High-stability and high-activity bdSENP1 protease mutant and application thereof

Abstract

The invention discloses a high-stability and high-activity bdSENP1 protease mutant and application thereof, the mutant is obtained by single mutation or multiple mutation of ubiquitin sample-protease structural domain bdSENP1 of a complete sequence of ubiquitin sample protease family structural domain protein, and compared with wild type bdSENP1, the mutant has higher stability and / or activity. The invention further provides a preferred bdSENP1 protease mutant bdSENP1-M1, the yield of the mutant is increased by about 2 times, the Tm value is increased by about 32 DEG C, the activity is increased by about 2 times compared with that of wild type bdSENP1, the mutant can still keep efficient SUMO cutting activity under the high-temperature condition, and the application range of the mutant in recombinant protein purification and industrial production is remarkably expanded.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a bdSENP1 protease mutant with high stability and high activity and an application thereof. Background Art

[0002] Sentrin-Specific Protease (SENP1) is a key member of the deSUMOylation enzyme family. It is responsible for specifically removing SUMO molecules from SUMO-modified target proteins, thereby regulating the protein's SUMOylation status. SUMOylation is an important post-translational modification of proteins involved in various cellular processes, including transcriptional regulation, DNA repair, cell cycle control, and stress response. Due to the widespread role of SUMOylation in cells, abnormal function of SENP1 is closely associated with various diseases, such as cancer, neurodegenerative diseases, and cardiovascular disease.

[0003] In the biotechnology and biopharmaceutical fields, SENP1 is widely used in recombinant protein purification due to its highly specific SUMO cleavage activity. The SUMO tagging system is a highly efficient tool for recombinant protein expression and purification, significantly improving the solubility and stability of target proteins. However, conventional SENP1 enzymes are susceptible to inactivation at high temperatures or under harsh conditions, limiting their application in industrial production. To improve the stability and applicability of SENP1, researchers have begun to modify it through protein engineering to develop mutants with higher thermal stability and catalytic efficiency. Summary of the Invention

[0004] In order to overcome the deficiencies of the prior art, the present invention aims to provide a bdSENP1 protease mutant with high stability and high activity and its application.

[0005] The present invention achieves the above-mentioned purpose through the following technical solutions: The present invention provides a bdSENP1 protease mutant with high stability and high activity. The mutant is obtained by single mutation or multiple mutation of the ubiquitin-like protease family domain protein of the full sequence of the ubiquitin-like protease family domain protein bdSENP1, wherein the bdSENP1 is located at positions 248-481 of the ubiquitin-like protease family domain protein, and the amino acid sequence thereof is shown in SEQ ID NO.1, wherein the single mutation comprises S274V, Q425M, T272I, N259R, T272V, H421S, E456P, Q425L, S274A, K294G, Q417D, H262R, N259K, A422D, G481E, M396K, G481D, G427C, G460S, K268N, A422N, G427V, A422 E, V458D, N259L, R269L, G265E, S465P, A422G, G427T, V458N, D258E, R267T, G265Y, G460D, G265N, D258A, and R267S; the mutant obtained by the multiple mutations is a bdSENP1-M1 mutant, and the amino acid sequence of the bdSENP1-M1 mutant is shown in SEQ ID NO.4.

[0006] As a further optimization scheme of the present invention, the mutation sites of the highly stable bdSENP1 protease mutant include at least one of S274V, Q425M, T272I, N259R, T272V, H421S, E456P, Q425L, S274A, K294G, Q417D, H262R, N259K, A422D, G481E, M396K, G481D, G427C, and G460S, or a bdSENP1-M1 mutant.

[0007] As a further optimization scheme of the present invention, the mutation sites of the highly active bdSENP1 protease mutant include at least one of Q425M, A422E, G417D, G427C, D258A, R267S, S274A, K268N, G265N, M396K, A422D, V458N, Q425L, G460D, G481E, S465P, H421S, R267T, G481S, H262R, or the bdSENP1-M1 mutant.

[0008] As a further optimization scheme of the present invention, the mutation sites of the highly stable and highly active bdSENP1 protease mutant include at least one of Q425M, H421S, Q425L, S274A, H262R, A422D, G481E, M396K, G427C, or a bdSENP1-M1 mutant.

[0009] The present invention also provides a polynucleotide encoding the bdSENP1 protease mutant.

[0010] As a further optimized solution of the present invention, the sequence of the polynucleotide is shown as SEQ ID NO. 5, which is used to encode the bdSENP1-M1 mutant.

[0011] The present invention also provides a recombinant plasmid, which is an expression vector containing the polynucleotide and capable of translating and expressing the bdSENP1 protease mutant.

[0012] As a further optimized solution of the present invention, the expression vector carries a bdSUMO-CGS-10His tag, the sequence of the bdSUMO-CGS-10His tag is shown in SEQ ID NO. 2, wherein the expression vector is a pET-28a vector.

[0013] The present invention also provides an affinity purification matrix, wherein the affinity purification matrix is an affinity matrix on which the bdSENP1 protease mutant is immobilized.

[0014] The present invention also provides a use of the bdSENP1 protease mutant in protein purification.

[0015] The present invention also provides a method for protein purification using the bdSENP1 protease mutant, comprising the following steps: (1) Immobilizing the bdSENP1 protease mutant onto an affinity matrix to obtain an affinity purification matrix; (2) Connecting the bdSUMO tag sequence containing the bdSENP1 protease mutant protease cleavage site to the target protein to be purified to construct a fusion protein, which is then expressed in a heterologous expression cell line; (3) harvesting cells containing the bdSUMO-tagged fusion protein and lysing the cells to obtain the cell supernatant containing the fusion protein; (4) Incubating the cell supernatant with the affinity purification matrix; (5) Collect the target protein after enzyme digestion from the affinity purification matrix; (6) The collected enzyme-digested samples are enriched using a nickel column to collect the highly purified target protein.

[0016] The present invention has the following beneficial effects: 1) The present invention provides a series of single mutation sites in bdSENP1. The resulting bdSENP1 mutants exhibit approximately 0.2-4°C higher thermal stability and 1.1- to approximately 2-fold higher activity than wild-type bdSENP1. The present invention also provides a bdSENP1 protease mutant with multiple mutation sites, bdSENP1-M1. Compared to wild-type bdSENP1, this mutant exhibits approximately 2-fold higher yield, an approximately 32°C higher Tm, and approximately 2-fold higher activity. This highly thermostable and active bdSENP1-M1 can also be prepared into a protease cleavage column. This column exhibits strong enzymatic cleavage, capable of cleaving tens of milligrams of target protein in just a few minutes. It can also be reused over a long period of time, has broader application prospects, and is more suitable for tag removal during protein purification, facilitating large-scale production and industrial applications.

[0017] 2) Based on the three-dimensional structure and thermostability mechanisms of SENP1, this study designed and developed a series of highly thermostable and active SENP1 mutant proteins, specifically the bdSENP1-M1 mutant. This mutant maintains efficient SUMO cleavage activity even under high-temperature conditions, significantly expanding its application in recombinant protein purification and industrial production. This achievement not only provides a new tool for optimizing SUMO tagging systems but also offers an important reference for thermostability engineering of other proteases. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The purification results of 40 single-point mutations of bdSENP1 provided by the present invention are shown.

[0019] Figure 2 The purification results of the recombinant bdSUMO substrate provided by the present invention; Figure 3 The bdSENP1 protease activity assay results provided by the present invention; Figure 4 The purification results of the wild type and bdSENP1-M1 mutant of the bdSENP1 protease provided by the present invention are as follows; Figure 5 The QC test results of the wild type and bdSENP1-M1 mutant of the bdSENP1 protease provided by the present invention are as follows; Figure 6 The Tm value and activity test results of the bdSENP1-M1 protease provided by the present invention; Figure 7 Application of the affinity purification medium prepared by the bdSENP1-M1 protease provided by the present invention in protease cleavage; Figure 8The stability of the affinity purification medium prepared by the bdSENP1-M1 protease provided by the present invention was tested in protease cleavage applications. DETAILED DESCRIPTION

[0020] The present application is described in further detail below in conjunction with the accompanying drawings. It is necessary to point out that the following specific implementation methods are only used to further illustrate the present application and cannot be understood as limiting the scope of protection of the present application. Technicians in this field can make some non-essential improvements and adjustments to the present application based on the above application content.

[0021] 1. Materials Unless otherwise specified, the methods used in the present invention are conventional methods known to those skilled in the art. If specific conditions are not specified, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments is not specified, they are all conventional products that can be purchased commercially.

[0022] 2. Methods 2.1 Construction of recombinant bdSUMO protease and its substrate plasmid The gene sequences of the recombinant SUMO protease and its substrate provided herein were obtained through gene synthesis. The wild-type SUMO enzyme is derived from Brachypodium distachyon. The ubiquitin-like protease domain (Ulp1) of the ubiquitin-like protease family domain protein (Uniprot: HYPERLINK "https: / / www.uniprot.org / uniprotkb / I1HF82 / entry" I1HF82) was synthesized through gene synthesis. The full sequence of the ubiquitin-like protease family domain protein is shown in SEQ ID NO. 6. The amino acid sequence of the Ulp1 domain extends from P248 to G481 of the full sequence (shown in SEQ ID NO. 1), and this protein is designated wild-type bdSENP1.

[0023] All mutants were constructed based on the wild-type bdSENP1 by designing corresponding mutant primers and molecular cloning methods, including S274V, Q425M, T272I, N259R, T272V, H421S, E456P, Q425L, S274A, K294G, Q417D, H262R, N259K, A422D, G481E, M396K, G481D, G427C, G460 S, K268N, A422N, G427V, A422E, V458D, N259L, R269L, G265E, N259A, S465P, A422G, G427T, V458N, D258E, R267T, G265Y, G460D, G265N, D258A, G481S, and R267S, a total of 40 single-point mutations (numbered according to the full sequence sites of the ubiquitin-like protease family domain protein).

[0024] Wild-type bdSENP1 and its mutant proteins were constructed in a modified pET-28a vector (GenScript). The T7 promoter of this vector was fused with a bdSUMO-CGS-10His tag sequence. The tag sequence is shown in SEQ ID NO. 2 (wherein, 10His is the tag sequence used for affinity purification, "bdSUMO" is the SUMO protease cleavage site used for tag removal during subsequent purification, and "CGS" is the linker sequence. In the sequence shown in SEQ ID NO. 2, "HHHHHHHHHH" is the 10His sequence, and "SDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPLRRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGG" is the bdSUMO sequence). The gene sequences of the constructed recombinant proteins were verified by sequencing companies.

[0025] The corresponding substrate for bdSENP1 was also synthesized and constructed in the pET-28a vector. To better detect enzyme activity, we designed a special substrate: 6His-mCherry-GG-TEV-GG-bdSUMO-GG-eGFP. This substrate contains a 6His tag, the red fluorescent protein mCherry, and a TEV protease cleavage site at the N-terminus, and the green fluorescent protein eGFP at the C-terminus. The "GG" connects the mCherry protein, the TEV protease cleavage site, the bdSUMO substrate sequence, and the eGFP protein. Its amino acid sequence is shown in SEQ ID NO. 3. In the sequence shown in SEQ ID NO.3, positions 1-6 "HHHHHH" are the 6His sequence, positions 7-241 are the mCherry sequence, positions 242-243 "GG" are the linker sequence, positions 244-250 "ENLYFQG" are the TEV restriction site sequence, positions 251-252 "GG" are the linker sequence, positions 253-329 are the bdSUMO substrate sequence, positions 330-331 "GG" are the linker sequence, and positions 332-569 are the eGFP sequence.

[0026] All recombinant plasmids were verified by sequencing to be completely consistent with the target sequence.

[0027] 2.2. Small-scale expression and purification of bdSENP1 mutant protein Using conventional molecular biological methods, the constructed bdSENP1 mutant plasmids were transformed into BL21 (DE3) E. coli competent cells in a clean bench and cultured at 37°C overnight. Monoclonal colonies from overnight culture were picked and transferred to 5 ml LB liquid medium and cultured at 37°C until the OD value of the bacterial solution reached 0. 600When the pH value is 0.6-0.8, take a small amount of bacterial solution and fix it with loading buffer. Take a small amount of bacterial solution and add glycerol to freeze it at -80℃. Add 0.5mM IPTG to the remaining bacterial solution and induce it at 15℃ for 16 hours before collecting the bacteria. The collected bacteria were dissolved in lysis buffer (50mM Tris-HCl (pH8.0), 500mM NaCl, 5% glycerol) and then ultrasonically disrupted. The supernatant was collected and added to 50μL NiBestarose FF filler treated with buffer (50mM Tris-HCl (pH8.0), 500mM NaCl, 5% glycerol) and incubated at 4℃ for 30 minutes. The incubated samples were centrifuged at 12000rpm at 4℃ for 10 minutes, 1mL of buffer was added, and after washing three times, 100μL of elution buffer (50mM Tris-HCl (pH8.0), 500mM NaCl, 5% glycerol, 500mM imidazole) was added and centrifuged at 12000rpm at 4℃ for 5 minutes. The eluted sample was collected and a small amount of the eluted sample was fixed with loading buffer and analyzed by SDS-PAGE. The experimental results are shown in Figure 2. Figure 1 AD, among which, all single-point mutant proteins among the 40 single-point mutations were significantly expressed, and all single-point mutant proteins could be eluted with high purity.

[0028] 2.3. Thermal stability test of bdSENP1 mutant protein The thermal stability of the bdSENP1 mutant protein was tested using the Protein Thermal Shift (ThermoFluor) technique. This technique exploits the structural characteristics of proteins: proteins have hidden hydrophobic regions. When the temperature rises, these regions open up, exposing the hydrophobic regions. The fluorescent dye SYPRO Orange binds to these regions and excites them to fluoresce. The change in fluorescence signal intensity forms a melting curve, and the temperature corresponding to the maximum value of the melting curve derivative is the melting point (Tm). The more stable the protein, the higher the measured Tm value.

[0029] The specific operations are as follows: 5 μg of bdSENP1 mutant protein was added to each well of a 96-well PCR plate. 10× SYPRO Orange fluorescent dye was then added to the corresponding wells. The 96-well PCR plate was then placed in a qPCR instrument. The instrument parameters were set to increase the temperature from 25°C to 99°C in a 1°C gradient over 1 minute. The protein melting curve was then calculated. The T values for all single mutant proteins are shown in Table 1. Table 1 Tm values of bdSENP1 single mutant proteins Serial number mutation site Tm (℃) ΔTm (℃) Serial number mutation site Tm (℃) ΔTm (℃) WT WT 40.85 0 21 A422N 41.82 0.97 1 S274V 44.91 4.06 22 G427V 41.76 0.91 2 Q425M 43.84 2.99 23 A422E 41.65 0.8 3 T272I 43.51 2.66 24 V458D 41.62 0.77 4 N259R 43.50 2.65 25 N259L 41.53 0.68 5 T272V 43.48 2.63 26 R269L 41.53 0.68 6 H421S 43.28 2.43 27 G265E 41.48 0.63 7 E456P 43.01 2.16 28 N259A 41.37 0.52 8 Q425L 42.68 1.83 29 S465P 41.34 0.49 9 S274A 42.52 1.67 30 A422G 41.28 0.43 10 K294G 42.52 1.67 31 G427T 41.27 0.42 11 Q417D 42.46 1.61 32 V458N 41.26 0.41 12 H262R 42.42 1.57 33 D258E 41.22 0.37 13 N259K 42.39 1.54 34 R267T 41.22 0.37 14 A422D 42.31 1.46 35 G265Y 41.18 0.33 15 G481E 42.30 1.45 36 G460D 41.13 0.28 16 M396K 42.23 1.38 37 G265N 41.12 0.27 17 G481D 42.18 1.33 38 D258A 41.09 0.24 18 G427C 42.15 1.3 39 G481S 41.09 0.24 19 G460S 41.95 1.1 40 R267S 41.06 0.21 20 K268N 41.84 0.99 As shown in Table 1 , the Tm values of S274V, Q425M, T272I, N259R, T272V, H421S, E456P, Q425L, S274A, K294G, Q417D, H262R, N259K, A422D, G481E, M396K, G481D, G427C, and G460S among the 40 single mutant proteins increased by 1°C–4.39°C. Therefore, the present invention screened out 19 mutants, including S274V, Q425M, T272I, N259R, T272V, H421S, E456P, Q425L, S274A, K294G, Q417D, H262R, N259K, A422D, G481E, M396K, G481D, G427C, and G460S, which can significantly improve the thermal stability of bdSENP1.

[0030] 2.4. Expression and purification of substrate proteins corresponding to bdSENP1 protease Using conventional molecular biology techniques, the constructed bdSENP1 substrate plasmids (6His-mCherry-GG-TEV-GG-bdSUMO-GG-eGFP) were transformed into BL21(DE3) competent E. coli cells in a cleanroom and cultured overnight at 37°C. Single colonies from the overnight culture were transferred to 5 ml of LB liquid medium and incubated at 37°C. When the OD600 of the culture reached 0.6-0.8, a small amount of the culture was fixed with loading buffer. A small amount of the culture was added to glycerol stock and frozen at -80°C. This glycerol stock was inoculated into 50 ml of LB liquid medium and cultured overnight at 37°C. The overnight culture was then inoculated into 1 L of LB liquid medium at a 1:100 ratio and incubated at 37°C until the OD600 reached 0.6-0.8. 0.5 mM IPTG was then added and cultured overnight at 15°C. The cells were then harvested by centrifugation at 5000 rpm.

[0031] The collected cells were weighed and the corresponding volume of lysis buffer (50 mM Tris-HCl (pH 7.5), 500 mM NaCl, 5% glycerol, 20 mM imidazole) was added at a 1:10 ratio. The cells were disrupted using a high-pressure homogenizer, and the supernatant was collected by high-speed centrifugation at 16,000 rpm. The protein was enriched and purified using a Ni Bestarose FF affinity chromatography column.

[0032] The specific process is as follows: first, wash and equilibrate the Ni Bestarose FF affinity chromatography column with lysis buffer for 10 column volumes, then load the lysis supernatant onto the Ni Bestarose FF affinity chromatography column, and elute with different gradients of imidazole solution. The proteins eluted with different gradients of imidazole are collected for SDS-PAGE detection, and the protein concentration is measured with Nanodrop to calculate the protein yield. The N-terminus of the substrate has a 6His tag, and the protein is enriched and purified using a Ni Bestarose FF affinity chromatography column. The corresponding purification results are shown in Figure 2 ,according to Figure 2 The results showed that the substrate could be obviously purified by Ni Bestarose FF affinity chromatography with good purity.

[0033] 2.5. Activity Assay of bdSENP1 Mutants The SUMO protease activity was detected using a fluorescence resonance energy transfer method. The principle of fluorescence resonance energy transfer is that when the emission spectrum of one fluorescent group (the donor) overlaps with the absorption spectrum of the other group (the acceptor), and the distance between the two fluorescent groups is less than 100 Å, fluorescence energy transfer from the donor to the acceptor occurs, making the donor fluorescence intensity much lower than when it exists alone (fluorescence quenching). The substrate provided in the present invention, such as 6His-mCherry-GG-TEV-GG-bdSUMO-GG-eGFP, contains a red fluorescent protein mCherry at its N-terminus (maximum excitation and emission light of 587 nm and 610 nm, respectively) and a green fluorescent protein eGFP at its C-terminus (maximum excitation and emission light of 484 nm and 507 nm, respectively). When the substrate bdSUMO is recognized and cleaved by its corresponding bdSENP1 protease, the mCherry at the N-terminus and the eGFP fluorescent protein at the C-terminus are separated, releasing a fluorescent signal. The higher the bdSENP1 protease activity, the more fluorescent groups are released and the greater the fluorescence intensity. Enzyme activity parameters were expressed as the fluorescence intensity absorbed per nanomole protein per second.

[0034] The specific operations are as follows: The substrate was prepared as a 1 mM stock solution and aliquoted for later use. The SUMO protease activity assay buffer consisted of 40 mM Tris-HCl (pH 7.0), 200 mM NaCl, 2 mM MgCl₂, 250 mM Sucrose, and 2 mM DTT. The protease concentration was diluted to 1000 nM using the buffer in a series of approximately two-fold dilutions, for a total of 12 dilutions. Thirty μL of substrate was transferred to a 384-well plate in duplicate. Thirty μL of the bdSENP1 protease to be tested was then transferred to the corresponding wells of the plate. Immediately centrifuge and shaken to mix thoroughly. Fluorescence signals were collected using a TECAN F200 microplate reader. Data were analyzed using Graph Pad Prism 9 software to determine the activity parameters of the tested protease. Figure 3 That is, different bdSENP1 protease activity parameters were obtained by analyzing the Graph Pad Prism9 software.

[0035] from Figure 3 It can be seen that the activities of all 38 single mutants maintained 80% or above, with only two mutants, G481D and N259A, showing a significant decrease in activity. Furthermore, the activities of nine bdSENP1 single mutants, including Q425M, A422E, G417D, G427C, D258A, R267S, S274A, K268N, and G265N, were approximately 2-fold higher than that of wild-type bdSENP1. Eleven mutants, including M396K, A422D, V458N, Q425L, G460D, G481E, S465P, H421S, R267T, G481S, and H262R, showed Tm values 1.5-1.8-fold higher than that of the wild-type. Therefore, the present invention screened out 20 mutant proteins, including Q425M, A422E, G417D, G427C, D258A, R267S, S274A, K268N, G265N, M396K, A422D, V458N, Q425L, G460D, G481E, S465P, H421S, R267T, G481S, and H262R.

[0036] 2.6. Expression and purification of bdSENP1-M1 protein To further investigate the function of high-quality bdSENP1 mutant proteins, the present invention also provides a multiple mutant, designated bdSENP1-M1, with 38 mutations compared to wild-type bdSENP1. Its amino acid sequence is shown in SEQ ID NO. 4, and its nucleotide sequence is shown in SEQ ID NO. 5. Protein properties of the wild-type bdSENP1 and bdSENP1-M1 mutants were compared. All plasmids were heterologously expressed in Escherichia coli, and the expressed proteins were purified.

[0037] 2.6.1. Purification of bdSENP1 wild-type and bdSENP1-M1 mutant proteins (1) Affinity chromatography Each collected bacterial mass was weighed and the corresponding volume of lysis buffer (50 mM Tris-HCl (pH 8.0), 500 mM NaCl, 5% glycerol, 1 mM TCEP) was added at a 1:10 ratio. The cells were disrupted using a high-pressure homogenizer, and the supernatant was collected by high-speed centrifugation at 16,000 rpm. Both the wild-type bdSENP1 and bdSENP1-M1 mutant proteins were 10-His-tagged and were enriched and purified using a Ni Bestarose FF affinity column. The specific procedure was as follows: the Ni Bestarose FF affinity column was first washed and equilibrated with lysis buffer for 10 column volumes. The lysate was then loaded onto the Ni Bestarose FF affinity column and eluted with lysis buffer containing 500 mM imidazole. The eluted protein was collected and analyzed by SDS-PAGE, and the protein concentration was determined using Nanodrop to calculate the protein yield.

[0038] Protein purification results are as follows Figure 4 Both wild-type bdSENP1 and bdSENP1-M1 mutant proteins could be enriched by NiBestarose FF affinity chromatography column with good purity.

[0039] (2) Gel filtration chromatography and QC testing The flowthrough was concentrated to approximately 2 mL and then subjected to gel filtration chromatography using a HiLoad 16 / 600 Superdex 200 pg column in a buffer of 50 mM HEPES (pH 8.0), 500 mM NaCl, 5% glycerol, and 1 mM TCEP. Samples were collected after gel filtration chromatography and subjected to protein quality analysis using SDS-PAGE purity, mass spectrometry, and analytical molecular sieve analysis.

[0040] Test results such as Figure 5 As shown in Figure 2, SDS-PAGE results indicate that the purity of both the bdSENP1 wild-type and bdSENP1-M1 proteins is greater than 80%. Calculated yields indicate that the wild-type yield is 6 mg / L, while the bdSENP1-M1 yield is 13 mg / L, approximately two-fold higher than the wild-type. Mass spectrometry results also indicate that the molecular weight of the tested samples is essentially consistent with that of the target protein, confirming that the purified proteins are the target proteins. Furthermore, analytical molecular sieve analysis revealed that all proteins were monomeric in solution.

[0041] 2.6.2. bdSENP1-M1 protein Tm value and activity detection In order to further verify the thermal stability and enzyme activity of the purified bdSENP1-M1 protein, the purified bdSENP1-M1 protein was subjected to Tm value and activity detection according to the similar methods in 2.3 and 2.5, respectively. The experimental results are shown in Figure 6 The Tm value test results showed that its Tm value was 72°C, which was about 32°C higher than that of the wild type, and its activity was about 2 times higher than that of the wild type.

[0042] 2.7 Application of bdSENP1-M1 protease digestion 2.7.1 Enzyme digestion test Because the bdSENP1-M1 protease has very strong enzymatic activity, in order to better improve its efficiency, the enzyme was immobilized for enzymatic cleavage testing. The bdSENP1-M1 mutant is fused with a bdSUMO-CGS-10His tag at its N-terminus, resulting in the bdSUMO-CGS-10His-bdSENP1-M1 mutant. Upon expression, the bdSUMO tag is recognized by the bdSENP1-M1 mutant, resulting in the removal of the N-terminal bdSUMO tag and the CGS-10His-bdSENP1-M1 mutant protein. The bdSENP1-M1 N-terminus exposes a Cys (cysteine) residue, which can be coupled to an iodoacetyl resin via a sulfhydryl group. The specific steps are as follows: SulfoLink™ packing (Thermo, 20404) was loaded into a 1 ml prepacked column and washed three times with buffer (50 mM HEPES (pH 8.0), 500 mM NaCl, 5% glycerol, 2 mM TCEP), each for three column volumes. Then, 50 mg of bdSENP1-M1 protein was loaded onto the resin and loaded overnight at 4°C at a low flow rate. The column was then washed six times with the same buffer, each for three column volumes. The SulfoLink™ resin was then blocked by adding a buffer containing 50 mM cysteine and incubating at 4°C for two hours. Finally, the column was washed with the same buffer for five column volumes. The immobilized amount of bdSENP1-M1 mutant protein on the resin was 17 mg / ml by subtraction, calculating the difference between the total amount of enzyme before coupling and the amount remaining after coupling.

[0043] 24 mg of substrate (concentration: 8 mg / ml, volume: 3 ml) was loaded onto the immobilized bdSENP1-M1 enzyme column at a flow rate of 1 ml per minute. After 3 minutes, the flow-through sample was collected for SDS-PAGE analysis. The SDS-PAGE analysis results are shown in Figure 7 From the SDS-PAGE test results, almost all substrates were cleaved into two separate proteins, mCherry and eGFP, by the immobilized enzyme column, indicating that the immobilized bdSENP1-M1 enzyme column has high enzymatic cleavage activity.

[0044] 2.7.2 Enzyme Stability Test To further test the long-term use effect of the immobilized enzyme column, we tested its actual enzymatic cleavage effect after the enzyme column was placed in a 4°C refrigerator for 1 month and 6 months. The specific operation is as follows: 10 mg (concentration: 9 mg / ml, volume: 1 ml) of substrate was loaded onto the immobilized bdSENP1-M1 enzyme column at a flow rate of 1 ml per minute. After 2 minutes, 5 ml of buffer was used to rinse the column and then 10 mg of substrate was reloaded onto the immobilized bdSENP1-M1 enzyme column at a flow rate of 1 ml per minute. The column was then washed with buffer. This process was repeated 10 times. Samples after each substrate flow-through were collected for SDS-PAGE analysis. The sample loading amount remained the same. The enzyme digestion results of the enzyme column after 1 month of storage are shown in Figure 2. Figure 8 Left: According to the results, the substrates can be completely digested by the enzyme column, and repeated use does not affect the digestion efficiency of the enzyme column. Figure 8 The right side shows the enzymatic digestion results of an enzyme column stored in a 4°C refrigerator for 6 months. The results are comparable to those of a column stored for 1 month, indicating that the enzyme column prepared with bdSENP1-M1 protein has high enzyme activity and stability and can be stored in a 4°C refrigerator for 6 months or even longer.

[0045] 3. Conclusion The above results indicate that the bdSENP1 mutant protein provided by the present invention has higher enzymatic activity and better thermal stability. Furthermore, the enzyme can be used to prepare a protease column, demonstrating strong enzymatic cleavage efficiency, enabling target protein cleavage in just a few minutes. Its high thermal stability allows for a longer shelf life, maintaining high enzymatic cleavage efficiency even after being stored at 4°C for six months or longer, and even after repeated cycles. This allows for a wider range of applications and greater practical value, making it more suitable for large-scale production and industrial use.

[0046] The above-described embodiments merely illustrate several implementations of the present invention. 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 a person skilled in the art would be able to make numerous variations and improvements without departing from the spirit of the present invention, and all such variations and improvements fall within the scope of protection of the present invention.

Claims

1. A highly stable and highly active bdSENP1 protease mutant, characterized in that: The mutant is a ubiquitin-like protease family domain protein with a complete sequence, wherein the ubiquitin-like protease domain bdSENP1 is obtained by single mutation or multiple mutation. The bdSENP1 is located at positions 248-481 of the ubiquitin-like protease family domain protein, and its amino acid sequence is shown in SEQ ID NO.

1. The single mutation includes S274V, Q425M, T272I, N259R, T272V, H421S, E456P, Q425L, S274A, K294G, Q417D, H262R, N259K, A422D, G481E, M396K, G481D, G427C, G460S, K268N, A422N, G427V, A422 E, V458D, N259L, R269L, G265E, S465P, A422G, G427T, V458N, D258E, R267T, G265Y, G460D, G265N, D258A, and R267S; the mutant obtained by the multiple mutations is a bdSENP1-M1 mutant, and the amino acid sequence of the bdSENP1-M1 mutant is shown in SEQ ID NO.

4.

2. The highly stable and highly active bdSENP1 protease mutant according to claim 1, characterized in that: The mutation sites of the highly stable bdSENP1 protease mutant include at least one of S274V, Q425M, T272I, N259R, T272V, H421S, E456P, Q425L, S274A, K294G, Q417D, H262R, N259K, A422D, G481E, M396K, G481D, G427C, and G460S, or the bdSENP1-M1 mutant.

3. The highly stable and highly active bdSENP1 protease mutant according to claim 1, characterized in that: The mutation sites of the highly active bdSENP1 protease mutant include at least one of Q425M, A422E, G417D, G427C, D258A, R267S, S274A, K268N, G265N, M396K, A422D, V458N, Q425L, G460D, G481E, S465P, H421S, R267T, G481S, H262R, or the bdSENP1-M1 mutant.

4. The highly stable and highly active bdSENP1 protease mutant according to claim 1, characterized in that: The mutation sites of the highly stable and highly active bdSENP1 protease mutant include at least one of Q425M, H421S, Q425L, S274A, H262R, A422D, G481E, M396K, and G427C, or a bdSENP1-M1 mutant.

5. A polynucleotide, characterized in that The polynucleotide encodes the bdSENP1 protease mutant according to any one of claims 1 to 4.

6. A polynucleotide according to claim 5, characterized in that The sequence of the polynucleotide is shown as SEQ ID NO. 5, which is used to encode the bdSENP1-M1 mutant.

7. A recombinant plasmid, characterized in that The recombinant plasmid is an expression vector containing the polynucleotide according to any one of claims 5 to 6 and capable of translating and expressing the bdSENP1 protease mutant according to any one of claims 1 to 4.

8. A recombinant plasmid according to claim 7, characterized in that, The expression vector carries a bdSUMO-CGS-10His tag, the sequence of which is shown in SEQ ID NO. 2, wherein the expression vector is a pET-28a vector.

9. An affinity purification matrix, characterized in that The affinity purification matrix is an affinity matrix immobilized with the bdSENP1 protease mutant according to any one of claims 1 to 4.

10. Use of the bdSENP1 protease mutant according to any one of claims 1 to 4 in protein purification.

11. A method for protein purification using the bdSENP1 protease mutant according to any one of claims 1 to 4, characterized in that: The following steps are involved: (1) Immobilizing the bdSENP1 protease mutant onto an affinity matrix to obtain an affinity purification matrix; (2) Connecting the bdSUMO tag sequence containing the bdSENP1 protease mutant protease cleavage site to the target protein to be purified to construct a fusion protein, which is then expressed in a heterologous expression cell line; (3) harvesting cells containing the bdSUMO-tagged fusion protein and lysing the cells to obtain the cell supernatant containing the fusion protein; (4) Incubating the cell supernatant with the affinity purification matrix; (5) Collect the target protein after enzyme digestion from the affinity purification matrix; (6) The collected enzyme-digested samples are enriched using a nickel column to collect the highly purified target protein.

Images