Enzyme digestion system applicable to eukaryotic and prokaryotic expression systems and application of enzyme digestion system

Abstract

The invention discloses an enzyme digestion system suitable for eukaryotic and prokaryotic expression systems and application thereof, and relates to the technical field of biology, the enzyme digestion system comprises SUMO protease and an enzyme digestion substrate; wherein the SUMO protease is a BioSENP1 protease, and the enzyme cutting substrate is a BioSUMO label. According to the BioSENP1 protease and the corresponding BioSUMO tag suitable for eukaryotic and prokaryotic expression systems, the situation that the SUMO tag is digested by endogenous Ulp1 enzyme in the eukaryotic expression system can be overcome, the yield of the BioSENP1 protease is high, the enzyme activity is relatively high, the enzyme digestion efficiency is high, the practical application effect of the BioSENP1 protease is equivalent to the effect of the Ulp1 enzyme sold in the market, and the BioSENP1 protease has a good application prospect. Wide application values and prospects are realized.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to an enzyme cutting system suitable for eukaryotic and prokaryotic expression systems and an application thereof. Background Art

[0002] Ubiquitin is a molecule widely involved in protein degradation and signal transduction in cells, while small ubiquitin-like modifier (SUMO) is another type of ubiquitin-like protein that participates in regulating a variety of biological processes in cells, such as nuclear protein transport, DNA repair, transcriptional regulation, cell cycle control, etc. SUMO modification, as a post-translational modification, has important biological significance and has been widely used in protein labeling, functional research, and protein purification.

[0003] SUMO tag has many advantages in protein purification. It can not only increase the solubility of the target protein, but also because it has a spatial conformation-specific SUMO protease Ulp1, which can be directly cleaved by Ulp1 and will not leave other amino acids on the target protein sequence, it is often used in protein purification. The most commonly used SUMO tag comes from yeast, and the corresponding Ulp1 enzyme in yeast is also sold as a common commercial enzyme. However, since both the SUMO tag and the Ulp1 enzyme are derived from eukaryotic organisms, endogenous Ulp1 enzymes are expressed in eukaryotic cells including insects and mammalian cells. Therefore, when expressing recombinant proteins in eukaryotic expression systems, if the SUMO tag is fused to the sequence of the recombinant protein, the SUMO tag may be cleaved by the endogenous Ulp1 enzyme.

[0004] At present, in order to solve the problem of SUMO tag being removed in eukaryotic systems, LifeSensors has designed a new SUMOstar tag based on the SUMO tag and the corresponding SUMOstar enzyme that removes the SUMOstar tag. This combination is the only SUMOstar tag and SUMOstar enzyme that have been reported to be suitable for eukaryotic expression systems. However, since the tag and enzyme have been patented, their application and development are subject to certain restrictions. In addition, there are still some problems that need to be solved in the existing technology, such as unstable tag removal efficiency, large impact on the target protein, and excessive dependence on the SUMO enzyme.

[0005] Therefore, it is necessary to develop new SUMO enzymes with better activity and corresponding SUMO tags suitable for eukaryotic and prokaryotic expression systems to improve the efficiency of tag removal and reduce interference with the target protein. More efficient and stable SUMO tags and SUMO enzyme systems can provide new research ideas for the use of SUMO tags and SUMO enzymes in the current biochemistry and molecular biology research fields. Summary of the invention

[0006] The purpose of the present invention is to provide an enzyme cutting system suitable for eukaryotic and prokaryotic expression systems and its application.

[0007] The present invention achieves the above-mentioned purpose through the following technical solutions:

[0008] As a first aspect of the present invention, a SUMO protease is provided, wherein the SUMO protease is BioSEN P1 protease, the amino acid sequence of the BioSENP1 protease is shown as SEQ ID NO.2, and the nucleotide sequence of the BioSENP1 protease is shown as SEQ ID NO.3.

[0009] A further improvement is that the method for preparing the BioSENP1 protease comprises the following steps:

[0010] (1) firstly adding a 10His tag to the N-terminus of the amino acid sequence of the BioSENP1 protease as shown in SEQ ID NO.2, then synthesizing a nucleotide sequence encoding the amino acid sequence according to the amino acid sequence after adding the 10His tag, and finally constructing the synthesized nucleotide sequence on the pET-28a vector to obtain a recombinant plasmid;

[0011] (2) First, the recombinant plasmid is expressed using an Escherichia coli prokaryotic expression system, cells containing the BioSENP1 protease are harvested, the cells are lysed to obtain a cell supernatant, and then the cell supernatant is 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 cleavage system suitable for eukaryotic and prokaryotic expression systems is also provided, the enzyme cleavage system comprising SUMO protease and an enzyme cleavage substrate, wherein the enzyme cleavage 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 present invention, there is also provided a use of any of the above-mentioned enzyme cleavage systems in expressing and purifying recombinant proteins in eukaryotic and prokaryotic expression systems.

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

[0015] (1) First, the target protein is connected to the amino acid sequence containing the enzyme cleavage substrate to construct a recombinant protein, and then the recombinant protein is constructed on an expression vector to obtain a recombinant plasmid;

[0016] (2) expressing the recombinant plasmid using a eukaryotic or prokaryotic expression system, harvesting cells containing the recombinant protein, and lysing the cells to obtain a cell supernatant containing the recombinant protein;

[0017] (3) First, the cell supernatant is incubated with the affinity purification matrix, and then a buffer of the SUMO protease corresponding to the cleavage substrate is added 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] The present invention provides an enzyme cleavage system adapted to eukaryotic and prokaryotic expression systems, comprising SUMO protease and an enzyme cleavage substrate; wherein the SUMO protease is BioSENP1 protease, and the enzyme cleavage substrate is a BioSUMO tag. The BioSENP1 protease provided by the present invention and the corresponding BioSUMO tag adapted to eukaryotic and prokaryotic expression systems can overcome the situation that the SUMO tag is cleaved by endogenous Ulp1 in the eukaryotic expression system, and the BioSENP1 protease has high yield, high enzyme activity, high enzyme cleavage efficiency, and the actual application effect of the BioSENP1 protease is equivalent to that of the Ulp1 enzyme sold on the market, and has broad application value and prospects. BRIEF DESCRIPTION OF THE DRAWINGS

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

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

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

[0024] Figure 4The enzymatic cleavage verification result of the BioSENP1 protease provided by the present invention;

[0025] Figure 5 The verification results of the usage scenarios of the BioSENP1 protease and the label BioSUMO provided by the present invention in the prokaryotic expression system;

[0026] Figure 6 This is the verification result of the usage scenario of the BioSENP1 protease and the label BioSUMO provided by the present invention in the eukaryotic expression system. DETAILED DESCRIPTION

[0027] The present application is further described in detail below in conjunction with the accompanying drawings. It is necessary to point out here 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. Technical personnel in this field 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, the methods used in the present invention are conventional methods known to those skilled in the art. If no specific conditions are 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.

[0030] 2. Methods

[0031] 2.1. Construction of recombinant SUMO protease and its substrate plasmid

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

[0033] The modified SUMO enzyme was mutated based on the BdSENP1 sequence and named BioSENP1. The 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. At the same time, the substrate corresponding to BioSENP1 was synthesized. In order to better detect the enzyme activity, we specially designed the substrate: 6His-mCherry-GG-TEV-GG-substrate-GG-eGFP, with a 6His tag, red fluorescent protein mCherry and TEV protease cleavage site at the N-terminus, and a green fluorescent protein eGFP at the C-terminus. "GG" connects the mCherry protein, TEV protease cleavage site, substrate sequence and 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, 1-6 "HHHHHH" is a 6His sequence, 7-241 is an mCherry sequence, 242-243 "GG" is a linker sequence, 244-250 "ENLYFQG" is a TEV restriction site sequence, 251-252 "GG" is a linker sequence, 253-329 is a bdSUMO tag sequence, 330-331 "GG" is a linker sequence, and 332-569 is an eGFP sequence.

[0035] The substrate corresponding to the modified BioSENP1 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" are 6His sequences, positions 7-241 are mCherry sequences, positions 242-243 "GG" are linker sequences, positions 244-250 "ENLYFQG" are TEV restriction site sequences, positions 251-252 "GG" are linker sequences, positions 253-329 are BioSUMO tags, positions 330-331 "GG" are linker sequences, and positions 332-569 are eGFP sequences. 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 pET-28a vectors, and all recombinant plasmids were verified by sequencing to be completely consistent with the target sequence.

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

[0038] Using conventional molecular biological methods, 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) Escherichia coli competent cells in a clean bench and cultured at 37°C overnight. The monoclonal colonies cultured overnight were picked and placed in 5 ml LB liquid medium and cultured at 37°C until the bacterial solution OD 600 When the OD 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 at -80℃. Inoculate the glycerol bacteria into 50ml LB liquid medium and culture at 37℃ overnight. Inoculate the bacteria cultured overnight into 1L LB liquid medium at a ratio of 1:100 and culture at 37℃ until the bacterial solution OD value reaches 0.6-0.8. 600 When the pH value was 0.6-0.8, 0.5 mM IPTG was added and cultured overnight at 15°C. The bacteria were collected by centrifugation at 5000 rpm.

[0039] 2.3. Purification of recombinant BdSENP1 and BioSENP1 protease

[0040] The collected cells were weighed, and the corresponding volume of lysis buffer (50mM Tris-HCl (pH7.5), 500mM NaCl, 5% glycerol, 20mM imidazole) was added at a ratio of 1:10. The cells were broken using a high-pressure homogenizer, and the supernatant was collected by high-speed centrifugation at 16000rpm. Both BioSENP1 and BdSENP1 proteases have 10His tags, and the proteins were enriched and purified using NiBestarose FF affinity chromatography columns.

[0041] 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, elute with different gradients of imidazole solution, collect the proteins eluted with different gradients of imidazole for SDS-PAGE detection, and use Nanodrop to measure the protein concentration and calculate the protein yield.

[0042] Protein purification results Figure 1According to the purification results, BioSENP1 and BdSENP1 can be purified by NiBestarose FF affinity chromatography, and the purified proteins have high purity. According to the concentration information measured by Nanodrop, the yield of BioSENP1 is about 63 mg / L, and the yield of BdSENP1 is 50 mg / L. In order to further compare the activity with the commercially available Ulp1 protease, 10His-Ulp1 protease was constructed according to the construction method of BioSENP1 and BdSENP1. The sequence of 10His-Ulp1 protease is shown in SEQ ID NO.8 (in the sequence, 'MGSS' is the sequence on the vector, and 'HHHHHHHHHH' is the 10His tag). The results show that its yield is about 53 mg / L.

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

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

[0045] 2.5. Determination of protease activity of recombinant BdSENP1 and BioSENP1

[0046] The SUMO protease activity was detected by fluorescence resonance energy transfer. The principle of fluorescence resonance energy transfer is that in two different fluorescent groups, the emission spectrum of one fluorescent group (donor) overlaps with the absorption spectrum of the other group (acceptor), and the distance between the two fluorescent groups is less than Fluorescence energy will be transferred from the donor to the acceptor, 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 red fluorescent protein mCherry at its N-terminus (maximum excitation light and emission light are 587nm and 610nm, respectively), and green fluorescent protein eGFP at its C-terminus (maximum excitation light and emission light are 484nm and 507nm, 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 the fluorescent signal. The higher the SUMO protease activity, the more fluorescent groups are released, and the greater the fluorescence intensity. The enzyme activity parameter is represented by the fluorescence intensity absorbed per nanomole of protein per second.

[0047] The specific operations are as follows:

[0048] Different substrates were prepared into 1mM stock solutions and divided for use. The SUMO protease activity test buffer was 40mM Tris-HCl pH 7.0, 200mM NaCl, 2mM MgCl2, 250mM Sucrose, and 2mM DTT. The concentration of the protease was diluted to 1000nM with a 2-fold gradient dilution using the buffer, with a total of 12 dilutions. 30μL of substrate was transferred to a 384-well plate and two replicate wells were set up. 30μL of the SUMO protease to be tested was transferred to the corresponding well plate, immediately centrifuged and oscillated to mix, and the fluorescence signal value generated by the reaction was collected using a TECANF200 microplate reader. Graph Pad Prism9 analysis software was used for data analysis, and the enzyme activity parameters of the protease to be tested were finally obtained. Figure 3 That is, different SUMO protease activity parameters were obtained by analyzing the Graph Pad Prism9 software.

[0049] from Figure 3 It can be seen that the activity of BioSENP1 for BioSUMO substrate was 0.001 (RFU / s / nM), the activity of BdSENP1 for BdSUMO substrate was 0.023 (RFU / s / nM), and the activity of Ulp1 for SUMO substrate was 0.002 (RFU / s / nM). From the enzyme activity data, the activity of BioSENP1 is 20 times lower than that of BdSENP1 and about 2 times lower than that of the commonly used Ulp1 enzyme on the market. Because the substrate also has a TEV cleavage site, the substrate can also be used to determine the activity of TEV protease. The TEV enzyme used in the present invention is a product of Biortus (Article No.: BP11748-17A). The enzyme activity parameter of TEV protease detected under the same conditions is 0.00004 (RFU / s / nM). The activity of BioSENP1 is 25 times higher than that of TEV protease, indicating that although the activity of BioSENP1 is lower than that before the modification, it is 25 times higher than the activity of TEV enzyme commonly used in protein purification, indicating that its protease cleavage activity is still very high and is used for protease cleavage in protein purification.

[0050] 2.5. BioSENP1 protease digestion verification application

[0051] In order to test the practical application effect of the modified BioSENP1 protease, the BioSENP1 protease and its corresponding substrate were tested for enzyme cleavage in vitro, and the enzyme cleavage effect was compared with the commercially commonly used Ulp1 enzyme and its corresponding substrate. The specific operation is as follows:

[0052] In this experiment, the reaction buffer was 50mM Tris-HCl (pH8.0), 500mM NaCl, and 5% glycerol. The enzyme digestion reaction was tested at 4°C. The mass ratio of BioSENP1 protease and Ulp1 protease to their corresponding substrates was 1:1000 for incubation. The samples after enzyme digestion at 7 different time points, including before the start of incubation, 5 minutes, 15 minutes, 30 minutes, 1 hour, 2 hours, and 4 hours, were collected for SDS-PAGE detection to compare the enzyme digestion effects of BioSENP1 and Ulp1.

[0053] Results Figure 4 , more than 90% of the substrates were cut by BioSENP1 enzyme after about 5 minutes of enzyme digestion, and all substrates were basically completely cut after about 1 hour. Compared with Ulp1 enzyme, more than 90% of the substrates were cut after 15 minutes of enzyme digestion, and all substrates were completely removed after about 1 hour of enzyme digestion. The actual application effect of the modified BioSENP1 is equivalent to that of Ulp1 sold on the market. In actual application, the amount of BioSENP1 protease required is only 1 / 1000 of the amount of substrate, which greatly reduces the cost of protease in enzyme digestion and reduces the problem of enzyme residue during use.

[0054] 2.6. BioSENP1 protease usage scenario verification

[0055] The biggest problem with SUMO enzyme during use is that its substrate will be removed by the Ulp1 enzyme endogenously expressed in eukaryotic cells when used in a eukaryotic expression system to express recombinant proteins, resulting in the inability of the recombinant protein to be enriched by the corresponding affinity chromatography column after expression.

[0056] In order to further verify the usage scenario of BioSENP1 protease, that is, whether its substrate BioSUMO tag will be removed by the Ulp1 enzyme endogenously expressed in eukaryotic cells, verification was performed in prokaryotic expression system and eukaryotic expression system respectively.

[0057] (A) Prokaryotic expression system verification

[0058] The test protein used in the prokaryotic expression system of the present invention is protein A, and the plasmid is constructed as 8His-eGFP-BioSUMO-protein A (molecular weight 49.9kDa), and the sequence of 8His-eGFP-BioSUMO-protein A is shown in SEQ ID NO.9, and the protein is constructed on the pET-28a vector. Its N-terminal is fused with 8His-eGFP-BioSUMO tag (positions 1-323 of the sequence), and protein A (positions 324-429 of the sequence) can be completely cut out of the BioSUMO tag by BioSENP1, leaving protein A (molecular weight 12.5kDa) and 8His-GFP-BioSUMO protein (molecular weight 37.5kDa) at the N-terminal. The correctly sequenced protein A was expressed in Escherichia coli. The expression was referred to step 2.2. 5 mL was expressed in a small amount. After induction with 0.5 mM IPTG at 15°C for 16 hours, the bacteria were collected and the induced bacterial solution was taken for SDS-PAGE detection. The bacteria were ultrasonically disrupted and the supernatant was collected by centrifugation. 25 μL of the lysate supernatant was taken for fluorescence-detection size-exclusion chromatography (FSEC). At the same time, a small amount of purification was performed. The lysate supernatant was incubated with Ni Bestarose FF filler and eluted with a buffer containing 300 mM imidazole, and a sample was taken for SDS-PAGE detection.

[0059] The verification results of SDS-PAGE are shown in Figure 5 , only a band with a molecular weight of about 50kDa was detected on SDS-PAGE, indicating that the tag contained in protein A was not cleaved by enzymes; FSEC detected a peak with a molecular weight of about 58kDa, which was close to the theoretical molecular weight of the complete recombinant protein A, and no peak of the tag 8His-eGFP-BioSUMO was detected. Therefore, the BioSUMO tag was not cleaved by enzymes in the prokaryotic expression system, and the BioSENP1 enzyme and the corresponding tag BioSUMO can be applied to recombinant proteins in the prokaryotic system.

[0060] (B) Eukaryotic expression system verification

[0061] The test protein used in the eukaryotic expression system of the present invention is protein B, and the sequence of protein B is shown in SEQ ID NO.10, and the molecular weight is about 18.2kDa. In order to better compare the application of different SUMO tags in eukaryotic systems, 6His-BioSUMO tags, i.e. 6His-BioSUMO-protein B, and the molecular weight of the fusion protein is about 28kDa, 6His-SUMO tags, i.e. 6His-SUMO-protein B, and the molecular weight of the fusion protein is about 30.2kDa and 6His-bdSUMO tags, i.e. 6His-bdSUMO-protein B, and the molecular weight of the fusion protein is about 28kDa for comparison. 6His-BioSUMO-protein B, 6His-SUMO-protein B, and 6His-bdSUMO-protein B are respectively constructed on pFastBac1 vector (GenScript, pFastBac1), and the sequences of all recombinant plasmids are synthesized by genes and verified to be correct by sequencing. The specific steps of the expression of recombinant protein B are:

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

[0063] The three plasmids of the above recombinant protein B were transformed into DH10Bac competent cells (Bomed, BC112-01) respectively, and cultured at 37°C for 48 hours. Positive clones were successfully obtained through blue-white screening, and positive clones were picked and cultured overnight in LB medium. Recombinant Bacmid (baculovirus plasmid) was extracted, and then the extracted Bacmid plasmid was transfected into insect cells to prepare baculovirus. The specific virus preparation process is: first dilute the insect Sf9 cells (Thermo, 11496015) to 1×10 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, and the mixture of recombinant Bacmid and transfection reagent was 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 supernatant was collected for 5 minutes as P1 virus; 4 mL of P1 virus was added to 200 ml of Sf9 cells and cultured for 3 days, and the supernatant was collected by centrifugation for 5 minutes as P2 virus.

[0064] (2) Small-scale expression and purification of recombinant protein B

[0065] The P2 generation virus of the prepared recombinant protein was transfected into Sf21 cells (Thermo, 11497013) and Hi-5 cells (Thermo, B85502) at a multiplicity of infection (MOI) of 1 for small-scale expression (10 mL). The cells were collected after culturing at 27°C for 48 hours, and the cells were ultrasonically disrupted and the supernatant was collected by centrifugation. 25 μL of the lysate supernatant was taken for FSEC detection. In addition, a small amount of purification was performed, and the lysate supernatant was incubated with NiBestarose FF filler and eluted with a buffer containing 300 mM imidazole, and a sample was taken for SDS-PAGE detection.

[0066] The eukaryotic expression system validation results are shown in Figure 6 According to the results of SDS-PAGE, after the N-terminal fusion tags 6His-SUMO and 6His-bdSUMO of protein B were detected, in addition to the target protein, a band with a molecular weight of about 12kDa and 10kDa respectively appeared. These bands correspond to the 6His-SUMO and 6His-bdSUMO tags, respectively, indicating that the fusion protein B was removed by the Ulp1 enzyme endogenously expressed in eukaryotic cells. However, when the N-terminus of recombinant protein B was fused with 6His-BioSUMO, there was only a band with a molecular weight of about 28kDa on its SDS-PAGE, and no separate tag band appeared, indicating that the 6His-BioSUMO tag would not be removed by the endogenous Ulp1 enzyme in eukaryotic cells.

[0067] Similarly, the FSEC detection results of the lysate supernatant after expression showed that after the N-terminal fusion tags 6His-SUMO and 6His-bdSUMO of protein B were added, only a small amount of target protein peaks were detected, and most of the peaks detected were peaks with molecular weights of 18kDa and 9kDa, respectively. This peak corresponds to the separate 6His-SUMO and 6His-bdSUMO tags. After the N-terminal fusion tag 6His-BioSUMO tag of protein B was added, only the peak of fusion protein B with a molecular weight of about 31kDa was detected on FSEC. The results of FSEC are consistent with those of SDS-PAGE, which also shows that 6His-BioSUMO-protein B is not removed by the Ulp1 enzyme endogenously expressed in eukaryotic cells. It can be seen that the BioSUMO tag will not be removed by the Ulp1 enzyme endogenously expressed in eukaryotic cells, and the BioSENP1 enzyme and the corresponding tag BioSUMO can be applied to recombinant proteins in eukaryotic systems.

[0068] 3. Conclusion

[0069] The above shows that the present invention provides an enzyme cleavage system adapted to eukaryotic and prokaryotic expression systems, including SUMO protease and enzyme cleavage substrate; wherein the SUMO protease is BioSENP1 protease, and the enzyme cleavage substrate is BioSUMO tag. The BioSENP1 protease provided by the present invention and the corresponding BioSUMO tag adapted to eukaryotic and prokaryotic expression systems can overcome the situation that the SUMO tag is cleaved by endogenous Ulp1 in the eukaryotic expression system, and the BioSENP1 protease has high yield, high enzyme activity, high enzyme cleavage efficiency, and the actual application effect of BioSENP1 is comparable to that of Ulp1 sold on the market, and has broad application value and prospects.

[0070] The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the present invention. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention.

Claims

1. A SUMO protease, characterized in that The SUMO protease is BioSENP1 protease, the amino acid sequence of the BioSENP1 protease is shown in SEQ ID NO.2, and the nucleotide sequence of the BioSENP1 protease is shown in SEQ ID NO.

3.

2. A SUMO protease according to claim 1, characterized in that The preparation method of the BioSENP1 protease comprises the following steps: (1) firstly adding a 10His tag to the N-terminus of the amino acid sequence of the BioSENP1 protease as shown in SEQ ID NO.2, then synthesizing a nucleotide sequence encoding the amino acid sequence according to the amino acid sequence after adding the 10His tag, and finally constructing the synthesized nucleotide sequence on the pET-28a vector to obtain a recombinant plasmid; (2) First, the recombinant plasmid is expressed using an Escherichia coli prokaryotic expression system, cells containing the BioSENP1 protease are harvested, the cells are lysed to obtain a cell supernatant, and then the cell supernatant is loaded onto a NiBestarose FF affinity chromatography column to enrich and purify the protein, thereby obtaining the BioSENP1 protease.

3. An enzyme digestion system suitable for eukaryotic and prokaryotic expression systems, characterized in that: The enzyme cleavage system comprises an enzyme cleavage substrate and the SUMO protease according to any one of claims 1 to 2, wherein the enzyme cleavage 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. Use of the enzyme cleavage system as claimed in claim 3 in expressing and purifying recombinant proteins in eukaryotic and prokaryotic expression systems.

5. The use according to claim 4, characterized in that: The method for expressing and purifying recombinant proteins in the eukaryotic and prokaryotic expression systems comprises the following steps: (1) First, the target protein is connected to the amino acid sequence containing the enzyme cleavage substrate to construct a recombinant protein, and then the recombinant protein is constructed on an expression vector to obtain a recombinant plasmid; (2) expressing the recombinant plasmid using a eukaryotic or prokaryotic expression system, harvesting cells containing the recombinant protein, and lysing the cells to obtain a cell supernatant containing the recombinant protein; (3) First, the cell supernatant is incubated with the affinity purification matrix, and then a buffer of the SUMO protease corresponding to the cleavage substrate is added to wash the affinity purification matrix, so that the target protein can be eluted from the affinity purification matrix.

6. The use according to claim 5, characterized in 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.

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