CN114539425A - Method for improving biological expression of linear polypeptide - Google Patents

Abstract

The invention discloses a method for improving linear polypeptide biological expression, which comprises the steps of carrying out fusion expression on EDDIE (C69E, C168E) -Sumo combined label and target polypeptide to obtain fusion protein; renaturing the fusion protein; the Sumo label in the renatured fusion protein is cut off by adopting the specific protease which is adaptive to the combined label, so that the biological expression of the linear polypeptide can be improved. The invention combines the advantages of two labels of EDDIE and SUMO protein, fuses target polypeptide at C end by using EDDIE (C69E, C168E) and SUMO combined label for the first time, cuts off the label by using SUMO protease, and purifies enzyme digestion product by using a solid phase extraction small column C8 reverse phase column to obtain polypeptide, thereby establishing an efficient expression system of escherichia coli, and obtaining the target polypeptide with high expression and high purity.

Description

Method for improving biological expression of linear polypeptide

Technical Field

The invention relates to the field of bioengineering, relates to a method for improving linear polypeptide biological expression, and particularly relates to a novel fusion expression tag and application thereof in promoting heterologous polypeptide expression.

Background

Coli expression systems have high expression levels, fast growth rates, and are suitable for continuous and high cell density culture processes, and therefore, e.coli is the most popular recombinant protein production host in biotechnology to date. However, there are still some disadvantages of E.coli expression systems, one of which is the production of small proteins and polypeptides. When the polypeptide with smaller segments is expressed by a direct expression mode, the degradation is easy to occur, so that the yield is low, and the expression of toxic peptide or antibacterial peptide which is unfavorable for the growth of thalli is not suitable. In addition, many recombinant polypeptides are prone to misfolding in bacteria during expression due to excessive translation rates and lack of homologous partners. To solve this problem, many tags are fused to the target protein, some of which increase the yield of the protein by forming inclusion body proteins. Compared with soluble expression, the inclusion body expression has the advantages of high yield, strong stability, easy separation and purification and the like, and reduces the toxic effect of target protein or polypeptide on host cells, but the inclusion body protein can obtain correctly folded functional protein through a complex renaturation process, thus offsetting the advantage of high expression of the inclusion body to a certain extent. In addition, the removal of protein tags is critical for biomedical applications.

Disclosure of Invention

The purpose of the invention is as follows: the technical problem to be solved by the invention is to provide a method for expressing polypeptide aiming at the defects of the prior art.

The technical problem to be solved by the present invention is to provide a method for preparing polypeptide.

The invention also aims to solve the technical problem of providing an EDDIE (C69E, C168E) -Sumo combined label.

The technical problem to be solved by the present invention is to provide a gene encoding the above-mentioned combination tag.

The technical problem to be solved by the invention is to provide a protein expression vector.

The technical problem to be solved by the invention is to provide a kit.

In order to solve the first technical problem, the invention discloses a method for expressing a polypeptide, which comprises the following steps: the EDDIE (C69E, C168E) -Sumo combined tag is fused with the target polypeptide to express the target polypeptide.

Wherein the EDDIE (C69E, C168E) -Sumo combined tag comprises an amino acid sequence shown in SEQ ID NO. 1.

The EDDIE label is an inclusion body label protein label; the EDDIE label is Npro mutein of Npro swine fever self-cleavage protein, and Cys at 69-position is mutated into Glu and Cys at 168-position is mutated into Glu respectively. This is mainly due to the many deficiencies of the purified proteins of natural Npro protein, compared to EDDIE, which is an Npro mutein provided by the present invention, with increased solubility, faster renaturation and higher cleavage efficiency, EDDIE still has certain deficiencies in the aspect of self-cleavage efficiency; after the EDDIE is mutated at 69 th and 168 th critical sites for the cleavage activity, the EDDIE does not have the self-cleavage capacity, but retains the capacity of forming inclusion bodies.

Wherein the target polypeptide is any one of antibacterial peptide, linear peptide and toxic peptide.

In order to solve the second technical problem, the invention discloses a method for preparing polypeptide, which adopts EDDIE (C69E, C168E) and Sumo protein tags to establish a C-terminal fusion expression system, simultaneously uses specific cutting of SUMO protease to remove tags, thereby obtaining target polypeptide, and finally enzyme digestion products are purified by a solid phase extraction method. According to the invention, through the established C-terminal fusion expression system, 69 th and 168 th key sites of the EDDIE shearing activity are mutated, so that the EDDIE does not have self-shearing capability, but retains the capability of forming an inclusion body; the ability of the SUMO protein tag to promote correct folding of the protein is also exploited to make it less prone to precipitation during EDDIE renaturation.

Specifically, the method comprises the following steps: carrying out fusion expression on the EDDIE (C69E, C168E) -Sumo combined label and the target polypeptide to obtain a fusion protein; renaturing the fusion protein; and (3) cutting the Sumo label in the renatured fusion protein by using specific protease which is adaptive to the combined label, and purifying to obtain the target polypeptide.

Wherein the EDDIE (C69E, C168E) -Sumo combined tag comprises an amino acid sequence shown in SEQ ID NO. 1.

Wherein the target polypeptide is a linear peptide including but not limited to any one of antibacterial peptide, linear peptide and toxic peptide.

Wherein the buffer solution adopted in the renaturation process is 1-1.5M Tris-HCl, the pH value is 7-8, 2.5-10% of glycerol and 1-10mM Tris (2-carboxyethyl) phosphine hydrochloride (Tcep hydrochloride), and the buffer solution is preferably 1M Tris-HCl, the pH value is 7.8, 5% of glycerol and 2mM Tcep hydrochloride.

Wherein the renaturation is dialysis renaturation.

Wherein, the specific protease matched with the combined label is SUMO protease, and the efficiency is higher.

Wherein the Sumo tag method in the fusion protein after excision renaturation is a shearing method: directly adding a corresponding amount of SUMO protease into renaturation fusion protein with the concentration of 0.8-1.2mg renaturation fusion protein/mL renaturation liquid renaturation fusion protein; the amount of SUMO protease used is the amount of protein to be cleaved x 0.007 SUMO protease concentration.

Wherein the enzyme digestion is carried out for 2.5 to 4.5 hours at the temperature of 25 to 35 ℃, and preferably for 3.5 hours at the temperature of 30 ℃.

Wherein the purification is performed by using a C8 reverse phase column.

Wherein the parameters of the C8 reversed phase column are carbon content: 9%, specific surface area: 280m²The grain diameter per gram: average pore diameter of 40-75 μm:

in order to solve the third technical problem, the invention discloses an EDDIE (C69E, C168E) -Sumo combined tag which comprises an amino acid sequence shown as SEQ ID NO. 1.

In order to solve the fourth technical problem, the invention discloses a gene for coding the EDDIE (C69E, C168E) -Sumo combined label, which comprises a nucleotide sequence shown in SEQ ID NO. 2.

In order to solve the fifth technical problem, the invention discloses a protein expression vector, which comprises the EDDIE (C69E, C168E) -Sumo combined label.

In order to solve the fifth technical problem, the invention discloses a kit comprising the EDDIE (C69E, C168E) -Sumo combined tag, the gene encoding the EDDIE (C69E, C168E) -Sumo combined tag, or the protein expression vector.

Has the advantages that: compared with the prior art, the invention has the following advantages:

the invention relates to a method for expressing and purifying linear polypeptide, which fuses the polypeptide with inclusion body protein EDDIE (C69E, C168E) and SUMO protein tag, the fused polypeptide can be expressed and purified in an inclusion body form in escherichia coli, the objective polypeptide is cut off by SUMO protease after dialysis and renaturation, finally, small peptide is purified by C8 solid phase extraction reverse phase column, and the purity is 80-90% by HPLC chromatography; the method can effectively solve the problems of difficult renaturation of the inclusion body and label removal.

Drawings

The foregoing and/or other advantages of the invention will become further apparent from the following detailed description of the invention when taken in conjunction with the accompanying drawings.

FIG. 1 shows the EDDIE (C69E) -Sumo renaturation cleavage, the control is non-renaturated EDDIE (C69E) -Sumo protein, and 1-4 are renaturated EDDIE (C69E) -Sumo protein, respectively.

FIG. 2 shows the EDDIE (C168E) -Sumo renaturation and cleavage conditions, the control is non-renaturated EDDIE (C168E) -Sumo protein, and 1-4 are renatured EDDIE (C168E) -Sumo protein respectively.

FIG. 3 is a vector construction RCR amplification electrophoresis chart.

FIG. 4 is a diagram showing the cleavage effect of the fusion polypeptide.

FIG. 5 is a diagram showing the purification effect of a solid phase extraction cartridge.

Fig. 6 is a diagram of a polypeptide freeze-dried finished product.

Detailed Description

The experimental methods described in the following examples are all conventional methods unless otherwise specified; the reagents and materials are commercially available, unless otherwise specified.

Example 1:

(1) construction of EDDIE (C69E, C168E) -Sumo Association tag with amino acid sequence shown as SEQ ID NO.1

The primers shown in SEQ ID NO.3-24 are used for gene synthesis of the gene sequence shown in SEQ ID NO.1, and the expected lengths of the sequences are respectively about 885 bp. The gene synthesis is carried out in 2 rounds: first round PCR reaction (50. mu.L): 10 μ L of Phusion Buffer (5X), 1 μ L of dNTP Mix (10mmol/L), 2 μ L of primer Mix (SEQ ID NO.3 and SEQ ID NO.24), 0.5 μ L of Phusion enzyme (5U/. mu.L), 36.5 μ L H₂And O. The first round of PCR reaction program is pre-denaturation at 98 ℃ for 30 sec; 18 cycles: 98 ℃ for 10 sec; 58 ℃, 20 sec; 72 ℃ for 1.5 min; 5min at 72 ℃. Second round PCR reaction (50. mu.L): 10 μ L of Phusion Buffer (5X), 1 μ L of dNTP mix (10mmol/L), EDDIE (C69EC168E) -Sumo-1: 1 μ L (10 μmol/L), EDDIE (C69EC168E) -Sumo-22: 1 μ L (10 μmol/L), 1 μ L of first round template, 0.5 μ L of Phusion enzyme (5U/μ L), 35.5 μ L H₂And O. The second round of PCR reaction program is pre-denaturation at 98 ℃ for 30 sec; 25 cycles: 98 ℃ for 10 sec; 58 ℃, 20 sec; 72 ℃ for 1.5 min; 72 ℃ for 5 min.

The PCR product is detected by 1.5 percent agarose gel electrophoresis and is recovered by a hanging column PCR product purification kit by adopting a method of adsorbing DNA by a silica gel membrane: 1. the PCR reaction was transferred to a clean 1.5mL centrifuge tube, 3 volumes of Buffer B3 were added and mixed well. 2. The whole mixture was transferred to an adsorption column 8000 Xg and centrifuged for 30 sec. 3. The liquid in the collection tube was decanted, and 500. mu.L of Wash Solution was added to the adsorption column and centrifuged at 9000 Xg for 30 sec. 4. Repeat step 3 once. 5. The empty adsorption column and collection tube were placed in a centrifuge and centrifuged at 9000 Xg for 1 min. 6. 40 μ L solution Buffer was added to the center of the adsorption membrane, left to stand at room temperature for l-2min, and centrifuged at 9000 Xg for 1 min. The Elution Buffer was 2.5mM Tris-HCI, pH 8.5. Then connecting the purified PCR product with pET-15bvector after NcoI/XhoI double enzyme digestion, transforming into Escherichia coli TOP10 competent cells, carrying out ampicillin resistance screening, extracting plasmids after selecting positive transformants and sending the plasmids to the company for sequencing, wherein the sequencing result shows that the sequence is correct, and the nucleotide sequence is shown as SEQ ID NO. 25.

In the combined tag, Cys at 69 th is mutated into Glu, Cys at 168 th is mutated into Glu, EDDIE (C69E) -Sumo and EDDIE (C168E) -Sumo renaturation and shearing conditions are respectively shown in figures 1 and 2, EDDIE (C69E) -Sumo and EDDIE (C168E) -Sumo proteins are dialyzed in 0.2M Arg, 20mM Tris, 0.01% Tween-20, 250mM NaCl, 5% glycerol, 5mM DTT, 10mM magnesium sulfate and pH 9.0, 20 mu L of the proteins are respectively taken after overnight at 4 ℃ and subjected to protein electrophoresis, and the control is dialyzed and untreated. The self-splicing reaction can not occur after the 69 th mutation and the 168 th mutation are proved, the two mutations play a key role in self-splicing, and both sites are mutated in the invention.

(2) Fusing the combined label vector and a target polypeptide to express the target polypeptide to obtain a fusion protein

EDDIE (C69E, C168E) -SUMO-conjugated tag vector (FIG. 3, lanes 1-2) was amplified to give linearized tag vector fragments: the amplification primers are as follows: f: 5'-taaCTCGAGGATCCGGCTGCTAAC-3' (SEQ ID NO.26), R: 5'-GCCTCCAATCTGTTCGCGGTGAGCCTCA-3' (SEQ ID NO.27), the expected length of the sequence is approximately 6497 bp. PCR reaction (50. mu.L): 10 μ L of Phusion Buffer (5X), 1 μ L of dNTP mix (10mmol/L), 1 μ L of forward primer (10 μmol/L), 1 μ L of reverse primer (10 μmol/L), PET15bEDDIE (C69E, C168E) -Sumo template 1. mu.L, 0.5. mu.L LPHUsion enzyme (5U/. mu.L), 35.5. mu. L H₂And O. The PCR reaction program is pre-denaturation at 98 ℃ for 30 sec; 25 cycles: at 98 ℃ for 10 sec; 58 ℃, 20 sec; 72 ℃ for 1.5 min; 72 ℃ for 5 min.

Amplifying a first target polypeptide, as shown in FIG. 3, wherein a lane 3 is B147-1(28aa), the nucleotide sequences of which are respectively shown as SEQ ID NO.28, and the amino acid sequence of the target polypeptide is shown as SEQ ID NO. 29; amplifying a second target polypeptide, as shown in FIG. 3, wherein a lane 4 is GLP1-1(31aa), the nucleotide sequences of which are respectively shown as SEQ ID NO.30, and the amino acid sequence of the target polypeptide is shown as SEQ ID NO. 31; amplifying a third target polypeptide, as shown in FIG. 3, wherein Lane 5 is Cherry50(51aa), the nucleotide sequences thereof are respectively shown as SEQ ID NO.32, and the amino acid sequence of the target polypeptide is shown as SEQ ID NO. 33; amplifying a fourth target polypeptide, as shown in FIG. 3, lane 6 is Cherry80(80aa), the nucleotide sequences are shown as SEQ ID NO.34, and the amino acid sequence of the target polypeptide is shown as SEQ ID NO. 35.

Respectively carrying out enzyme coupling (20 min at 50 ℃) on the gene segments of the target polypeptides 1-4 and a linearized label carrier, wherein the enzyme coupling system is a 20 mu L system: gibson recombinase: 5 μ L, target polypeptide gene fragment: 10ng, linearized tag vector: 50ng, H₂O: make up to 20. mu.L. After expression identification, the DNA is sent to the company for sequencing, and the sequencing result shows that the sequence is correct; successfully constructs a recombinant fusion polypeptide vector.

Inducing the fusion polypeptide vector to express the target protein in escherichia coli, and 1) activating: the fusion polypeptide vector was spread on an LB plate (Amp +), and incubated overnight at 37 ℃ in a constant temperature incubator. The next morning a clone was picked up into a tube containing 2ml LB solution (Amp +) and shaken at 37 ℃ and 200rpm to an OD600 of about 0.6. 2) 2ml of the inoculum solution was added to 200ml of LB at 37 ℃ and shaken at 200rpm to an OD600 of about 0.6. 3) Inducing expression: IPTG was added to a final concentration of 1mM and shaking was continued for 3-4 h. 4) Cell collection: centrifuge (5000rpm, 5min, 4 ℃). 5) Resuspension washing: using washing buffer: bacteria were suspended in 50mM Tris-HCl, 5mM EDTA, 1% Triton X-100, pH 8.0. 6) Cells were disrupted by sonication. 7) Collecting an inclusion body precipitate: the supernatant was removed by centrifugation (12,000rpm, 15min, 4 ℃). 8) Washing of inclusion bodies: washing buffer: 50mM Tris-HCl, 5mM EDTA, 1% Triton X-100, pH 8.0 heavy suspension wash. 9) Dissolving the inclusion body: solubilization with inactivation buffer (50mM Tris-HCl, 150mM NaCl, 5mM imidazole, 8M urea, pH 8.0) gave a fusion protein solution.

(3) Renaturation of fusion proteins

Preparing 4 different renaturation buffers which are respectively as follows: buffer 1: 1M Tris-HCl, pH 7.8, 5% glycerol, 2mM Tcep; buffer 2: 500mM NaCL, 20mM Tris-HCl, pH 9.5, 2mM EDTA, 5% glycerol, 2mM Tcep; buffer 3: 0.2M Arg, 500mM NaCL, 20mM Tris-HCl, pH 9.0,2mM EDTA, 5% glycerol, 2mM Tcep, 0.01% Tween-20; buffer 4: 0.2M Arg, 500mM NaCl, 20mM Tris-HCl, pH 9.0,2mM EDTA, 5% glycerol, 2mM Tcep, 0.01% Tween-20, 800mM urea.

Respectively carrying out dialysis renaturation on the 4 renaturation buffers and the fusion protein: 1) the 2.5K dialysis bag is cut into small sections with a proper length of about 10 cm. 2) The dialysis bag was placed in boiling water to boil for 10 minutes. 3) And (3) taking out the dialysis bag, discharging water, sealing one end of the dialysis bag by using a dialysis clamp, adding the fusion protein solution obtained in the step (2) into the dialysis bag, discharging air bubbles, and sealing two ends of the dialysis bag by uniformly dialyzing the clamp. 4) And (3) putting the dialysis bags filled with different proteins into 5L renaturation buffer solution for dialysis, and replacing the dialysate every 30min until urea is dialyzed completely to obtain the renatured fusion protein.

Among them, the present test found that the fusion protein (inclusion body protein of the fusion polypeptide) was not easily precipitated under the conditions of high Tris (1M Tris) and high urea (800mM urea), but urea affected the cleavage efficiency of SUMO protease, while high Tris (1M) had no effect on the cleavage of SUMO protease. Buffer1 is therefore preferred: 1M Tris-HCl, pH 7.8, 5% glycerol, 2mM Tcep. Therefore, the problems that because EDDIE has more hydrophobic amino acids, the EDDIE is easy to precipitate and difficult to renature after urea is removed by dialysis can be effectively solved.

(4) And (3) removing the SUMO label in the fusion protein by adopting SUMO protease shearing:

taking the fusion protein renatured in the step (3) as a substrate, adding SUMO protease, controlling the substrate concentration at 1mg of renatured fusion protein/mL of renatured buffer solution, and reacting at 30 ℃ for 3.5h to obtain an enzyme digestion product; meanwhile, the non-renatured corresponding proteins were used as negative controls, respectively (FIG. 4).

Wherein the volume (ml) of the SUMO protease is the mass (mg) of the renaturation fusion protein to be cut x 0.007 ÷ Sumo protease concentration (mg/ml), and the Sumo protease is ThermoFisher 12588018.

(5) And (3) enzyme digestion product purification: the polypeptide is purified by hydrophobic interaction of the polypeptide and C8 reverse column purification

Adding 5mL of the enzyme digestion product obtained in the step (4) to a 200mg/3mL C8 small column activated by 100% methanol for 2 times, automatically flowing by gravity, wherein the tag protein does not completely flow out of the C8 column, the polypeptide can be combined to a C8 column, washing by 3mL of 5% acetonitrile after no liquid flows out of the tail end of the column, completely draining the liquid to be washed by gravity, and eluting the polypeptide by 2mL of 95% acetonitrile to obtain the purified target polypeptide.

The purity of the eluted polypeptide is high by HPLC detection, and the purity of B147-1 is as follows: 93%, Glp-1 purity 83%, cherry50 purity: 91%, cherry80 purity: 86% (fig. 5), and the lyophilized powder was white (fig. 6). Therefore, the method is convenient to operate, the purified polypeptide has high purity, and the method can remove salt and has high universality.

Wherein the parameters of the C8 reversed phase column are carbon content: 9%, specific surface area: 280m²The particle diameter per gram: average pore diameter of 40-75 μm:

to sum up, the invention combines the advantages of two tags of EDDIE and SUMO protein, the EDDIE (C69E, C168E) and SUMO combined tag are firstly utilized to fuse target polypeptide at the C end, SUMO protease is utilized to cut off the tag, a small solid-phase extraction column C8 reverse phase column is adopted to purify enzyme digestion products to obtain the polypeptide, and an efficient escherichia coli expression system is established, so that the high-expression and high-purity target polypeptide is obtained. The method of the invention solves the problem that the small peptide is easy to degrade in the biological expression process and meets the requirements of expressing toxic peptide or antibacterial peptide.

The present invention provides a method for improving the biological expression of linear polypeptides, and a plurality of methods and ways for implementing the method, and the above description is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, a plurality of modifications and amendments can be made without departing from the principle of the present invention, and these modifications and amendments should also be regarded as the protection scope of the present invention. All the components not specified in the present embodiment can be realized by the prior art.

Sequence listing

<110> Cheng quan peptide Biochemical technology Co., Ltd in Hunan province

<120> a method for improving the biological expression of linear polypeptides

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<211> 6497

<212> DNA

<213> PET15b-EDDIE(C69E、C168E)-Sumo

<400> 25

ttcttgaaga cgaaagggcc tcgtgatacg cctattttta taggttaatg tcatgataat 60

aatggtttct tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg 120

tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat 180

gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat 240

tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt 300

aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag 360

cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa 420

agttctgcta tgtggcgcgg tattatcccg tgttgacgcc gggcaagagc aactcggtcg 480

ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct 540

tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac 600

tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca 660

caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat 720

accaaacgac gagcgtgaca ccacgatgcc tgcagcaatg gcaacaacgt tgcgcaaact 780

attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc 840

ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga 900

taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg 960

taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg 1020

aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca 1080

agtttactca tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta 1140

ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca 1200

ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg 1260

cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga 1320

tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa 1380

tactgtcctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc 1440

tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg 1500

tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac 1560

ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct 1620

acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc 1680

ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg 1740

gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg 1800

ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct 1860

ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga 1920

taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg 1980

cagcgagtca gtgagcgagg aagcggaaga gcgcctgatg cggtattttc tccttacgca 2040

tctgtgcggt atttcacacc gcatatatgg tgcactctca gtacaatctg ctctgatgcc 2100

gcatagttaa gccagtatac actccgctat cgctacgtga ctgggtcatg gctgcgcccc 2160

gacacccgcc aacacccgct gacgcgccct gacgggcttg tctgctcccg gcatccgctt 2220

acagacaagc tgtgaccgtc tccgggagct gcatgtgtca gaggttttca ccgtcatcac 2280

cgaaacgcgc gaggcagctg cggtaaagct catcagcgtg gtcgtgaagc gattcacaga 2340

tgtctgcctg ttcatccgcg tccagctcgt tgagtttctc cagaagcgtt aatgtctggc 2400

ttctgataaa gcgggccatg ttaagggcgg ttttttcctg tttggtcact gatgcctccg 2460

tgtaaggggg atttctgttc atgggggtaa tgataccgat gaaacgagag aggatgctca 2520

cgatacgggt tactgatgat gaacatgccc ggttactgga acgttgtgag ggtaaacaac 2580

tggcggtatg gatgcggcgg gaccagagaa aaatcactca gggtcaatgc cagcgcttcg 2640

ttaatacaga tgtaggtgtt ccacagggta gccagcagca tcctgcgatg cagatccgga 2700

acataatggt gcagggcgct gacttccgcg tttccagact ttacgaaaca cggaaaccga 2760

agaccattca tgttgttgct caggtcgcag acgttttgca gcagcagtcg cttcacgttc 2820

gctcgcgtat cggtgattca ttctgctaac cagtaaggca accccgccag cctagccggg 2880

tcctcaacga caggagcacg atcatgcgca cccgtggcca ggacccaacg ctgcccgaga 2940

tgcgccgcgt gcggctgctg gagatggcgg acgcgatgga tatgttctgc caagggttgg 3000

tttgcgcatt cacagttctc cgcaagaatt gattggctcc aattcttgga gtggtgaatc 3060

cgttagcgag gtgccgccgg cttccattca ggtcgaggtg gcccggctcc atgcaccgcg 3120

acgcaacgcg gggaggcaga caaggtatag ggcggcgcct acaatccatg ccaacccgtt 3180

ccatgtgctc gccgaggcgg cataaatcgc cgtgacgatc agcggtccag tgatcgaagt 3240

taggctggta agagccgcga gcgatccttg aagctgtccc tgatggtcgt catctacctg 3300

cctggacagc atggcctgca acgcgggcat cccgatgccg ccggaagcga gaagaatcat 3360

aatggggaag gccatccagc ctcgcgtcgc gaacgccagc aagacgtagc ccagcgcgtc 3420

ggccgccatg ccggcgataa tggcctgctt ctcgccgaaa cgtttggtgg cgggaccagt 3480

gacgaaggct tgagcgaggg cgtgcaagat tccgaatacc gcaagcgaca ggccgatcat 3540

cgtcgcgctc cagcgaaagc ggtcctcgcc gaaaatgacc cagagcgctg ccggcacctg 3600

tcctacgagt tgcatgataa agaagacagt cataagtgcg gcgacgatag tcatgccccg 3660

cgcccaccgg aaggagctga ctgggttgaa ggctctcaag ggcatcggtc gagatcccgg 3720

tgcctaatga gtgagctaac ttacattaat tgcgttgcgc tcactgcccg ctttccagtc 3780

gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt 3840

gcgtattggg cgccagggtg gtttttcttt tcaccagtga gacgggcaac agctgattgc 3900

ccttcaccgc ctggccctga gagagttgca gcaagcggtc cacgctggtt tgccccagca 3960

ggcgaaaatc ctgtttgatg gtggttaacg gcgggatata acatgagctg tcttcggtat 4020

cgtcgtatcc cactaccgag atatccgcac caacgcgcag cccggactcg gtaatggcgc 4080

gcattgcgcc cagcgccatc tgatcgttgg caaccagcat cgcagtggga acgatgccct 4140

cattcagcat ttgcatggtt tgttgaaaac cggacatggc actccagtcg ccttcccgtt 4200

ccgctatcgg ctgaatttga ttgcgagtga gatatttatg ccagccagcc agacgcagac 4260

gcgccgagac agaacttaat gggcccgcta acagcgcgat ttgctggtga cccaatgcga 4320

ccagatgctc cacgcccagt cgcgtaccgt cttcatggga gaaaataata ctgttgatgg 4380

gtgtctggtc agagacatca agaaataacg ccggaacatt agtgcaggca gcttccacag 4440

caatggcatc ctggtcatcc agcggatagt taatgatcag cccactgacg cgttgcgcga 4500

gaagattgtg caccgccgct ttacaggctt cgacgccgct tcgttctacc atcgacacca 4560

ccacgctggc acccagttga tcggcgcgag atttaatcgc cgcgacaatt tgcgacggcg 4620

cgtgcagggc cagactggag gtggcaacgc caatcagcaa cgactgtttg cccgccagtt 4680

gttgtgccac gcggttggga atgtaattca gctccgccat cgccgcttcc actttttccc 4740

gcgttttcgc agaaacgtgg ctggcctggt tcaccacgcg ggaaacggtc tgataagaga 4800

caccggcata ctctgcgaca tcgtataacg ttactggttt cacattcacc accctgaatt 4860

gactctcttc cgggcgctat catgccatac cgcgaaaggt tttgcgccat tcgatggtgt 4920

ccgggatctc gacgctctcc cttatgcgac tcctgcatta ggaagcagcc cagtagtagg 4980

ttgaggccgt tgagcaccgc cgccgcaagg aatggtgcat gcaaggagat ggcgcccaac 5040

agtcccccgg ccacggggcc tgccaccata cccacgccga aacaagcgct catgagcccg 5100

aagtggcgag cccgatcttc cccatcggtg atgtcggcga tataggcgcc agcaaccgca 5160

cctgtggcgc cggtgatgcc ggccacgatg cgtccggcgt agaggatcga gatctcgatc 5220

ccgcgaaatt aatacgactc actatagggg aattgtgagc ggataacaat tcccctctag 5280

aaataatttt gtttaacttt aagaaggaga tataccttaa gaaggagata taccatggag 5340

ttaaaccatt tcgagcttct gtataaaaca tctaagcaaa agccagttgg agtggaggag 5400

ccagtgtatg ataccgctgg ccgcccgctg tttggcaacc cgtcggaagt tcatcctcaa 5460

tcaaccctga aattgccaca tgatcgtggc gaagatgaca ttgaaaccac actgcgtgat 5520

ttgccacgca aaggtgacga acgtagtggt aaccatcttg ggcctgtcag cggcatctac 5580

atcaagcccg gccccgtgta ttaccaggat tacaccggac cggtttacca ccgtgcaccg 5640

ctggaattct ttgacgaaac gcagttcgag gagactacta aacgcatcgg gcgcgttacc 5700

ggctctgacg gaaagctgta ccacatttac gtcgaagttg acggtgagat cttactgaag 5760

caagcgaaac gcggcacacc acgtacattg aaatggacgc gcaatacgac gaactgtccc 5820

ttatgggtaa cgagcgaaag tggtggccat catcatcatc atcatggcgg cagctcggac 5880

tcagaagtca atcaagaagc taagccagag gtcaagccag aagtcaagcc tgagactcac 5940

atcaatttaa aggtgtccga tggatcttca gagatcttct tcaagatcaa aaagaccact 6000

cctttaagaa ggctgatgga agcgttcgct aaaagacagg gtaaggaaat ggactcctta 6060

agattcttgt acgacggtat tagaattcaa gctgatcagg cccctgaaga tttggacatg 6120

gaggataacg atattattga ggctcaccgc gaacagattg gaggctaact cgaggatccg 6180

gctgctaaca aagcccgaaa ggaagctgag ttggctgctg ccaccgctga gcaataacta 6240

gcataacccc ttggggcctc taaacgggtc ttgaggggtt ttttgctgaa aggaggaact 6300

atatccggat atcccgcaag aggcccggca gtaccggcat aaccaagcct atgcctacag 6360

catccagggt gacggtgccg aggatgacga tgagcgcatt gttagatttc atacacggtg 6420

cctgactgcg ttagcaattt aactgtgata aactaccgca ttaaagctta tcgatgataa 6480

gctgtcaaac atgagaa 6497

<210> 26

<211> 24

<212> DNA

<213> primer (F)

<400> 26

taactcgagg atccggctgc taac 24

<210> 27

<211> 28

<212> DNA

<213> primer (R)

<400> 27

gcctccaatc tgttcgcggt gagcctca 28

<210> 28

<211> 130

<212> DNA

<213> B147-1DNASEQ

<400> 28

ctcaccgcga acagattgga ggctttcacc accatcatca tcacggggta aatgcaggcg 60

gttggcaaac atctagaggg agtgagacaa agcaaataaa ttgggattaa ctcgaggatc 120

cggctgctaa 130

<210> 29

<211> 28

<212> PRT

<213> B147-1

<400> 29

Phe His His His His His His Gly Val Asn Ala Gly Gly Trp Gln Thr

1 5 10 15

Ser Arg Gly Ser Glu Thr Lys Gln Ile Asn Trp Asp

20 25

<210> 30

<211> 139

<212> DNA

<213> GLP-1DNASEQ

<400> 30

ctcaccgcga acagattgga ggccatgcgg aaggcacctt taccagcgat gtgagcagct 60

atctggaagg ccaggcggcg aaagaattta ttgcgtggct ggtgaaaggc cgcggctaac 120

tcgaggatcc ggctgctaa 139

<210> 31

<211> 31

<212> PRT

<213> GLP-1

<400> 31

His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly

1 5 10 15

Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly

20 25 30

<210> 32

<211> 199

<212> DNA

<213> Cherry50DNASEQ

<400> 32

ctcaccgcga acagattgga ggcatggtgg gacaaccctt atgggacgtt agccgttcat 60

caactggtaa tcttgtcgaa tggttccttt tgcgcaaagc aaagctgtta gcgaactctt 120

tttatgggta ttacgggtac gctaagggtg gctctagtca tcatcaccat caccattaac 180

tcgaggatcc ggctgctaa 199

<210> 33

<211> 51

<212> PRT

<213> Cherry50

<400> 33

Met Val Gly Gln Pro Leu Trp Asp Val Ser Arg Ser Ser Thr Gly Asn

1 5 10 15

Leu Val Glu Trp Phe Leu Leu Arg Lys Ala Lys Leu Leu Ala Asn Ser

20 25 30

Phe Tyr Gly Tyr Tyr Gly Tyr Ala Lys Gly Gly Ser Ser His His His

35 40 45

His His His

50

<210> 34

<211> 286

<212> DNA

<213> Cherry80DNASEQ

<400> 34

ctcaccgcga acagattgga ggcgtgaagc accccgccga catccccgac tacttgaagc 60

tgtccttccc cgagggcttc aagtgggagc gcgtgatgaa cttcgaggac ggcggcgtgg 120

tgaccgtgac ccaggactcc tccctgcagg acggcgagtt catctacaag gtgaagctgc 180

gcggcaccaa cttcccctcc gacggccccg taatgcagaa gaagaccatg ggcggtggct 240

ctagtcatca tcaccatcac cattaactcg aggatccggc tgctaa 286

<210> 35

<211> 80

<212> PRT

<213> Cherry80

<400> 35

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

1 5 10 15

Glu Gly Phe Lys Trp Glu Arg Val Met Asn Phe Glu Asp Gly Gly Val

20 25 30

Val Thr Val Thr Gln Asp Ser Ser Leu Gln Asp Gly Glu Phe Ile Tyr

35 40 45

Lys Val Lys Leu Arg Gly Thr Asn Phe Pro Ser Asp Gly Pro Val Met

50 55 60

Gln Lys Lys Thr Met Gly Gly Gly Ser Ser His His His His His His

65 70 75 80

Claims (10)

1. A method of polypeptide expression comprising the steps of:

fusing EDDIE (C69E, C168E) -Sumo combined label with the target polypeptide to express the target polypeptide;

wherein the EDDIE (C69E, C168E) -Sumo combined tag comprises an amino acid sequence shown in SEQ ID NO. 1.

2. A method of producing a polypeptide comprising the steps of:

carrying out fusion expression on the EDDIE (C69E, C168E) -Sumo combined label and the target polypeptide to obtain a fusion protein; renaturing the fusion protein; cutting the Sumo label in the renatured fusion protein by using specific protease which is adaptive to the combined label to obtain the target polypeptide;

wherein the EDDIE (C69E, C168E) -Sumo combined tag comprises an amino acid sequence shown in SEQ ID NO. 1.

3. The method according to claim 1 or 2, wherein the target polypeptide is any one of an antibacterial peptide, a linear peptide and a toxic peptide.

4. The method of claim 2, wherein the buffer used in the renaturation process is 1-1.5M Tris-HCl, pH 7-8, 2.5-10% glycerol, 1-10mM tris (2-carboxyethyl) phosphine hydrochloride.

5. The method of claim 2, wherein the specific protease to which the combination tag is adapted is SUMO protease; the Sumo label method in the fusion protein after excision renaturation is to add SUMO protease into renaturation solution with the concentration of 0.8-1.2mg renaturation fusion protein/mL.

6. The method of claim 2, comprising the steps of:

carrying out fusion expression on the EDDIE (C69E, C168E) -Sumo combined label and the target polypeptide to obtain a fusion protein; renaturing the fusion protein; removing the Sumo tag in the renatured fusion protein by using specific protease adapted to the combined tag, and purifying to obtain the target polypeptide;

wherein the purification is a purification by using a C8 reverse phase column; the parameters of the C8 reversed phase column are carbon content: 9%, specific surface area: 280m²The particle diameter per gram: average pore diameter of 40-75 μm:

7. an EDDIE (C69E, C168E) -Sumo combined tag is characterized by comprising an amino acid sequence shown as SEQ ID NO. 1.

8. A gene encoding the EDDIE (C69E, C168E) -Sumo combination tag of claim 7, comprising the nucleotide sequence shown in SEQ ID No. 2.

9. A protein expression vector comprising the EDDIE (C69E, C168E) -Sumo combination tag of claim 7.

10. A kit comprising the EDDIE (C69E, C168E) -Sumo combination tag of claim 6, or the gene encoding the EDDIE (C69E, C168E) -Sumo combination tag of claim 7, or the protein expression vector of claim 8.

Images