An enzyme digestion system suitable for eukaryotic and prokaryotic expression systems and its use

Abstract

The application discloses an enzyme cutting system suitable for eukaryotic and prokaryotic expression systems and application thereof, relates to the technical field of biology, and comprises a SUMO protease and an enzyme cutting substrate; wherein the SUMO protease is a BioSENP1 protease, and the enzyme cutting substrate is a BioSUMO label. The BioSENP1 protease and the corresponding BioSUMO label suitable for eukaryotic and prokaryotic expression systems can overcome the case that the SUMO label is cut by an endogenous Ulp1 in a eukaryotic expression system, and the BioSENP1 protease has high yield, high enzyme activity and high enzyme cutting efficiency; the actual application effect of the BioSENP1 protease is equivalent to that of the Ulp1 enzyme sold on the market, and the BioSENP1 protease has wide application value and prospect.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to an enzyme digestion system adapted to eukaryotic and prokaryotic expression systems and its applications. Background Technology

[0002] Ubiquitins are molecules widely involved in protein degradation and signal transduction within cells. Small ubiquitin-like modifiers (SUMOs) are another class of ubiquitin-like proteins that participate in regulating various biological processes in cells, such as nuclear protein transport, DNA repair, transcriptional regulation, and cell cycle control. SUMO modification, as a post-translational modification, has significant biological importance and has been widely applied in protein labeling, functional studies, and protein purification.

[0003] SUMO tags offer numerous advantages for protein purification. They not only increase the solubility of the target protein but also, due to the conformationally specific SUMO protease Ulp1, can be directly cleaved by Ulp1 without leaving any residual amino acids in the target protein sequence, making them frequently used in protein purification. The most commonly used SUMO tag originates from yeast, and its corresponding Ulp1 enzyme is also sold as a common commercial enzyme. However, since both SUMO tags and the Ulp1 enzyme are derived from eukaryotes, and endogenous Ulp1 enzyme expression exists in eukaryotic cells, including insect and mammalian cells, when expressing recombinant proteins in eukaryotic expression systems, fusing a SUMO tag to the recombinant protein sequence can lead to the SUMO tag being cleaved by endogenous Ulp1.

[0004] Currently, to address the issue of SUMO tag removal in eukaryotic systems, LifeSensors has designed a new SUMOstar tag and a corresponding SUMOstar enzyme for juvenating the SUMOstar tag. This combination is currently the only reported SUMOstar tag and SUMOstar enzyme suitable for eukaryotic expression systems. However, because the tag and enzyme are patented, their application and development are somewhat limited. Furthermore, existing technologies still face several unresolved challenges, such as unstable tag removal efficiency, significant impact on the target protein, and excessive dependence on the SUMO enzyme.

[0005] Therefore, developing novel SUMOases with better activity and corresponding SUMO tags suitable for eukaryotic and prokaryotic expression systems is crucial to improving tag removal efficiency while reducing interference with target proteins. More efficient and stable SUMO tags and SUMOase systems can provide new research ideas for the use of SUMO tags and SUMOases in current biochemical and molecular biology research. Summary of the Invention

[0006] The purpose of this invention is to provide an enzyme digestion system adapted to eukaryotic and prokaryotic expression systems and its applications.

[0007] The present invention achieves the above objectives through the following technical solutions:

[0008] As a first aspect of the present invention, a SUMO protease is provided, wherein the SUMO protease is a BioSEN P1 protease, the amino acid sequence of the BioSEN P1 protease is shown in SEQ ID NO.2, and the nucleotide sequence of the BioSEN P1 protease is shown in SEQ ID NO.3.

[0009] A further improvement is that the preparation method of the BioSENP1 protease includes the following steps:

[0010] (1) First, add a 10His tag to the N-terminus of the amino acid sequence of the BioSENP1 protease as shown in SEQ ID NO.2. Then, synthesize a nucleotide sequence that can encode the amino acid sequence according to the amino acid sequence after adding the 10His tag. Finally, construct the synthesized nucleotide sequence on the pET-28a vector to obtain a recombinant plasmid.

[0011] (2) First, the recombinant plasmid was expressed using the Escherichia coli prokaryotic expression system. Cells containing BioSENP1 protease were harvested, and the cells were lysed to obtain cell supernatant. Then, the cell supernatant was loaded onto a Ni Bestarose FF affinity chromatography column to enrich and purify the protein, thereby obtaining the BioSENP1 protease.

[0012] As a second aspect of the present invention, an enzyme digestion system adapted to eukaryotic and prokaryotic expression systems is also provided, the enzyme digestion system comprising a SUMO protease and a digestion substrate, wherein the digestion substrate is a BioSUMO tag, the amino acid sequence of the BioSUMO tag is shown in SEQ ID NO.4, and the nucleotide sequence of the BioSUMO tag is shown in SEQ ID NO.5.

[0013] As a third aspect of the invention, the application of the enzyme digestion system as described above in the expression and purification of recombinant proteins in eukaryotic and prokaryotic expression systems is also provided.

[0014] A further improvement is that the method for expressing and purifying recombinant proteins in the eukaryotic and prokaryotic expression systems includes the following steps:

[0015] (1) First, the target protein is linked with an amino acid sequence containing enzyme digestion substrate to construct a recombinant protein. Then, the recombinant protein is constructed on an expression vector to obtain a recombinant plasmid.

[0016] (2) Express recombinant plasmids using eukaryotic or prokaryotic expression systems, harvest cells containing recombinant proteins, and lyse the cells to obtain cell supernatant containing recombinant proteins.

[0017] (3) First, incubate the cell supernatant with the affinity purification matrix, then add the buffer of SUMO protease corresponding to the enzyme digestion substrate to wash the affinity purification matrix, so that the target protein can be eluted from the affinity purification matrix.

[0018] A further improvement is that the prokaryotic expression system is an Escherichia coli prokaryotic expression system, and the eukaryotic expression system is an insect cell eukaryotic expression system or a mammalian cell eukaryotic expression system.

[0019] The present invention has the following beneficial effects:

[0020] This invention provides an enzyme digestion system adapted to eukaryotic and prokaryotic expression systems, comprising a SUMO protease and a digestion substrate; wherein the SUMO protease is a BioSENP1 protease, and the digestion substrate is a BioSUMO tag. The BioSENP1 protease and the corresponding BioSUMO tag adapted to eukaryotic and prokaryotic expression systems provided by this invention can overcome the problem of SUMO tags being digested by endogenous Ulp1 in eukaryotic expression systems. Furthermore, the BioSENP1 protease exhibits high yield, high enzyme activity, and high digestion efficiency. The practical application effect of the BioSENP1 protease is comparable to that of commercially available Ulp1 enzymes, demonstrating broad application value and promising prospects. Attached Figure Description

[0021] Figure 1 The purification results of BioSENP1, BdSENP1 and Ulp1 proteases provided by this invention;

[0022] Figure 2 The purification results of the recombinant BioSUMO and bdSUMO substrates provided by this invention;

[0023] Figure 3 The results of the BioSENP1 and BdSENP1 protease activity assays provided by this invention;

[0024] Figure 4The enzyme digestion verification results of the BioSENP1 protease provided by this invention;

[0025] Figure 5 The results validate the application scenarios of the BioSENP1 protease and tag BioSUMO provided by this invention in prokaryotic expression systems.

[0026] Figure 6 The results validate the application scenarios of the BioSENP1 protease and the tag BioSUMO provided by this invention in eukaryotic expression systems. 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 SUMO protease and its substrate particle

[0032] The gene sequences of the recombinant SUMO protease and its substrate provided by this invention were obtained through gene synthesis. The wild-type SUMO enzyme was derived from Brachypodium distachyon. Its ubiquitin-like protease domain (Ulp1) was synthesized using gene synthesis technology and named BdSENP1. The sequence of BdSENP1 is shown in SEQ ID NO.1. An additional 10His tag was added to the N-terminus of the sequence shown in SEQ ID NO.1 to facilitate subsequent protein purification. The synthesized sequence was constructed on the pET-28a vector.

[0033] The modified SUMO enzyme is a mutation of the BdSENP1 sequence, named BioSENP1. Its protein sequence is shown in SEQ ID NO.2, and the corresponding gene sequence is shown in SEQ ID NO.3. A 10His tag was added to the N-terminus of the sequence shown in SEQ ID NO.2 to facilitate subsequent protein purification. The substrate corresponding to BioSENP1 was also synthesized. To better detect enzyme activity, the substrate was specially designed: 6His-mCherry-GG-TEV-GG-substrate-GG-eGFP. The N-terminus contains a 6His tag, red fluorescent protein mCherry, and a TEV protease cleavage site. The C-terminus contains green fluorescent protein eGFP. The "GG" link is between the mCherry protein, the TEV protease cleavage site, the substrate sequence, and the eGFP protein.

[0034] Specifically, the substrate of BdSENP1 is 6His-mCherry-GG-TEV-GG-bdSUMO-GG-eGFP, and its amino acid sequence is SEQ ID NO.6. In the sequence shown in SEQ ID NO.6, positions 1-6 "HHHHHH" is the 6His sequence, positions 7-241 is the mCherry sequence, positions 242-243 "GG" is the linker sequence, positions 244-250 "ENLYFQG" is the TEV restriction site sequence, positions 251-252 "GG" is the linker sequence, positions 253-329 is the bdSUMO tag sequence, positions 330-331 "GG" is the linker sequence, and positions 332-569 is the eGFP sequence.

[0035] The modified BioSENP1 substrate is 6His-mCherry-GG-TEV-GG-BioSUMO-GG-eGFP, and its amino acid sequence is shown in SEQ ID NO.7. In the sequence shown in SEQ ID NO.7, positions 1-6 "HHHHHH" is the 6His sequence, positions 7-241 are the mCherry sequence, positions 242-243 "GG" is the linker sequence, positions 244-250 "ENLYFQG" is the TEV restriction site sequence, positions 251-252 "GG" is the linker sequence, positions 253-329 are the BioSUMO tag, positions 330-331 "GG" is the linker sequence, and positions 332-569 are the eGFP sequence. Specifically, the amino acid sequence of the BioSUMO tag is shown in SEQ ID NO.4, and the gene sequence of the BioSUMO tag is shown in SEQ ID NO.5.

[0036] All recombinant SUMO proteases and their substrates were constructed on the pET-28a vector, and all recombinant plasmids were sequenced and verified to be completely consistent with the target sequence.

[0037] 2.2 Expression of recombinant SUMO protease and its substrate proteins

[0038] Using conventional molecular biology techniques, the constructed BdSENP1, BioSENP1, and their corresponding substrates 6His-mCherry-GG-TEV-GG-bdSUMO-GG-eGFP and 6His-mCherry-GG-TEV-GG-BioSUMO-GG-eGFP plasmids were 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 cultured at 37°C until the bacterial culture showed an OD value. 600 When the bacterial concentration is 0.6-0.8, take a small amount of bacterial suspension and fix it with loading buffer. Add a small amount of bacterial suspension to glycerol and freeze to -80°C. Inoculate the glycerol-treated bacteria into 50 ml of LB liquid medium and incubate overnight at 37°C. Dilute the overnight cultured bacteria 1:100 with 1 L of LB liquid medium and incubate at 37°C until the bacterial OD reaches 0.5. 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.

[0039] 2.3 Purification of recombinant BdSENP1 and BioSENP1 proteases

[0040] The collected bacterial cells were weighed and 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 then homogenized using a high-pressure homogenizer, and the supernatant was collected by centrifugation at 16,000 rpm. Both BioSENP1 and BdSENP1 proteases have a 10His tag, and the proteins were enriched and purified using a NiBestarose FF affinity chromatography column.

[0041] The specific procedure is as follows: First, wash and equilibrate the Ni Bestarose FF affinity chromatography column to 10 column volumes with lysis buffer. Then, load the lysis supernatant onto the Ni Bestarose FF affinity chromatography column and elute with imidazole solutions of different gradients. Collect the proteins eluted by imidazole solutions of different gradients for SDS-PAGE detection, and use Nanodrop to determine the protein concentration and calculate the protein yield.

[0042] Protein purification results are as follows Figure 1The purification results showed that BioSENP1 and BdSENP1 could be effectively purified by NiBestarose FF affinity chromatography, resulting in high protein purity. According to Nanodrop concentration data, the yield of BioSENP1 was approximately 63 mg / L, and the yield of BdSENP1 was 50 mg / L. To further compare its activity with that of the commercially available Ulp1 protease, a 10His-Ulp1 protease was constructed simultaneously using the same methods as BioSENP1 and BdSENP1. The sequence of the 10His-Ulp1 protease is shown in SEQ ID NO. 8 (where 'MGSS' is the vector sequence and 'HHHHHHHHHH' is the 10His tag). The results showed that its yield was approximately 53 mg / L.

[0043] 2.4 Purification of recombinant bdSUMO and BioSUMO substrate proteins

[0044] The substrate has a 6His tag at its N-terminus, and the protein was enriched and purified using a Ni Bestarose FF affinity chromatography column. The purification procedure was the same as that for the protease in section 2.3. The corresponding purification results are shown below. Figure 2 ,according to Figure 2 The results showed that all substrates could be effectively purified by Ni Bestarose FF affinity chromatography with good purity.

[0045] 2.5 Assay of recombinant BdSENP1 and BioSENP1 protease activities

[0046] The activity of the SUMO protease was detected using fluorescence resonance energy transfer (FRET). The principle of FRET is that, among 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 information]. Fluorescent energy transfer occurs from the donor to the acceptor, resulting in a significantly lower fluorescence intensity in the donor compared to its solitary state (fluorescence quenching). The substrates provided in this invention, such as 6His-mCherry-GG-TEV-GG-bdSUMO-GG-eGFP, contain red fluorescent protein mCherry at the N-terminus (maximum excitation and emission wavelengths of 587 nm and 610 nm, respectively) and green fluorescent protein eGFP at the C-terminus (maximum excitation and emission wavelengths of 484 nm and 507 nm, respectively). When the substrate bdSUMO is recognized and cleaved by its corresponding BdSENP1 protease, the N-terminal mCherry and C-terminal eGFP fluorescent proteins separate, releasing a fluorescent signal. Higher SUMO protease activity results in the release of more fluorescent groups and a greater fluorescence intensity. Enzyme activity parameters are expressed as the fluorescence intensity absorbed per nanomolar of protein per second.

[0047] The specific steps are as follows:

[0048] Different substrates were prepared into 1 mM stock solutions and aliquoted for 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 serially diluted 2-fold to 1000 nM using the buffer, resulting in 12 concentrations. 30 μL of substrate was transferred to a 384-well plate with two replicates. 30 μL of the SUMO protease to be tested was transferred to the corresponding well, and the plates were immediately centrifuged and vortexed to mix. The fluorescence signal values ​​generated by the reaction were collected using a TECANF200 microplate reader. Data analysis was performed using Graph Pad Prism9 software to obtain the enzyme activity parameters of the test protease. Figure 3 The activity parameters of different SUMO proteases were obtained by analyzing the Graph Pad Prism9 software.

[0049] from Figure 3 As can be seen, the activity of BioSENP1 for the BioSUMO substrate was 0.001 (RFU / s / nM), the activity of BdSENP1 for the BdSUMO substrate was 0.023 (RFU / s / nM), and the activity of Ulp1 for the SUMO substrate was 0.002 (RFU / s / nM). Based on the enzyme activity data, the activity of BioSENP1 was 20 times lower than that of BdSENP1 and approximately twice as low as that of the commonly used Ulp1 enzyme. Because the substrate also has a TEV cleavage site, it can also be used to determine the activity of TEV protease. The TEV enzyme used in this invention is Biortus (catalog number: BP11748-17A). The enzyme activity parameter of the TEV protease under the same conditions was 0.00004 (RFU / s / nM). The activity of BioSENP1 was 25 times higher than that of the TEV protease, indicating that although the activity of BioSENP1 was lower than that of the enzyme before modification, it was still 25 times higher than that of the TEV enzyme commonly used in protein purification. This shows that its protease cleavage activity is still very high and it can be used for protease cleavage in protein purification.

[0050] 2.5. Application of BioSENP1 protease digestion verification

[0051] To test the practical application effect of the modified BioSENP1 protease, the BioSENP1 protease and its corresponding substrate were subjected to in vitro enzyme digestion verification tests. Simultaneously, the digestion effect was compared with that of the commercially available Ulp1 enzyme and its corresponding substrate. The specific procedures are as follows:

[0052] 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. Both BioSENP1 and Ulp1 proteases were incubated at a mass ratio of 1:1000 to their corresponding substrates. Samples were collected at seven different time points—before incubation, at 5 minutes, 15 minutes, 30 minutes, 1 hour, 2 hours, and 4 hours—for SDS-PAGE analysis to compare the digestion efficiency of BioSENP1 and Ulp1.

[0053] See results Figure 4 The BioSENP1 enzyme cleaves over 90% of the substrate within approximately 5 minutes of digestion, and almost all substrate is completely cleaved within about 1 hour. In contrast, the Ulp1 enzyme cleaves over 90% of the substrate within 15 minutes and completely removes all substrate within about 1 hour. The modified BioSENP1 performs comparably to commercially available Ulp1 in practical applications. Furthermore, the BioSENP1 protease requires only 1 / 1000th the amount of substrate, significantly reducing protease usage costs and minimizing enzyme residue issues during digestion.

[0054] 2.6 Validation of BioSENP1 protease application scenarios

[0055] The biggest problem with SUMO enzymes is that when their substrates are used to express recombinant proteins in eukaryotic expression systems, they are cleaved by the Ulp1 enzyme expressed endogenously in eukaryotic cells, resulting in the recombinant proteins being unable to be enriched by the corresponding affinity chromatography columns after expression.

[0056] To further verify the application scenario of the BioSENP1 protease, namely whether its substrate BioSUMO tag can be cleaved by the Ulp1 enzyme expressed endogenously in eukaryotic cells, the verification was carried out in both prokaryotic and eukaryotic expression systems.

[0057] (A) Validation of prokaryotic expression system

[0058] The test protein used in the prokaryotic expression system of this invention is protein A. The plasmid is constructed as 8His-eGFP-BioSUMO-protein A (molecular weight 49.9 kDa). The sequence of 8His-eGFP-BioSUMO-protein A is shown in SEQ ID NO.9. The protein is constructed in the pET-28a vector. An 8His-eGFP-BioSUMO tag (positions 1-323) is fused to its N-terminus. Protein A (positions 324-429) can be completely cleaved by BioSENP1 to remove the BioSUMO tag, leaving protein A (molecular weight 12.5 kDa) and the N-terminal 8His-GFP-BioSUMO protein (molecular weight 37.5 kDa). Protein A, which was correctly sequenced, was expressed in *E. coli* according to step 2.2. A small amount of 5 mL was expressed and induced with 0.5 mM IPTG at 15°C for 16 hours. After induction, the bacterial cells were collected and the induced bacterial solution was analyzed by SDS-PAGE. After sonication and centrifugation, the supernatant was collected. 25 μL of the lysis supernatant was used for fluorescence-detection size-exclusion chromatography (FSEC). Simultaneously, a small amount of purification was performed. The lysis supernatant was incubated with Ni Bestarose FF packing material and eluted with buffer containing 300 mM imidazole. The sample was then analyzed by SDS-PAGE.

[0059] The verification results of SDS-PAGE can be found in [link to SDS-PAGE]. Figure 5 SDS-PAGE only detected a band with a molecular weight of approximately 50 kDa, indicating that the tag contained in protein A was not cleaved. FSEC detected a peak with a molecular weight of approximately 58 kDa, which is close to the theoretical molecular weight of the intact recombinant protein A, but no peak of the tag 8His-eGFP-BioSUMO was detected. Therefore, the BioSUMO tag was not cleaved in the prokaryotic expression system, and the BioSENP1 enzyme and the corresponding tag BioSUMO can be applied to recombinant proteins in prokaryotic systems.

[0060] (B) Validation of eukaryotic expression system

[0061] The test protein used in this invention for the eukaryotic expression system is protein B, the sequence of which is shown in SEQ ID NO.10, with a molecular weight of approximately 18.2 kDa. To better compare the application of different SUMO tags in the eukaryotic system, three fusion tags were used for comparison: a 6His-BioSUMO tag (6His-BioSUMO-protein B, with a molecular weight of approximately 28 kDa), a 6His-SUMO tag (6His-SUMO-protein B, with a molecular weight of approximately 30.2 kDa), and a 6His-bdSUMO tag (6His-bdSUMO-protein B, with a molecular weight of approximately 28 kDa). The 6His-BioSUMO-protein B, 6His-SUMO-protein B, and 6His-bdSUMO-protein B were constructed on the pFastBac1 vector (GenScript, pFastBac1). The sequences of all recombinant plasmids were synthesized by the gene and verified by sequencing. The specific steps for expressing recombinant protein B are as follows:

[0062] (1) Viral preparation of recombinant protein B

[0063] The three plasmids of recombinant protein B were transformed into DH10Bac competent cells (Bomaid, BC112-01) and cultured at 37°C for 48 hours. Positive clones were successfully obtained through blue-white screening. These positive clones were then cultured overnight in LB medium, and recombinant Bacmid (baculovirus plasmid) was extracted. The extracted Bacmid plasmid was then transfected into insect cells to prepare baculovirus. The specific virus preparation process was as follows: Insect Sf9 cells (Thermo, 11496015) were first diluted to 1×10⁻⁶. 6 Cells / mL, 15 μL of recombinant Bacmid was added to 100 μL of Grace insect medium (Thermo, 10902104) containing 7 μL of X-treme (Roche) transfection reagent, mixed and incubated for 15 minutes. The mixture of recombinant Bacmid and transfection reagent was then transferred into Sf9 cells. After culturing in an incubator for 4 days, P0 recombinant baculovirus was obtained. 1 mL of the harvested P0 virus was added to 50 mL of Sf9 cells, and after culturing in an incubator for 3 days, the cells were centrifuged for 5 minutes, and the supernatant was collected as P1 virus. 4 mL of P1 virus was added to 200 mL of Sf9 cells, and after culturing for 3 days, the cells were centrifuged for 5 minutes, and the supernatant was collected as P2 virus.

[0064] (2) Low-level expression and purification of recombinant protein B

[0065] The prepared recombinant protein P2 generation virus was transfected into Sf21 cells (Thermo, 11497013) and Hi-5 cells (Thermo, B85502) at a multiplicity of infection (MOI) ratio of 1 for small-scale expression (10 mL). After culturing at 27°C for 48 hours, the cells were collected, sonicated, and centrifuged to collect the supernatant. 25 μL of the lysis supernatant was taken for FSEC analysis. In addition, a small-scale purification was performed. The lysis supernatant was incubated with NiBestarose FF packing material and eluted with buffer containing 300 mM imidazole. Samples were taken for SDS-PAGE analysis.

[0066] The validation results of the eukaryotic expression system are shown in Figure 6 According to the SDS-PAGE results, after the N-terminus of protein B was fused with the tags 6His-SUMO and 6His-bdSUMO, in addition to detecting the target protein, bands with molecular weights of approximately 12 kDa and 10 kDa appeared, respectively. These bands correspond to the 6His-SUMO and 6His-bdSUMO tags, indicating that the fusion protein B was cleaved by the endogenously expressed Ulp1 enzyme in eukaryotic cells. However, when the N-terminus of recombinant protein B was fused with 6His-BioSUMO, only a band with a molecular weight of approximately 28 kDa appeared on the SDS-PAGE, without a separate tag band, indicating that the 6His-BioSUMO tag was not cleaved by the endogenous Ulp1 enzyme in eukaryotic cells.

[0067] Similarly, FSEC analysis of the post-expression lysate supernatant showed that after the N-terminal fusion tags 6His-SUMO and 6His-bdSUMO of protein B, only a small number of peaks of the target protein were detected; the majority of the detected peaks were those with molecular weights of 18 kDa and 9 kDa, respectively. These peaks corresponded to the individual 6His-SUMO and 6His-bdSUMO tags. However, after the N-terminal fusion tag 6His-BioSUMO of protein B, only a peak of approximately 31 kDa of the fusion protein B was detected on FSEC. The FSEC results were consistent with the SDS-PAGE results, indicating that 6His-BioSUMO-protein B was not cleaved by the Ulp1 enzyme expressed endogenously in eukaryotic cells. Therefore, the BioSUMO tag is not cleaved by the Ulp1 enzyme expressed endogenously in eukaryotic cells, and the BioSENP1 enzyme and its corresponding tag BioSUMO can be applied to recombinant proteins in eukaryotic systems.

[0068] 3. Conclusion

[0069] The above description indicates that the present invention provides an enzyme digestion system adapted to eukaryotic and prokaryotic expression systems, comprising a SUMO protease and a digestion substrate; wherein the SUMO protease is a BioSENP1 protease, and the digestion substrate is a BioSUMO tag. The BioSENP1 protease and the corresponding BioSUMO tag adapted to eukaryotic and prokaryotic expression systems provided by the present invention can overcome the problem of SUMO tags being digested by endogenous Ulp1 in eukaryotic expression systems. Furthermore, the BioSENP1 protease has high yield, high enzyme activity, and high digestion efficiency. The practical application effect of BioSENP1 is comparable to that of commercially available Ulp1, demonstrating broad application value and prospects.

[0070] 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 SUMO protease, characterized in that, The SUMO protease is the BioSENP1 protease, the amino acid sequence of which is shown in SEQ ID NO.2, and the nucleotide sequence of which is shown in SEQ ID NO.

3.

2. A SUMO protease, characterized in that, The method for preparing the SUMO protease includes the following steps: (1) First, add a 10His tag to the N-terminus of the amino acid sequence of BioSENP1 protease as shown in SEQ ID NO.

2. Then, synthesize a nucleotide sequence that can encode the amino acid sequence according to the amino acid sequence after adding the 10His tag. Finally, construct the synthesized nucleotide sequence on the pET-28a vector to obtain a recombinant plasmid. (2) First, use the Escherichia coli prokaryotic expression system to express the recombinant plasmid, harvest cells containing SUMO protease, lyse the cells to obtain cell supernatant, and then load the cell supernatant onto a Ni Bestarose FF affinity chromatography column to enrich and purify the protein, thereby obtaining the SUMO protease.

3. An enzyme digestion system adapted to eukaryotic and prokaryotic expression systems, characterized in that, The enzyme digestion system includes an enzyme digestion substrate and a SUMO protease as described in any one of claims 1-2, wherein the enzyme digestion substrate is a BioSUMO tag, the amino acid sequence of the BioSUMO tag is shown in SEQ ID NO.4, and the nucleotide sequence of the BioSUMO tag is shown in SEQ ID NO.

5.

4. The application of the enzyme digestion system as described in claim 3 in the expression and purification of recombinant proteins in eukaryotic and prokaryotic expression systems.

5. The application according to claim 4, characterized in that, The method for expressing and purifying recombinant proteins using the eukaryotic and prokaryotic expression systems includes the following steps: (1) First, the target protein is linked with the amino acid sequence containing the enzyme digestion substrate to construct a recombinant protein, and then the recombinant protein is constructed on an expression vector to obtain a recombinant plasmid; (2) Express recombinant plasmids using eukaryotic or prokaryotic expression systems, harvest cells containing recombinant proteins, and lyse the cells to obtain cell supernatant containing recombinant proteins; (3) First, incubate the cell supernatant with the affinity purification matrix, then add the buffer of SUMO protease corresponding to the enzyme digestion substrate to wash the affinity purification matrix, so that the target protein can be eluted from the affinity purification matrix.

6. The application according to claim 5, characterized in that, The prokaryotic expression system is the Escherichia coli prokaryotic expression system, and the eukaryotic expression system is the insect cell eukaryotic expression system or the mammalian cell eukaryotic expression system.

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