CN117625656A - SUMO protease gene, recombinant expression vector, engineering bacterium and application thereof - Google Patents
SUMO protease gene, recombinant expression vector, engineering bacterium and application thereof Download PDFInfo
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Classifications
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention provides a SUMO protease gene, a recombinant expression vector, engineering bacteria and application thereof. The nucleotide sequence of the SUMO protease gene is shown as SEQ ID No. 2. The invention has the following technical effects: 1) The SUMO tag can promote the folding of target proteins and improve the protein yield. The SUMO protease DNA with the SUMO tag can promote the sufficient folding of the SUMO protease in the escherichia coli cell, improve the solubility of the SUMO protease protein in the supernatant after bacterial disruption, and can be automatically digested to finally obtain the SUMO protease without the SUMO tag; 2) Besides the SUMO label, the constructed SUMO protease DNA also has His affinity label, and the SUMO protease protein with the purity of more than 95% can be obtained simply through affinity chromatography.
Description
Technical Field
The invention relates to the technical field of genetic engineering. In particular to a SUMO protease gene, a recombinant expression vector containing the SUMO protease gene, engineering bacteria for expressing the SUMO protease and application thereof.
Background
Small molecule ubiquitin-related modified proteins (SUMO) are a class of ubiquitin-related proteins that modulate the function of a target protein by binding to lysine side chains. In recent years, SUMO tags have become an effective biotechnology tool, and fusion of SUMO and target proteins can promote protein folding, enhance soluble expression of proteins, protect proteins from protease hydrolysis, improve stability of proteins, and successfully express various heterologous proteins. The traditional fusion tag protein only recognizes the primary amino acid sequence by the corresponding protease in the subsequent cutting process, and the recognition mode can lead to the low cutting efficiency of the cutting protease due to space repression. However, the SUMO tag protein can be specifically identified by SUMO protease Ulp1 to have a tertiary structure and accurately cut, and no amino acid residue exists after cutting, so that the SUMO tag is one of the most ideal fusion tags for fusion expression of the current procaryote and is widely used.
SUMO proteases are one of the members of the cysteine egg Bai Meichao family, and in eukaryotes mainly perform two physiological functions: (1) Excision of the C-terminal-GG-ATY sequence of the SUMO precursor to a mature-state-GG; (2) SUMO is cleaved from the active target protein by SUMO removal. To date, two SUMO proteases are found in yeast: ulp1 and Ulp2; the catalytic domain of about 200 amino acids common to both Ulp is called ULP, namely Ulp1 (403-621), which shows complete protease activity and is found in humans as a total of 7 SUMO-specific proteases.
ULP has been attracting attention due to the wide application of SUMO tags in bioengineering, and as research proceeds, ULP has been found to have an insurmountable advantage as a protease over conventional proteases. A systematic study of ULP was performed by the Tauseef R.Butt team of Lifestors, USA. The ULP has high activity in a wide temperature range, a wide pH range and various buffer conditions, is resistant to a plurality of chemical reagents, can keep high activity even in a high-concentration denaturant environment, and has no influence on ULP enzyme cutting SUMO labels due to other amino acids except Pro of the N-terminal residue of the target protein fused with SUMO.
Unlike traditional sequence-specific proteases, ULP has very high enzyme activity, and generally ULP and substrate can be cleaved very well at a ratio of 1:5000, even at a ratio of 1:10000. On the other hand, even if the ULP to substrate ratio is adjusted to 1:1, no non-specific cleavage of ULP occurs.
As both SUMO and ULP are derived from yeast, recombinant expression can be realized in E.coli, but due to the heterologous protein, the expression level is relatively low, thus limiting the expansion of SUMO fusion system.
In Chinese patent application with publication number of CN102234640A, named recombinant small molecule ubiquitin-like modifier protease, preparation method and application thereof, construction of SUMO protease with GST tag and preparation method thereof are provided; in patent CN201610560872.2, construction of SUMO protease genes directly expressing SUMO protease genes and SUMO protease genes containing 6 tags, respectively, is disclosed.
Although the above patents can obtain better expression of SUMO protease, the following technical problems exist:
1) The tag proteins used in the above patents have the main effect of promoting the soluble expression of the proteins, and the SUMO protease can be purified by using a specific affinity chromatography method aiming at the tag proteins, but the use of protein tags has no positive effect on the improvement of the activity of the SUMO protease;
2) Generally, if a SUMO enzyme protein without a tag is obtained in the above patent, the corresponding tag protein is excised by using a corresponding protease, which inevitably increases the purification steps and purification costs.
Aiming at the problems, the label adopted by the invention is SUMO label, high-purity protein can be obtained through simple affinity chromatography in purification, and self-digestion can be carried out in the expression and purification process to obtain SUMO protease without the label, and the purity of the obtained SUMO protease is high, and the activity of the SUMO protease is about 50 percent higher than that of a commercial product. The expression mode of the escherichia coli strain constructed by the invention is an engineering strain of SUMO protease with intracellular soluble expression and more than 25% of expression quantity, so that the SUMO protease ULP is expressed at a high level in escherichia coli, and the production cost of the SUMO protease is reduced.
Disclosure of Invention
The invention aims to solve the technical problems in the prior art and provides a SUMO protease recombinant expression vector, an engineering bacterium for expressing SUMO protease, a construction method and application thereof.
In order to achieve the above object, the present invention adopts the following technical scheme:
in a first aspect of the present invention, there is provided a SUMO protease gene consisting of a SUMO tag, a Ulp1 sequence and a polyhistidine tag, wherein the nucleotide sequence of the SUMO protease gene is shown in SEQ ID No. 2. The SUMO protease gene is used for encoding a novel high-activity SUMO protease.
In the invention, the SUMO label plays a role in promoting the folding of SUMO protease through fusion of the label and the SUMO protease, enhancing the soluble expression of the SUMO protease, protecting the SUMO protease from being hydrolyzed by the protease and improving the stability of the SUMO protease; the polyhistidine tag is used as a tag for affinity chromatography to facilitate protein purification.
In a second aspect of the present invention, there is provided a recombinant expression vector comprising the SUMO protease gene of the first aspect.
The expression vector is constructed by introducing the SUMO protease gene with the nucleotide sequence shown in SEQ ID No.2 in the first aspect into an expression plasmid,
the amino acid sequence of the Ulp1 is the 402 th-621 th amino acid (SEQ ID No. 1) of a SUMO enzyme Ulp1 protein sequence (GenBank: KAF 1908775.1) derived from Saccharomyces cerevisiae.
The amino acid sequence expressed by the expression vector is shown as SEQ ID No. 3.
Preferably, the expression plasmid is pET28, pET21, pET24 or pET31.
In a third aspect of the present invention, there is provided an engineering bacterium for efficiently expressing SUMO protease, the recombinant engineering strain being produced by transforming a host bacterium with the expression vector of the second aspect.
Preferably, the host bacteria are E.coli BL21 (DE 3), E.coli DH5a or E.coli Rosetta.
In a fourth aspect of the present invention, there is provided a method for constructing an engineering bacterium highly expressing SUMO protease, comprising the steps of:
1) Obtaining a SUMO protease gene consisting of a SUMO tag, a Ulp1 sequence and a polyhistidine tag by a PCR amplification mode, wherein the nucleotide sequence of the SUMO protease gene is shown as SEQ ID No. 2;
2) The SUMO protease gene obtained in the step 1) is respectively connected with the expression plasmid after double enzyme digestion, so as to obtain a recombinant plasmid containing the SUMO protease gene;
3) Preparing host bacterium competent cells, transforming the host bacterium by using the plasmid in the step 2), and screening positive clones to obtain recombinant engineering bacteria for efficiently expressing SUMO protease.
Preferably, the expression plasmid is pET28, pET21, pET24 or pET31.
Preferably, the host bacteria are E.coli BL21 (DE 3), E.coli DH5a or E.coli Rosetta.
In a preferred mode of the present invention, the method for constructing the engineering bacteria of the efficient SUMO protease comprises the following steps:
1) Designing an upstream primer F1 (SEQ ID No. 4) and a downstream primer R1 (SEQ ID No. 5) respectively, carrying out PCR (polymerase chain reaction) amplification by taking a SUMO protease gene with a nucleotide sequence of SEQ ID No.2 as a template to obtain a SUMO protease gene amplification product consisting of a SUMO tag, a Ulp1 sequence and a polyhistidine tag, wherein the amplification product is named 1901-1;
2) The PCR amplification product 1901-1 and pET28a plasmid of the step 1) are respectively subjected to double enzyme digestion by using restriction enzymes NcoI and BamHI and then are connected to obtain a recombinant plasmid for encoding SUMO protease genes, and the connection product is named as pET28-1901-1;
3) Transforming competent cells of the escherichia coli BL21 (DE 3) by using the connection product pET28-1901-1 obtained in the step 2) to obtain a recombinant engineering strain capable of efficiently expressing SUMO protease, wherein the recombinant engineering strain is named BL21 (DE 3)/pET 28a;
in the step 1), the primer sequences involved are shown in the following table:
in a fifth aspect of the present invention, there is provided an application of the engineering bacterium for efficiently expressing SUMO protease in the fourth aspect in preparing SUMO protease.
The invention has the following technical effects:
1) The SUMO tag can promote the folding of target proteins and improve the protein yield. The SUMO protease gene with the SUMO tag can promote the sufficient folding of SUMO protease in escherichia coli cells, and improves the solubility of SUMO protease protein in the supernatant after bacterial disruption; meanwhile, in the expression and purification process, the SUMO label can be automatically cut by the SUMO protease and purified and removed, and finally the SUMO protease without the SUMO label is obtained.
2) Besides the SUMO label, the constructed SUMO protease gene also has His affinity label, and the SUMO protease protein with the purity of more than 95% can be obtained simply through affinity chromatography.
3) According to the embodiment of the invention, the obtained engineering bacteria containing SUMO protease genes with SUMO labels are fermented and cultured, and supernatant is subjected to ion exchange, affinity and other purification modes after bacteria breaking and centrifugation to obtain the SUMO protease proteins. SDS-PAGE and N-terminal sequence detection are carried out on the protein, and the electrophoresis purity reaches more than 95%, and the molecular weight and the N-terminal sequence are correct. Through activity detection, the product activity is higher than that of commercial SUMO protease.
Drawings
FIG. 1 is a schematic diagram of expression vector construction.
FIG. 2 shows the results of induction expression of SUMO protease engineering bacteria:
lane 1: no control bacteria were induced;
lane 2: inducing for 1h;
lane 3: inducing for 2h;
lane 4: inducing for 3h;
lane 5: standard protein molecular weight.
FIG. 3 shows the purification results of SUMO protease.
Lane 1: standard protein molecular weight;
lane 2: SUMO protein zymogen fluid;
lane 3: SUMO protease affinity chromatography;
lane 4: SUMO protease Q chromatography;
lane 5: SUMO protease osteoclast supernatant.
FIG. 4 shows the results of fermentation of a plurality of batches of SUMO protease species.
Lane 1: standard protein molecular weight;
lane 2: SUMO protease fermentation results;
lane 3: SUMO protease fermentation results;
lane 4: SUMO protease fermentation results;
lane 5: SUMO protease fermentation results.
FIG. 5 is an electrophoretogram of SUMO protease activity assay.
Lane 1: the result of enzyme electrophoresis is not added;
lane 2: adding 5 μl of SUMO protease for cleavage;
lane 3: adding 10 μl of SUMO protease for cleavage;
lane 4: adding 15 μl of SUMO protease for cleavage;
lane 5: adding 20 μl of SUMO protease for cleavage;
lane 6: adding 25 μl of SUMO protease for cleavage;
lane 7: adding 30 μl of SUMO protease for cleavage;
lane 8: the result of enzyme electrophoresis is not added;
lane 9: adding 10 μl of SUMO protease control sample;
lane 10: adding 15 μl of SUMO protease control;
lane 11: adding 20 μl of SUMO protease control sample;
lane 12: adding 25 μl of SUMO protease control;
lane 13: adding 30 μl of SUMO protease control sample;
lanes 14: standard protein molecular weight.
Detailed Description
The present invention will be further described in detail below with reference to the drawings and examples, in order to make the objects, technical solutions and advantages of the present invention more apparent, and it is apparent that the described examples are only some of the examples of the present invention, but not all of the examples.
Example one construction of an expression Strain containing the Gene of the invention
(1) Primer design and PCR amplification of genes
The 402 th to 621 th amino acids (SEQ ID No. 1) of the Saccharomyces cerevisiae-derived SUMO enzyme Ulp1 protein sequence (GenBank: KAF 1908775.1) are intercepted, the amino terminal is added with an SUMO tag amino acid sequence, and the carboxyl terminal is added with a polyhistidine tag. The relevant DNA sequence (SEQ ID No. 2) was synthesized after codon optimization according to E.coli codon preference.
The upstream and downstream primers F1 (SEQ ID No. 4) and R1 (SEQ ID No. 5) were designed, respectively, and PCR amplification was performed using a TwitAmp kit (TwitDX) with the synthesized sequence as a template, to give an amplification product designated as 1901-1.
(2) Construction of expression vectors
The PCR amplification product 1901-1 obtained in step (1) was subjected to double cleavage treatment with pET28a plasmid (Novagen), respectively. 2 sterile microcentrifuge tubes, designated as tube A and tube B, were taken and 2. Mu.L of 10 XBuffer was added to each tube, with 1. Mu.L of restriction enzymes NcoI and BamHI. To the A tube, 1. Mu.L of amplification product and 16. Mu.L of nuclease-free ultrapure water were added, and to the B tube, 1. Mu.L of plasmid and 16. Mu.L of nuclease-free ultrapure water were added. The centrifuge tube was placed in a constant temperature water bath at 37℃for 15min and subjected to double digestion. The cut pieces were then recovered from the 2 centrifuge tubes, respectively, using the GeneJET PCR purification kit (Thermo Fisher).
The enzyme fragments were ligated with T4 DNA ligase. The following cleavage system was established: 1. Mu.L of plasmid fragment, 10. Mu.L of gene fragment, 5. Mu. L T4, 4 DNA Ligase Master Mix (Invitrogen), and ligation for 15min at 25℃with water without nuclease was performed to give ligation product pET28-1901-1.
The construction schematic of the expression vector is shown in FIG. 1.
Coli BL21 (DE 3) competent cells were transformed with the ligation product pET28-1901-1 to give the original strain.
The obtained original strain is added to an agar plate with kanamycin resistance, inoculated, cultured at a constant temperature of 37 ℃ overnight, and refrigerated for preservation, thus obtaining a screening plate. And (3) selecting single bacterial colony from the screening plate, extracting plasmids, and sequencing to verify whether the sequences are correct, wherein the bacterial colony with the correct sequence is the engineering bacterial strain BL21 (DE 3)/pET 28a containing the gene of the invention.
Example two expression and protein purification of engineering bacteria containing the Gene of the invention
The engineering strain BL21 (DE 3)/pET 28a containing the gene of the present invention obtained in example one was used. Inoculating to 200mL LB liquid medium containing kanamycin sulfate, and culturing at 37deg.C and 250rpm until OD600 is about 0.6; IPTG was added to a final concentration of 0.5mM for induction, and the fermentation broth was collected after further culturing at 37℃and 250rpm for 3 hours. The fermentation broth was centrifuged at 5000rpm at 4℃and the cells were collected and weighed, and the fermentation cells were subjected to SDS-PAGE electrophoresis, the results of which are shown in FIG. 2.
Adding the wet thalli into the thalli in a proportion of 10mL of the bacteria breaking buffer solution, adding the thalli into the bacteria breaking buffer solution precooled at 4 ℃, and stirring the thalli by a glass rod to fully suspend the thalli. The cells were broken by sonication for 45min under ice bath conditions, centrifuged at 8000rpm at 4℃for 30min, and the supernatant was collected.
The supernatant was purified by Capto Q (GE company), and the eluent was 100mM sodium chloride (Guozhen) solution, and the elution peak was collected to give a Q purified product. Purifying DEAE purified product by Ni-Chelating Sepharose Fast Flow chromatography packing (GE company), eluting with 160mM imidazole (raw) solution, and collecting eluting peak to obtain target protein.
The obtained affinity chromatography peak was concentrated by a membrane pack (MILLIPORE) with a molecular weight cut-off of 5kDa, desalted by Sephadex G-25 (GE company), and equilibrated solution of 20mmol/L PB (national drug) at pH 7.6. The sample was concentrated and desalted to obtain a SUMO protease zymogen solution, and the purity of the product was analyzed by SDS-PAGE, and the result is shown in FIG. 3.
As can be seen from FIG. 3, the molecular weight of the SUMO protease produced by the strain constructed by the invention is about 25.5kDa, and the molecular weight is consistent with the theoretical molecular weight of the SUMO protease, which indicates that the SUMO tag is automatically cleaved by the SUMO protease in the process of protein expression and purification, and the purified SUMO protease does not contain the SUMO tag.
As can be seen from FIG. 3, the SUMO protease produced by the strain constructed according to the present invention can reach an electrophoretic purity of 95% or more after being purified to a stock solution.
The SUMO protease produced by the strain constructed by the invention is subjected to N-terminal amino acid sequencing, and the first five amino acid residues at the N-terminal are LVPEL, so that the theory is consistent.
Conclusion: the purity of the target protein in the stock solution can reach more than 95%, and the molecular weight and the N-terminal sequence are correct, thereby meeting the expectations.
After four consecutive fermentations, the post-disruption supernatants were detected by SDS-PAGE electrophoresis, the results are shown in FIG. 4.
As can be seen from FIG. 4, the target protein bands were 25% or more in four consecutive batches of the culture supernatants.
Conclusion: the expression level of the target protein in the four batches of supernatants is more than 25%.
Example three, SUMO protease Activity assay
In 13 Eppendorf tubes of 1.5mL were each charged with 49.9. Mu.g of a substrate protein (the substrate protein was SUMO-1801 fusion protein containing a SUMO tag, purchased from Shanghai offshore technology Co., ltd.), 30. Mu.L, 25. Mu.L, 20. Mu.L, 15. Mu.L, 10. Mu.L, 5. Mu.L and 0 of SUMO protease (SUMO protease obtained in example two) were sequentially added to 7 tubes, and in the other 6 tubes, 30. Mu.L, 25. Mu.L, 20. Mu.L, 15. Mu.L, 10. Mu.L and 0 of SUMO protease control (commercially available, new sea gene C0801 (5U/. Mu.L)) were sequentially added, and 13 reactions were performed in an enzyme-digested buffer in a total volume of 150. Mu.L. After 1h of reaction at 30 ℃, the digestion reaction was stopped with 5×SDS small molecule loading buffer, 16% tricine-SDS-PAGE was performed, and after coomassie blue staining, the result of thin layer gel scanning analysis of the electrophoresis was shown in FIG. 5.
As can be seen from FIG. 5, construction of strain-purified SUMO protease cleaved SUMO tag from fusion protein, and the enzyme activity of SUMO enzyme was calculated by taking the amount of enzyme required for cleavage of more than 85% of 2. Mu.g SUMO-fusion protein at 30℃in 1 hour as one enzyme unit according to the enzyme activity definition, and the results are shown in Table 1
TABLE 1SUMO protease Activity
| Sample name | SUMO protease Activity (U/. Mu.L) |
| SUMO protease (present product) | 7.5 |
| SUMO protease control (commercially available) | 5 |
The results show that the SUMO protease purified by the strain can cut the SUMO label from the fusion protein, and the activity is about 50% higher than that of a commercial product, and the strain is constructed according to the expectations.
The foregoing is merely exemplary embodiments of the present invention and are not intended to limit the scope of the present invention, and all equivalent modifications made by the present invention or direct or indirect application in other related technical fields are included in the scope of the present invention.
Claims (10)
1. The SUMO protease gene is characterized in that the nucleotide sequence of the SUMO protease gene is shown as SEQ ID No. 2.
2. The recombinant expression vector containing the SUMO protease gene as claimed in claim 1, wherein the expression vector is constructed by introducing the SUMO protease gene with a nucleotide sequence shown in SEQ ID No.2 into an expression plasmid.
3. The recombinant expression vector of claim 2, wherein the expression plasmid is pET28, pET21, pET24, or pET31.
4. An engineering bacterium for efficiently expressing SUMO protease, which is characterized in that the recombinant engineering strain is prepared by transforming host bacteria with the recombinant expression vector of claim 2.
5. The engineering bacterium according to claim 4, wherein the host bacterium is E.coli BL21 (DE 3), E.coli DH5a or E.coli Rosetta.
6. The construction method of the engineering bacteria for efficiently expressing SUMO protease is characterized by comprising the following steps of:
1) Obtaining a SUMO protease gene consisting of a SUMO tag, a Ulp1 sequence and a polyhistidine tag by a PCR amplification mode, wherein the nucleotide sequence of the SUMO protease gene is shown as SEQ ID No. 2;
2) The SUMO protease gene obtained in the step 1) is respectively connected with the expression plasmid after double enzyme digestion, so as to obtain a recombinant plasmid containing the SUMO protease gene;
3) Preparing host bacterium competent cells, transforming the host bacterium by using the plasmid in the step 2), and screening positive clones to obtain recombinant engineering bacteria for efficiently expressing SUMO protease.
7. The method of claim 6, wherein the step of providing the first layer comprises,
in the step 2), the expression plasmid is pET28, pET21, pET24 or pET31;
in the step 3), the host bacteria are E.coli BL21 (DE 3), E.coli DH5a or E.coli Rosetta.
8. The method of claim 6, wherein the construction method of the engineering bacteria for efficiently expressing SUMO protease comprises the following steps:
1) Designing an upstream primer F1 and a downstream primer R1 respectively, and carrying out PCR amplification by taking a SUMO protease gene with a nucleotide sequence of SEQ ID No.2 as a template to obtain a SUMO protease gene amplification product consisting of a SUMO tag, a Ulp1 sequence and a polyhistidine tag;
2) The PCR amplified product of the step 1) and pET28a plasmid are respectively subjected to double enzyme digestion by using restriction enzymes NcoI and BamHI and then are connected to obtain recombinant plasmid of encoding SUMO protease gene;
3) Transforming competent cells of the escherichia coli BL21 (DE 3) by using the connection product of the step 2) to obtain a recombinant engineering strain capable of efficiently expressing SUMO protease, wherein the recombinant engineering strain is named BL21 (DE 3)/pET 28a;
the primers F1 and R1 are designed in the step 1), and are characterized in that the sequence F1 is shown as SEQ ID No.4, and the sequence R1 is shown as SEQ ID No. 5.
9. The use of the engineered bacterium of claim 4 or 5 in the preparation of SUMO protease.
10. The use of an engineered bacterium constructed according to the method of any one of claims 6-8 in the preparation of SUMO protease.
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