CN113025598A

CN113025598A - Method for preparing recombinant heparinase III by utilizing SUMO fusion expression system and SUMO _ heparinase III fusion protein prepared by method

Info

Publication number: CN113025598A
Application number: CN202110098828.5A
Authority: CN
Inventors: 蒋建华; 张亮; 邢岭; 吴双; 高伟
Original assignee: Shanghai Baowei Medicine Technology Co ltd
Current assignee: Shanghai Baowei Medicine Technology Co ltd
Priority date: 2021-01-25
Filing date: 2021-01-25
Publication date: 2021-06-25

Abstract

The invention discloses a method for preparing recombinant heparinase III by utilizing an SUMO fusion expression system and the prepared heparinase III, and the preparation method comprises the following steps: selecting a heparinase III sequence from Flavobacterium heparinum (Pedobacter heparinus), wherein the amino acid sequence of the heparinase III sequence is shown as SEQ ID No.1 and has 659 aa; removing the signal peptide sequence to obtain the DNA sequence of heparinase III, such as SEQ ID No. 2; inserting the DNA sequence of the heparinase III into a pSMART vector plasmid with an N-terminal SUMO protein tag; transforming the correct plasmid into BL21(DE3) escherichia coli competent cells, picking a single clone, and obtaining a fusion protein through fermentation and purification; and (3) cutting the SUMO tag protein from the fusion protein by using SUMO protease to obtain heparinase III. The invention has the advantages that: the solubility of the target protein is improved, the correct folding of the target protein is promoted, and the formation of an inclusion body is avoided; the purification cost is reduced, and the purification efficiency is improved; the SUMO protein label has small molecular weight and small influence on the enzymatic activity of the heparinase III, and the purified heparinase III is obtained.

Description

Method for preparing recombinant heparinase III by utilizing SUMO fusion expression system and SUMO _ heparinase III fusion protein prepared by method

The invention relates to the technical field of biology, in particular to a method for preparing recombinant heparinase III (heparinase III) by utilizing a SUMO fusion expression system.

Background

Heparinase is mainly separated from some bacteria using heparin as a carbon source, and is originally derived from Flavobacterium heparinum (Pedobacter heparinus), and three kinds of heparinase are separated and purified from the Flavobacterium heparinum and are respectively heparinase I, heparinase II and heparinase III. The heparinase has the main function of degrading heparin and is widely applied to the production of low molecular heparin and ultra-low molecular heparin by an enzyme degradation method.

There are two main sources of heparinase products currently used for the production of low molecular weight heparin: firstly, the heparinase is directly fermented and extracted from the microorganism for producing the heparinase (Shenzhen Shanghai Prorey pharmaceutical Co., Ltd., CN 102286448B); secondly, recombinant heparinase engineering bacteria are constructed by a gene engineering technology and fermented to extract the recombinant heparinase.

The heparinase III is directly extracted from microorganisms (such as Flavobacterium heparinum and the like) producing the heparinase, the fermentation condition and the purification process are relatively complex, and the potential for improving the yield by strain breeding is limited.

Genetic engineering recombination technology has been widely applied to protein preparation, and Maltose Binding Protein (MBP) labels and heparinase III are applied to form fusion protein at present. The adoption of genetic engineering recombination technology to produce exogenous proteins such as heparinase III in an escherichia coli system faces two challenges: firstly, the expression level of the protein expression system is low; secondly, the expressed protein is folded into insoluble aggregates, inclusion bodies, by mistake, and the extraction and renaturation are difficult.

At present, the solubility of the recombinant product can be obviously improved by adopting a soluble protein label and heparinase III to form fusion protein. However, fusion protein tags such as glutathione mercaptotransferase Tag (GST-Tag), maltose binding protein Tag (MBP-Tag), transcription anti-termination factor A Tag (NusA-Tag) interfere with the normal function of the target protein due to the large molecular weight (26-55kDa), and the yield of the target protein is relatively reduced by removing the Tag during expression and subsequent purification.

Disclosure of Invention

In order to solve the defects and shortcomings of the prior art, the present invention aims to provide a method for preparing recombinant heparinase III (heparinase III) by using a SUMO fusion expression system, so as to greatly improve the stability and solubility of a target protein and improve the activity and yield of the target protein.

In order to achieve the purpose, the technical scheme of the invention is as follows:

according to one aspect of the present invention, there is provided a method for preparing recombinant heparinase iii (heparinase iii) using SUMO fusion expression system, comprising the steps of:

s01: selecting a heparinase III sequence from Flavobacterium heparinum (Pedobacter heparinus), wherein the amino acid sequence of the heparinase III sequence is shown as SEQ ID No.1 and has 659 aa;

s02: removing the signal peptide sequence to obtain the DNA sequence of heparinase III, such as SEQ ID No. 2;

s03: inserting the DNA sequence of the heparinase III into a pSMART vector plasmid with an N-terminal SUMO protein tag;

s04: transforming the correct plasmid into BL21(DE3) escherichia coli competent cells, picking a single clone, and fermenting and purifying to obtain SUMO _ heparinase III fusion protein;

s05: and (4) cutting the SUMO tag protein from the SUMO _ heparinase III fusion protein in the step S04 by using SUMO protease to obtain heparinase III.

Further, the method also comprises an optimization step of the expression condition of the fusion protein.

Further, the optimal conditions for the fusion protein are that the fusion protein is induced to express at 0.2mmol/l isopropyl thiogalactoside (IPTG) at 15 ℃.

Further, the method also comprises a purification step of the fusion protein.

Further, the purification step of the fusion protein adopts a Ni column affinity chromatography method for separation and purification.

Further, the cutting of the SUMO tag protein from the SUMO _ heparinase III fusion protein of step S04 includes the following steps: dialyzing the fusion protein to replace buffer solution, adding a proper amount of SUMO protease, uniformly mixing, placing at 4 ℃ for reacting overnight, and separating a SUMO protein label (capable of being combined on a Ni column) with 6 histidine fragments and heparinase III (incapable of being combined on the Ni column) from a hydrolyzed fusion protein sample by using a Ni column affinity chromatography method.

According to another aspect of the present invention, there is also provided a SUMO _ heparinase III fusion protein prepared according to the above method, wherein the amino acid sequence of the fusion protein is shown as seq id No. 3.

According to a further aspect of the invention there is also provided a heparinase III prepared according to the above method.

According to the technical scheme, the recombinant heparinase III prepared by the SUMO fusion expression system is fused with a small ubiquitin-like protein modifier (SUMO) tag protein at the N end of the target protein, the molecular weight of the SMMO tag protein is about 11kDa, the stability and the solubility of the target protein can be greatly improved, and the influence on the activity of the target protein is small. In addition, SUMO Protease (SUMO Protease) is a cysteine Protease having a high activity, which recognizes the tertiary structure of SUMO tag protein, not the amino acid sequence, and thus can cleave SUMO tag protein from recombinant fusion protein efficiently and specifically to obtain heparinase III.

The advantages of preparing the recombinant heparinase III by adopting the SUMO protein tag fusion expression system are as follows:

1) the solubility of the target protein is improved, the correct folding of the target protein is promoted, and the formation of an inclusion body is avoided;

2) the near N end of the SUMO protein label is provided with 6 histidine fragments, so that the fusion protein can be purified by one step by using a Ni column affinity chromatography method, the purification cost is reduced, and the purification efficiency is improved;

3) the SUMO protein tag has small molecular weight and small influence on the enzymatic activity of the heparinase III, and the heparinase III with high activity can be obtained even if the protein tag in the fusion protein is not cut off;

4) the SUMO protease can recognize the tertiary structure of the SUMO tag protein, and the SUMO tag protein can be efficiently and specifically cut from the recombinant fusion protein, so that the purified heparinase III is obtained.

The following describes embodiments of the present invention in further detail with reference to the accompanying drawings. The above and other objects, features and advantages of the present invention will be apparent to those skilled in the art from the detailed description of the present invention.

Drawings

FIG. 1 is a SDS-PAGE electrophoretic analysis of heparinase III expression identification in one embodiment of the invention;

FIG. 2 is a SDS-PAGE analysis of expression optimization of heparinase III according to one embodiment of the invention;

FIG. 3 is a SDS-PAGE electrophoretic analysis of heparinase III solubility analysis in an embodiment of the invention;

FIG. 4 is a SDS-PAGE analysis of the affinity purification of heparinase III according to one embodiment of the invention;

FIG. 5 is a SDS-PAGE electrophoretic analysis of a Western Blot of heparinase III according to one embodiment of the invention;

FIG. 6 is an SDS-PAGE analysis of the tag removal of heparinase III according to one embodiment of the invention.

Detailed Description

The present invention will be described in further detail below with reference to specific embodiments and examples and the accompanying drawings.

According to an exemplary embodiment of the present invention, there is provided a method for preparing recombinant heparinase iii (heparinase iii) using a SUMO fusion expression system, comprising the steps of:

s04: transforming the correct plasmid into BL21(DE3) escherichia coli competent cells, selecting a single clone, and obtaining a fusion protein through fermentation and purification;

s05, cutting the SUMO tag protein from the fusion protein in the step S04 by using SUMO protease to obtain heparinase III.

Preferably, the method further comprises the step of optimizing the expression conditions of the fusion protein.

Preferably, the fusion protein is optimized under the condition that the fusion protein is induced to express at 0.2mmol/l isopropyl thiogalactoside (IPTG) at 15 ℃.

Preferably, a purification step of the fusion protein is also included.

Preferably, the purification step of the fusion protein adopts a Ni column affinity chromatography method for separation and purification.

Preferably, the cutting of the SUMO tag protein from the SUMO _ heparinase III fusion protein of step S04 comprises the following steps: dialyzing the fusion protein to replace buffer solution, adding a proper amount of SUMO protease, uniformly mixing, placing at 4 ℃ for reacting overnight, and separating a SUMO protein label (capable of being combined on a Ni column) with 6 histidine fragments and heparinase III (incapable of being combined on the Ni column) from a hydrolyzed fusion protein sample by using a Ni column affinity chromatography method.

According to an exemplary embodiment of the present invention, the SUMO _ heparinase III fusion protein prepared according to the above method is further provided, and the amino acid sequence of the fusion protein is shown as seq id No. 3.

According to an exemplary embodiment of the present invention, there is also provided a heparinase III prepared according to the above method.

The sequence of heparinase III from Flavobacterium heparinum (Pedobacter heparinus) is selected to be 659aa altogether, the amino acid sequence is shown as SEQ ID No.1, the inventor finds through protein sequence analysis that the heparinase III has no transmembrane structure, the 1 st to 24 th amino acids are signal peptide MTTKIFKRIIVFAVIALSSGNILA, and the last 635 th amino acids are the heparinase III sequence. Preparing heparinase III by adopting an escherichia coli intracellular expression mode, and removing a signal peptide sequence in advance when a sequence transferred into the engineering bacteria is obtained. The DNA sequence of heparinase III is shown as SEQ ID No.2, and is inserted into pSMART carrier plasmid with an N-terminal SUMO protein tag. The correct plasmid was transformed into BL21(DE3) E.coli competent cells, and a single clone was picked up and the expression of the fusion protein was confirmed to be normal by SDS-PAGE. The fusion protein consists of a SUMO protein tag of 109aa and a heparinase III of 635aa, and the amino acid sequence of the SUMO-heparinase III fusion protein is shown as SEQ ID No. 3.

Through optimization, the fusion protein is determined to be optimally induced and expressed at 15 ℃ with 0.2mmol/l isopropyl thiogalactoside (IPTG).

6 histidine (H) sequences are designed near the N end of the fusion protein, supernatant is removed from engineering bacteria fermentation liquor, and the fusion protein is separated and purified by adopting a Ni column affinity chromatography method after thalli are crushed.

Dialyzing the purified fusion protein sample, replacing buffer solution, adding a proper amount of SUMO protease, uniformly mixing, and reacting at 4 ℃ overnight. The hydrolyzed fusion protein sample was separated from heparinase III (not bound to Ni column) by Ni column affinity chromatography using a SUMO protein tag with 6 histidine fragments (bound to Ni column).

The following examples are provided to further illustrate the advantageous effects of the present invention.

Example one

1. Transformation of the plasmid into BL21(DE3) competent cells

1.1 adding 2 μ L plasmid into 100 μ L competent bacteria, and placing on ice for 30 min;

heat shock at 1.242 deg.C for 90s, and rapidly placing in ice for 5 min; adding 500 mu L of LB culture solution; 1.337 ℃, shaking at 220rpm for 1h, centrifuging, spreading all the mixture on an LB plate containing resistance, and culturing in an inverted way at 37 ℃ overnight.

2. Expression characterization

2.1 selecting 5 plates to be singly inoculated in a test tube containing 4mL of LB culture solution with proper resistance;

shaking at 2.237 deg.C and 220rpm until the OD600 of thallus is 0.6-0.8;

2.3 taking out 1mL of culture, centrifuging at 12000g for 5min at room temperature, discarding the supernatant, resuspending the bacterial pellet with 80. mu.L of 1 XPBS Buffer solution and adding 20. mu.L of 5 XPoading Buffer;

2.4 adding IPTG to the remaining culture to a final concentration of 0.5mM, shaking at 37 ℃ and 220rpm for 4h, inducing expression of the fusion protein;

2.5 taking out 0.5mL of culture, centrifuging at 12000g for 5min at room temperature, discarding the supernatant, resuspending the bacterial pellet with 80. mu.L of 1 XPBS Buffer solution and adding 20. mu.L of 5 XPoading Buffer;

2.6 analysis by 12% SDS-PAGE and expression identification is shown in FIG. 1, where M: protein molecular weight Marker (Protein Marker), 1: pre-induction sample, 2: post-induction samples i, 3: post-induction samples ii, 4: post-induction sample iii, 5: post-induction sample iv, 6: sample v after induction.

3. Expression optimization

3.1 picking the single clone on the streak plate and inoculating the single clone in 13 test tubes containing 4mL LB culture solution with proper resistance;

shaking at 220rpm at 3.237 deg.C until the OD600 of the thallus is 0.6-0.8;

3.3 take out 0.4mL culture, 12000g room temperature centrifugation for 5min, abandon the supernatant, use 80 u L1 x PBS Buffer solution heavy suspension thalli precipitation adding 20 u L5 x Loading Buffer;

3.4 adding IPTG to final concentrations of 0.2mM and 1mM respectively to the remaining culture, shaking at 37 ℃ and 15 ℃ for 4h and 16h at 220rpm respectively, inducing expression of the fusion protein;

3.5 take out 0.2mL culture, 12000g room temperature centrifugation for 5min, abandon the supernatant, with 80 u L1 x PBS Buffer heavy suspension bacterial precipitation and 20L 5 x Loading Buffer.

3.6 SDS-PAGE analysis, expression optimization is shown in FIG. 2, in which

M: protein molecular weight markers (Protein markers);

1: (ii) an uninduced sample;

2-4: after induction with 1.0mM IPTG at 37 ℃;

5-7: samples after induction with 0.2mM IPTG at 37 ℃;

8-10: after induction with 1.0mM IPTG at 15 ℃;

11-13: samples after induction with 0.2mM IPTG at 15 ℃.

4. Solubility assay

Centrifuging 2mL of the induced bacterial liquid at 37 ℃ and 2mL of the induced bacterial liquid at 15 ℃ for 5min at room temperature, discarding the supernatant, resuspending the centrifuged precipitate with 1mL of Buffer A (20mM Tris, 300mM NaCl, 10% Glycerol, pH8.0), ultrasonically crushing the precipitate (phi 3, 15%, 2s/8s, 10min), sampling the supernatant respectively, performing 12% SDS-PAGE analysis, and determining that the fusion protein is soluble under 15 ℃ induction, wherein the results of the solubility analysis are shown in FIG. 3

M: protein molecular weight markers (Protein markers);

1: (ii) an uninduced sample;

2: post-induction samples;

3: precipitating the sample after induction by 1.0mM IPTG at 37 ℃;

4: inducing with 1.0mM IPTG and then sampling the supernatant;

5: precipitating the sample after induction with 0.2mM IPTG at 37 ℃;

6: inducing the supernatant with 0.2mM IPTG at 37 ℃;

7: precipitating the sample after induction by 1.0mM IPTG at 15 ℃;

8: inducing with 1.0mM IPTG at 15 deg.C, and collecting supernatant;

9: precipitating the sample after induction with 0.2mM IPTG at 15 ℃;

10: after induction with 0.2mM IPTG, the supernatant was sampled at 15 ℃.

5. Amplified expression and affinity purification

5.1 the best clone was inoculated into 1L LB medium containing the appropriate antibiotics, shaken at 37 ℃ and 220rpm until OD600 was 0.6-0.8, added to a final concentration of 0.2mM IPTG, and shaken at 15 ℃ and 220rpm for 16 h.

5.2 centrifugation at 8000rpm for 10min, discarding the supernatant, and collecting all the cells.

5.3 resuspend the cells using Buffer A (20mM Tris, 300mM NaCl, 10% Glycerol, pH8.0) and sonicate (. PHI.10, 15%, 2s/8s, 30 min). 16000rpm for 10min, and collecting the supernatant.

5.4 Add 2mL Ni-NTA to the supernatant, mix well and incubate at 4 ℃ for 1 h.

5.5 add the incubation to the empty column and collect the effluent.

5.6 Wash the packing with Buffer B (20mM Tris, 300mM NaCl, 10% Glycerol, 20mM Imidazole, pH8.0) and Buffer C (20mM Tris, 300mM NaCl, 10% Glycerol, 40mM Imidazole, pH8.0), respectively, and collect the washes.

5.7 elute with Buffer D (20mM Tris, 300mM NaCl, 10% Glycerol, 500mM Imidazole, pH8.0) and collect the eluate.

5.8SDS-PAGE electrophoresis detection, the result of affinity purification is shown in FIG. 4, wherein

M: protein molecular weight Marker (Protein Marker)

1: precipitating after crushing

2: supernatant after crushing

3: effluent after Ni-NTA incubation

4: buffer B Wash sample

5: buffer C Wash sample

6: buffer D eluted.

6. Immunoblot (Western Blot) analysis

6.1SDS-PAGE

Electrophoresis sample: prestained Protein molecular weight markers (Protein markers), Buffer D eluates

Electrophoresis conditions: 200V, 60min

6.2 semi-dry electrotransfer

a. During the running of electrophoresis, the PVDF membrane is cut out to have the same size as the gel, a small angle is cut off to help the direction judgment, and the PVDF membrane is placed in methanol for soaking for 30s and then transferred to an electrotransformation liquid.

b. The thin filter paper and the thick filter paper are cut out and slightly larger than the gel, and two pieces of the thin filter paper and two pieces of the thick filter paper are soaked in the transfer buffer.

c. After electrophoresis, the gel concentrate was cut off and placed in an electrotransfer solution. The thick filter paper, the thin filter paper, the NC membrane, the glue, the thin filter paper and the thick filter paper are sequentially placed on the plane of the electric rotating apparatus.

d. The air bubbles between each layer are all removed. One end can be lightly pressed by hand, then a 50mL centrifuge tube is used for gently moving the upper layer to one side to remove air bubbles, one end is replaced by hand, and the centrifuge tube is used for removing air bubbles from the other end.

e. Linking the electrotransformation instrument to the instrument for 30mins with the protein of less than 50KDa and the protein of 30V; the protein is larger than 50KDa, and 45mins is used.

6.3 blocking of membranes and antibody incubation and Final treatment

a. Washing the transfer printing film: rinse 3 times with 1 XPBST for 5min at room temperature, without adding any solution to the membrane, slowly along the corner of the box to avoid washing away the bound material on the membrane, and rotate the shaker at 40 rpm.

b. PBST was poured off, placed in a 5% skim milk powder confining liquid, the shaker turned to the lowest speed, and confined at room temperature for 2 h.

c. Rinse with 1 × PBST for 5min at room temperature for 3 times.

d. Adding antibody (1: 2000 to 5% skimmed milk powder), incubating for 1h, and recovering to-20 deg.C after use.

e. Rinse with 1 × PBST for 5min at room temperature for 3 times.

f. Slowly adding a small amount of ECL solution to cover the membrane, standing for 2min, and taking a picture, as shown in FIG. 5, wherein M: protein molecular weight Marker (Protein Marker), Sample: buffer D eluted.

7. Tag removal proteins

7.1 the eluted sample was dialyzed into Buffer A (20mM Tris, 300mM NaCl, 10% Glycerol, pH 8.0).

7.2 after dialysis, the fusion protein was added with a suitable amount of SUMO Protease (SUMO Protease), mixed well, and left to react overnight at 4 ℃.

7.3 adding the product after the reaction into a Ni-NTA purification column, slowly loading the sample, and collecting the effluent.

7.4 Wash the column with Buffer A (20mM Tris, 300mM NaCl, 10% Glycerol, pH 8.0).

7.5 elution with Buffer D (20mM Tris, 300mM NaCl, 10% Glycerol, 500mM Imidazole, pH 8.0).

7.6 SDS-PAGE, as shown in FIG. 6, wherein:

m: protein molecular weight Marker (Protein Marker)

1: sample before enzyme digestion reaction

2: sample after enzyme digestion reaction

3: flow out of the column after enzyme digestion (heparinase III)

4: after enzyme digestion, passing through a column for cleaning

5: after cleavage, the column was eluted (SUMO protein tag).

In conclusion, the correct plasmid was transformed into BL21(DE3) E.coli competent cells, and a single clone was picked up and the expression of the fusion protein was normal as verified by SDS-PAGE. Under each optimized condition, the fusion protein is obviously expressed, the solubility of the fusion protein is better at the induction temperature of 15 ℃, 0.2mM IPTG induction is carried out at the temperature of 15 ℃, and the target protein heparinase III without the fusion tag is obtained by removing the tag after Ni-NTA purification.

The present invention is capable of other embodiments, and various changes and modifications can be made by one skilled in the art without departing from the spirit and scope of the invention.

SEQ ID No.1:

Amino acid sequence of heparinase III (heparinase III):

MTTKIFKRIIVFAVIALSSGNILAQSSSITRKDFDHINLEYSGLEKVNKAVAAGNYDDAA KALLAYYREKSKAREPDFSNAEKPADIRQPIDKVTREMADKALVHQFQPHKGYGYFDYGK DINWQMWPVKDNEVRWQLHRVKWWQAMALVYHATGDEKYAREWVYQYSDWARKNPLGLSQ DNDKFVWRPLEVSDRVQSLPPTFSLFVNSPAFTPAFLMEFLNSYHQQADYLSTHYAEQGN HRLFEAQRNLFAGVSFPEFKDSPRWRQTGISVLNTEIKKQVYADGMQFELSPIYHVAAID IFLKAYGSAKRVNLEKEFPQSYVQTVENMIMALISISLPDYNTPMFGDSWITDKNFRMAQ FASWARVFPANQAIKYFATDGKQGKAPNFLSKALSNAGFYTFRSGWDKNATVMVLKASPP GEFHAQPDNGTFELFIKGRNFTPDAGVFVYSGDEAIMKLRNWYRQTRIHSTLTLDNQNMV ITKARQNKWETGNNLDVLTYTNPSYPNLDHQRSVLFINKKYFLVIDRAIGEATGNLGVHW QLKEDSNPVFDKTKNRVYTTYRDGNNLMIQSLNADRTSLNEEEGKVSYVYNKELKRPAFV FEKPKKNAGTQNFVSIVYPYDGQKAPEISIRENKGNDFEKGKLNLTLTINGKQQLVLVP SEQ ID No.2:

heparinase III (heparinase III) DNA:

CAAAGCAGCAGCATAACAAGAAAAGACTTCGACCACATAAACCTAGAATACAGCGGACTAGAA AAAGTAAACAAAGCAGTAGCAGCAGGAAACTACGACGACGCAGCAAAAGCACTACTAGCATAC TACAGAGAAAAAAGCAAAGCAAGAGAACCAGACTTCAGCAACGCAGAAAAACCAGCAGACATA AGACAACCAATAGACAAAGTAACAAGAGAAATGGCAGACAAAGCACTAGTACACCAATTCCAA CCACACAAAGGATACGGATACTTCGACTACGGAAAAGACATAAACTGGCAAATGTGGCCAGTA AAAGACAACGAAGTAAGATGGCAACTACACAGAGTAAAATGGTGGCAAGCAATGGCACTAGTA TACCACGCAACAGGAGACGAAAAATACGCAAGAGAATGGGTATACCAATACAGCGACTGGGCA AGAAAAAACCCACTAGGACTAAGCCAAGACAACGACAAATTCGTATGGAGACCACTAGAAGTA AGCGACAGAGTACAAAGCCTACCACCAACATTCAGCCTATTCGTAAACAGCCCAGCATTCACA CCAGCATTCCTAATGGAATTCCTAAACAGCTACCACCAACAAGCAGACTACCTAAGCACACAC TACGCAGAACAAGGAAACCACAGACTATTCGAAGCACAAAGAAACCTATTCGCAGGAGTAAGC TTCCCAGAATTCAAAGACAGCCCAAGATGGAGACAAACAGGAATAAGCGTACTAAACACAGAA ATAAAAAAACAAGTATACGCAGACGGAATGCAATTCGAACTAAGCCCAATATACCACGTAGCA GCAATAGACATATTCCTAAAAGCATACGGAAGCGCAAAAAGAGTAAACCTAGAAAAAGAATTC CCACAAAGCTACGTACAAACAGTAGAAAACATGATAATGGCACTAATAAGCATAAGCCTACCA GACTACAACACACCAATGTTCGGAGACAGCTGGATAACAGACAAAAACTTCAGAATGGCACAA TTCGCAAGCTGGCAAGAGTATTCCCAGCAAACCAAGCAATAAAATACTTCGCAACAGACGGAA AACAAGGAAAAGCACCAAACTTCCTAAGCAAAGCACTAAGCAACGCAGGATTCTACACATTCA GAAGCGGATGGGACAAAAACGCAACAGTAATGGTACTAAAAGCAAGCCCACCAGGAGAATTCC ACGCACAACCAGACAACGGAACATTCGAACTATTCATAAAAGGAAGAAACTTCACACCAGACG CAGGAGTATTCGTATACAGCGGAGACGAAGCAATAATGAAACTAAGAAACTGGTACAGACAAA CAAGAATACACAGCACACTAACACTAGACAACCAAAACATGGTAATAACAAAAGCAAGACAAA ACAAATGGGAAACAGGAAACAACCTAGACGTACTAACATACACAAACCCAAGCTACCCAAACC TAGACCACCAAAGAAGCGTACTATTCATAAACAAAAAATACTTCCTAGTAATAGACAGAGCAA TAGGAGAAGCAACAGGAAACCTAGGAGTACACTGGCAACTAAAAGAAGACAGCAACCCAGTAT TCGACAAAACAAAAAACAGAGTATACACAACATACAGAGACGGAAACAACCTAATGATACAAA GCCTAAACGCAGACAGAACAAGCCTAAACGAAGAAGAAGGAAAAGTAAGCTACGTATACAACA AAGAACTAAAAAGACCAGCATTCGTATTCGAAAAACCAAAAAAAAACGCAGGAACACAAAACT TCGTAAGCATAGTATACCCATACGACGGACAAAAAGCACCAGAAATAAGCATAAGAGAAAACA AAGGAAACGACTTCGAAAAAGGAAAACTAAACCTAACACTAACAATAAACGGAAAACAACAAC TAGTACTAGTACCATAG

SEQ ID No.3:

amino acid sequence of SUMO-Heparinase III fusion protein (SUMO-Heparinase III):

MGHHHHHHMSDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPLRRLMEAF AKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGGQGSQSSSITRKDFDHINL EYSGLEKVNKAVAAGNYDDAAKALLAYYREKSKAREPDFSNAEKPADIRQPIDKVTREMADKAL VHQFQPHKGYGYFDYGKDINWQMWPVKDNEVRWQLHRVKWWQAMALVYHATGDEKYAREWVYQY SDWARKNPLGLSQDNDKFVWRPLEVSDRVQSLPPTFSLFVNSPAFTPAFLMEFLNSYHQQADYL STHYAEQGNHRLFEAQRNLFAGVSFPEFKDSPRWRQTGISVLNTEIKKQVYADGMQFELSPIYH VAAIDIFLKAYGSAKRVNLEKEFPQSYVQTVENMIMALISISLPDYNTPMFGDSWITDKNFRMA QFASWARVFPANQAIKYFATDGKQGKAPNFLSKALSNAGFYTFRSGWDKNATVMVLKASPPGEF HAQPDNGTFELFIKGRNFTPDAGVFVYSGDEAIMKLRNWYRQTRIHSTLTLDNQNMVITKARQN KWETGNNLDVLTYTNPSYPNLDHQRSVLFINKKYFLVIDRAIGEATGNLGVHWQLKEDSNPVFD KTKNRVYTTYRDGNNLMIQSLNADRTSLNEEEGKVSYVYNKELKRPAFVFEKPKKNAGTQNFVS IVYPYDGQKAPEISIRENKGNDFEKGKLNLTLTINGKQQLVLVP 。

sequence listing

<110> Shanghai Baowei medicine technology Limited

<120> method for preparing recombinant heparinase III by using SUMO fusion expression system and SUMO _ heparinase III fusion protein prepared by same

<130> 2020

<160> 3

<170> SIPOSequenceListing 1.0

<210> 1

<211> 659

<212> PRT

<213> Artificial sequence ()

<400> 1

Met Thr Thr Lys Ile Phe Lys Arg Ile Ile Val Phe Ala Val Ile Ala

1 5 10 15

Leu Ser Ser Gly Asn Ile Leu Ala Gln Ser Ser Ser Ile Thr Arg Lys

20 25 30

Asp Phe Asp His Ile Asn Leu Glu Tyr Ser Gly Leu Glu Lys Val Asn

35 40 45

Lys Ala Val Ala Ala Gly Asn Tyr Asp Asp Ala Ala Lys Ala Leu Leu

50 55 60

Ala Tyr Tyr Arg Glu Lys Ser Lys Ala Arg Glu Pro Asp Phe Ser Asn

65 70 75 80

Ala Glu Lys Pro Ala Asp Ile Arg Gln Pro Ile Asp Lys Val Thr Arg

85 90 95

Glu Met Ala Asp Lys Ala Leu Val His Gln Phe Gln Pro His Lys Gly

100 105 110

Tyr Gly Tyr Phe Asp Tyr Gly Lys Asp Ile Asn Trp Gln Met Trp Pro

115 120 125

Val Lys Asp Asn Glu Val Arg Trp Gln Leu His Arg Val Lys Trp Trp

130 135 140

Gln Ala Met Ala Leu Val Tyr His Ala Thr Gly Asp Glu Lys Tyr Ala

145 150 155 160

Arg Glu Trp Val Tyr Gln Tyr Ser Asp Trp Ala Arg Lys Asn Pro Leu

165 170 175

Gly Leu Ser Gln Asp Asn Asp Lys Phe Val Trp Arg Pro Leu Glu Val

180 185 190

Ser Asp Arg Val Gln Ser Leu Pro Pro Thr Phe Ser Leu Phe Val Asn

195 200 205

Ser Pro Ala Phe Thr Pro Ala Phe Leu Met Glu Phe Leu Asn Ser Tyr

210 215 220

His Gln Gln Ala Asp Tyr Leu Ser Thr His Tyr Ala Glu Gln Gly Asn

225 230 235 240

His Arg Leu Phe Glu Ala Gln Arg Asn Leu Phe Ala Gly Val Ser Phe

245 250 255

Pro Glu Phe Lys Asp Ser Pro Arg Trp Arg Gln Thr Gly Ile Ser Val

260 265 270

Leu Asn Thr Glu Ile Lys Lys Gln Val Tyr Ala Asp Gly Met Gln Phe

275 280 285

Glu Leu Ser Pro Ile Tyr His Val Ala Ala Ile Asp Ile Phe Leu Lys

290 295 300

Ala Tyr Gly Ser Ala Lys Arg Val Asn Leu Glu Lys Glu Phe Pro Gln

305 310 315 320

Ser Tyr Val Gln Thr Val Glu Asn Met Ile Met Ala Leu Ile Ser Ile

325 330 335

Ser Leu Pro Asp Tyr Asn Thr Pro Met Phe Gly Asp Ser Trp Ile Thr

340 345 350

Asp Lys Asn Phe Arg Met Ala Gln Phe Ala Ser Trp Ala Arg Val Phe

355 360 365

Pro Ala Asn Gln Ala Ile Lys Tyr Phe Ala Thr Asp Gly Lys Gln Gly

370 375 380

Lys Ala Pro Asn Phe Leu Ser Lys Ala Leu Ser Asn Ala Gly Phe Tyr

385 390 395 400

Thr Phe Arg Ser Gly Trp Asp Lys Asn Ala Thr Val Met Val Leu Lys

405 410 415

Ala Ser Pro Pro Gly Glu Phe His Ala Gln Pro Asp Asn Gly Thr Phe

420 425 430

Glu Leu Phe Ile Lys Gly Arg Asn Phe Thr Pro Asp Ala Gly Val Phe

435 440 445

Val Tyr Ser Gly Asp Glu Ala Ile Met Lys Leu Arg Asn Trp Tyr Arg

450 455 460

Gln Thr Arg Ile His Ser Thr Leu Thr Leu Asp Asn Gln Asn Met Val

465 470 475 480

Ile Thr Lys Ala Arg Gln Asn Lys Trp Glu Thr Gly Asn Asn Leu Asp

485 490 495

Val Leu Thr Tyr Thr Asn Pro Ser Tyr Pro Asn Leu Asp His Gln Arg

500 505 510

Ser Val Leu Phe Ile Asn Lys Lys Tyr Phe Leu Val Ile Asp Arg Ala

515 520 525

Ile Gly Glu Ala Thr Gly Asn Leu Gly Val His Trp Gln Leu Lys Glu

530 535 540

Asp Ser Asn Pro Val Phe Asp Lys Thr Lys Asn Arg Val Tyr Thr Thr

545 550 555 560

Tyr Arg Asp Gly Asn Asn Leu Met Ile Gln Ser Leu Asn Ala Asp Arg

565 570 575

Thr Ser Leu Asn Glu Glu Glu Gly Lys Val Ser Tyr Val Tyr Asn Lys

580 585 590

Glu Leu Lys Arg Pro Ala Phe Val Phe Glu Lys Pro Lys Lys Asn Ala

595 600 605

Gly Thr Gln Asn Phe Val Ser Ile Val Tyr Pro Tyr Asp Gly Gln Lys

610 615 620

Ala Pro Glu Ile Ser Ile Arg Glu Asn Lys Gly Asn Asp Phe Glu Lys

625 630 635 640

Gly Lys Leu Asn Leu Thr Leu Thr Ile Asn Gly Lys Gln Gln Leu Val

645 650 655

Leu Val Pro

<210> 2

<211> 1907

<212> DNA

<213> Artificial sequence ()

<400> 2

caaagcagca gcataacaag aaaagacttc gaccacataa acctagaata cagcggacta 60

gaaaaagtaa acaaagcagt agcagcagga aactacgacg acgcagcaaa agcactacta 120

gcatactaca gagaaaaaag caaagcaaga gaaccagact tcagcaacgc agaaaaacca 180

gcagacataa gacaaccaat agacaaagta acaagagaaa tggcagacaa agcactagta 240

caccaattcc aaccacacaa aggatacgga tacttcgact acggaaaaga cataaactgg 300

caaatgtggc cagtaaaaga caacgaagta agatggcaac tacacagagt aaaatggtgg 360

caagcaatgg cactagtata ccacgcaaca ggagacgaaa aatacgcaag agaatgggta 420

taccaataca gcgactgggc aagaaaaaac ccactaggac taagccaaga caacgacaaa 480

ttcgtatgga gaccactaga agtaagcgac agagtacaaa gcctaccacc aacattcagc 540

ctattcgtaa acagcccagc attcacacca gcattcctaa tggaattcct aaacagctac 600

caccaacaag cagactacct aagcacacac tacgcagaac aaggaaacca cagactattc 660

gaagcacaaa gaaacctatt cgcaggagta agcttcccag aattcaaaga cagcccaaga 720

tggagacaaa caggaataag cgtactaaac acagaaataa aaaaacaagt atacgcagac 780

ggaatgcaat tcgaactaag cccaatatac cacgtagcag caatagacat attcctaaaa 840

gcatacggaa gcgcaaaaag agtaaaccta gaaaaagaat tcccacaaag ctacgtacaa 900

acagtagaaa acatgataat ggcactaata agcataagcc taccagacta caacacacca 960

atgttcggag acagctggat aacagacaaa aacttcagaa tggcacaatt cgcaagctgg 1020

caagagtatt cccagcaaac caagcaataa aatacttcgc aacagacgga aaacaaggaa 1080

aagcaccaaa cttcctaagc aaagcactaa gcaacgcagg attctacaca ttcagaagcg 1140

gatgggacaa aaacgcaaca gtaatggtac taaaagcaag cccaccagga gaattccacg 1200

cacaaccaga caacggaaca ttcgaactat tcataaaagg aagaaacttc acaccagacg 1260

caggagtatt cgtatacagc ggagacgaag caataatgaa actaagaaac tggtacagac 1320

aaacaagaat acacagcaca ctaacactag acaaccaaaa catggtaata acaaaagcaa 1380

gacaaaacaa atgggaaaca ggaaacaacc tagacgtact aacatacaca aacccaagct 1440

acccaaacct agaccaccaa agaagcgtac tattcataaa caaaaaatac ttcctagtaa 1500

tagacagagc aataggagaa gcaacaggaa acctaggagt acactggcaa ctaaaagaag 1560

acagcaaccc agtattcgac aaaacaaaaa acagagtata cacaacatac agagacggaa 1620

acaacctaat gatacaaagc ctaaacgcag acagaacaag cctaaacgaa gaagaaggaa 1680

aagtaagcta cgtatacaac aaagaactaa aaagaccagc attcgtattc gaaaaaccaa 1740

aaaaaaacgc aggaacacaa aacttcgtaa gcatagtata cccatacgac ggacaaaaag 1800

caccagaaat aagcataaga gaaaacaaag gaaacgactt cgaaaaagga aaactaaacc 1860

taacactaac aataaacgga aaacaacaac tagtactagt accatag 1907

<210> 3

<211> 744

<212> PRT

<213> Artificial sequence ()

<400> 3

Met Gly His His His His His His Met Ser Asp Ser Glu Val Asn Gln

1 5 10 15

Glu Ala Lys Pro Glu Val Lys Pro Glu Val Lys Pro Glu Thr His Ile

20 25 30

Asn Leu Lys Val Ser Asp Gly Ser Ser Glu Ile Phe Phe Lys Ile Lys

35 40 45

Lys Thr Thr Pro Leu Arg Arg Leu Met Glu Ala Phe Ala Lys Arg Gln

50 55 60

Gly Lys Glu Met Asp Ser Leu Arg Phe Leu Tyr Asp Gly Ile Arg Ile

65 70 75 80

Gln Ala Asp Gln Thr Pro Glu Asp Leu Asp Met Glu Asp Asn Asp Ile

85 90 95

Ile Glu Ala His Arg Glu Gln Ile Gly Gly Gln Gly Ser Gln Ser Ser

100 105 110

Ser Ile Thr Arg Lys Asp Phe Asp His Ile Asn Leu Glu Tyr Ser Gly

115 120 125

Leu Glu Lys Val Asn Lys Ala Val Ala Ala Gly Asn Tyr Asp Asp Ala

130 135 140

Ala Lys Ala Leu Leu Ala Tyr Tyr Arg Glu Lys Ser Lys Ala Arg Glu

145 150 155 160

Pro Asp Phe Ser Asn Ala Glu Lys Pro Ala Asp Ile Arg Gln Pro Ile

165 170 175

Asp Lys Val Thr Arg Glu Met Ala Asp Lys Ala Leu Val His Gln Phe

180 185 190

Gln Pro His Lys Gly Tyr Gly Tyr Phe Asp Tyr Gly Lys Asp Ile Asn

195 200 205

Trp Gln Met Trp Pro Val Lys Asp Asn Glu Val Arg Trp Gln Leu His

210 215 220

Arg Val Lys Trp Trp Gln Ala Met Ala Leu Val Tyr His Ala Thr Gly

225 230 235 240

Asp Glu Lys Tyr Ala Arg Glu Trp Val Tyr Gln Tyr Ser Asp Trp Ala

245 250 255

Arg Lys Asn Pro Leu Gly Leu Ser Gln Asp Asn Asp Lys Phe Val Trp

260 265 270

Arg Pro Leu Glu Val Ser Asp Arg Val Gln Ser Leu Pro Pro Thr Phe

275 280 285

Ser Leu Phe Val Asn Ser Pro Ala Phe Thr Pro Ala Phe Leu Met Glu

290 295 300

Phe Leu Asn Ser Tyr His Gln Gln Ala Asp Tyr Leu Ser Thr His Tyr

305 310 315 320

Ala Glu Gln Gly Asn His Arg Leu Phe Glu Ala Gln Arg Asn Leu Phe

325 330 335

Ala Gly Val Ser Phe Pro Glu Phe Lys Asp Ser Pro Arg Trp Arg Gln

340 345 350

Thr Gly Ile Ser Val Leu Asn Thr Glu Ile Lys Lys Gln Val Tyr Ala

355 360 365

Asp Gly Met Gln Phe Glu Leu Ser Pro Ile Tyr His Val Ala Ala Ile

370 375 380

Asp Ile Phe Leu Lys Ala Tyr Gly Ser Ala Lys Arg Val Asn Leu Glu

385 390 395 400

Lys Glu Phe Pro Gln Ser Tyr Val Gln Thr Val Glu Asn Met Ile Met

405 410 415

Ala Leu Ile Ser Ile Ser Leu Pro Asp Tyr Asn Thr Pro Met Phe Gly

420 425 430

Asp Ser Trp Ile Thr Asp Lys Asn Phe Arg Met Ala Gln Phe Ala Ser

435 440 445

Trp Ala Arg Val Phe Pro Ala Asn Gln Ala Ile Lys Tyr Phe Ala Thr

450 455 460

Asp Gly Lys Gln Gly Lys Ala Pro Asn Phe Leu Ser Lys Ala Leu Ser

465 470 475 480

Asn Ala Gly Phe Tyr Thr Phe Arg Ser Gly Trp Asp Lys Asn Ala Thr

485 490 495

Val Met Val Leu Lys Ala Ser Pro Pro Gly Glu Phe His Ala Gln Pro

500 505 510

Asp Asn Gly Thr Phe Glu Leu Phe Ile Lys Gly Arg Asn Phe Thr Pro

515 520 525

Asp Ala Gly Val Phe Val Tyr Ser Gly Asp Glu Ala Ile Met Lys Leu

530 535 540

Arg Asn Trp Tyr Arg Gln Thr Arg Ile His Ser Thr Leu Thr Leu Asp

545 550 555 560

Asn Gln Asn Met Val Ile Thr Lys Ala Arg Gln Asn Lys Trp Glu Thr

565 570 575

Gly Asn Asn Leu Asp Val Leu Thr Tyr Thr Asn Pro Ser Tyr Pro Asn

580 585 590

Leu Asp His Gln Arg Ser Val Leu Phe Ile Asn Lys Lys Tyr Phe Leu

595 600 605

Val Ile Asp Arg Ala Ile Gly Glu Ala Thr Gly Asn Leu Gly Val His

610 615 620

Trp Gln Leu Lys Glu Asp Ser Asn Pro Val Phe Asp Lys Thr Lys Asn

625 630 635 640

Arg Val Tyr Thr Thr Tyr Arg Asp Gly Asn Asn Leu Met Ile Gln Ser

645 650 655

Leu Asn Ala Asp Arg Thr Ser Leu Asn Glu Glu Glu Gly Lys Val Ser

660 665 670

Tyr Val Tyr Asn Lys Glu Leu Lys Arg Pro Ala Phe Val Phe Glu Lys

675 680 685

Pro Lys Lys Asn Ala Gly Thr Gln Asn Phe Val Ser Ile Val Tyr Pro

690 695 700

Tyr Asp Gly Gln Lys Ala Pro Glu Ile Ser Ile Arg Glu Asn Lys Gly

705 710 715 720

Asn Asp Phe Glu Lys Gly Lys Leu Asn Leu Thr Leu Thr Ile Asn Gly

725 730 735

Lys Gln Gln Leu Val Leu Val Pro

740

Claims

1. A method for preparing recombinant heparinase III by utilizing an SUMO fusion expression system is characterized by comprising the following steps:

s01: selecting a heparinase III sequence from Flavobacterium heparinum (Pedobacter heparinus), wherein the amino acid sequence of the heparinase III sequence is shown as SEQ ID No.1 and is 659aa in total;

2. The method of claim 1, further comprising the step of optimizing the expression conditions of the fusion protein.

3. The method according to claim 2, wherein the optimized condition of the fusion protein is that the fusion protein is induced to express at 0.2mmol/l isopropyl thiogalactoside (IPTG) at 15 ℃.

4. The method of claims 1-3, further comprising a purification step of the fusion protein.

5. The method according to claim 4, wherein the step of purifying the fusion protein is performed by Ni column affinity chromatography.

6. The method of claim 1, wherein the SUMO tag protein is cleaved from the SUMO heparinase III fusion protein of step S04 by the following steps: dialyzing the fusion protein to replace buffer solution, adding a proper amount of SUMO protease, uniformly mixing, placing at 4 ℃ for reacting overnight, and separating a SUMO protein label (capable of being combined on a Ni column) with 6 histidine fragments and heparinase III (incapable of being combined on the Ni column) from a hydrolyzed fusion protein sample by using a Ni column affinity chromatography method.

7. The SUMO heparanase III fusion protein produced by the method of any one of claims 1 to 5, wherein the amino acid sequence of said fusion protein is as set forth in SEQ ID No. 3.

8. Heparinase III prepared by a method according to any one of claims 1 to 5.