amino acid sequence of 3C protease-M1, 3C protease-M2, or 3C protease-M3, and the expression products were purified using a Strep-Tactin XT affinity chromatography column.

9. The application of the 3C protease mutant as described in claim 1 as a proteolytic enzyme in protein purification.

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