CN114539425B - Method for improving biological expression of linear polypeptide - Google Patents

Abstract

The invention discloses a method for improving the biological expression of linear polypeptide, which fuses EDDIE (C69E, C168E) -Sumo joint label and target polypeptide to obtain fusion protein; renaturation of the fusion protein; and (3) removing the Sumo tag in the renaturated fusion protein by adopting a specific protease which is suitable for the combined tag, so that the biological expression of the linear polypeptide can be improved. The invention combines the advantages of EDDIE and SUMO protein, fuses target polypeptide at the C end by combining EDDIE (C69E, C E) and SUMO, cuts off the tag by SUMO protease, adopts a solid phase extraction small column C8 reverse phase column to purify enzyme cut product, and obtains the polypeptide, thereby establishing an escherichia coli high-efficiency expression system, further obtaining the target polypeptide with high expression and high purity, being mainly used for biosynthesis of linear polypeptide, and having the advantages of high yield, low cost and easy purification.

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 biological expression of linear polypeptides, in particular to a novel fusion expression tag and application thereof in promoting expression of heterologous polypeptides.

Background

Coli expression systems have higher expression levels, faster growth rates, and are suitable for continuous and high cell density culture methods, and thus, e.coli is the most popular recombinant protein production host in biotechnology to date. However, there are still some disadvantages to E.coli expression systems, one of which is the production of small proteins and polypeptides. Degradation occurs easily when smaller fragments of the polypeptide are expressed by direct expression, resulting in low yields, and are also unsuitable for expression of toxic or antimicrobial peptides that are detrimental to bacterial growth. In addition, many recombinant polypeptides are prone to misfolding in bacteria during expression due to excessive translational speed and the lack of homologous partners. To address this problem, a number of tags are fused to the target protein, some of which increase protein production 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 proteins or polypeptides on host cells, but the inclusion body protein can obtain correctly folded functional proteins through a complex renaturation process, which counteracts the high expression advantage of the inclusion body to a certain extent. In addition, removal of protein tags is critical for biomedical applications.

Disclosure of Invention

The invention aims to: the invention aims to solve the technical problem of providing a polypeptide expression method aiming at the defects in the prior art.

The invention also solves the technical problem of providing a method for preparing polypeptide.

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

The invention also solves the technical problem of providing a gene for encoding the above combined tag.

The invention also solves the technical problem of providing a protein expression vector.

The invention also solves the technical problem of providing a kit.

In order to solve the first technical problem, the invention discloses a method for expressing polypeptide, which comprises the following steps: EDDIE (C69E, C168E) -Sumo combined tag and target polypeptide fusion 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 an Npro mutant protein of Npro swine fever self-shearing protein, and Cys at 69 position is mutated into Glu and Cys at 168 position is mutated into Glu respectively. The invention is mainly characterized in that the purified protein of the natural Npro protein has a plurality of defects, compared with the purified protein of the natural Npro protein, the EDDIE of the mutant protein of the invention has increased solubility, faster renaturation and higher shearing efficiency, but the EDDIE still has a certain defect in self-shearing efficiency; and after mutating the cleavage active key sites 69 and 168 of EDDIE, it is made to have no self-cleavage ability, but remains to form inclusion body ability.

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 two protein tags of EDDIE (C69E, C E) and Sumo to establish a C-terminal fusion expression system, and simultaneously uses SUMO protease specificity to cleave to remove the tag, thereby obtaining target polypeptide, and finally enzyme digestion products are purified by a solid phase extraction method. The invention mutates 69 and 168 positions of the shearing active key site of EDDIE through the established C-terminal fusion expression system, so that the EDDIE does not have self-shearing capability, but remains to form inclusion body capability; the ability of the SUMO protein tag to promote proper folding of the protein is also utilized to make EDDIE less prone to precipitation during renaturation.

Specifically, the method comprises the steps of: fusion expression of EDDIE (C69E, C168E) -Sumo combined tag and target polypeptide to obtain fusion protein; renaturation of the fusion protein; and (3) adopting specific protease which is matched with the combined tag to cut off the Sumo tag in the renaturated fusion protein, 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 an antibacterial peptide, a linear peptide and a toxic peptide.

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 (Tcep hydrochloride), preferably 1M Tris-HCl, pH 7.8,5% glycerol, 2mM Tcep hydrochloride.

Wherein the renaturation is dialysis renaturation.

The specific protease suitable for the combined tag is SUMO protease, and the efficiency is high.

The Sumo label method in the fusion protein after excision and renaturation comprises the following steps: directly adding corresponding amount of SUMO protease into renaturation fusion protein with concentration of 0.8-1.2 mg/mL renaturation solution renaturation fusion protein; the amount of SUMO protease used is the amount of protein to be sheared x 0.007 ≡sumo protease concentration.

Wherein the cleavage is carried out at 25-35℃for 2.5-4.5h, preferably at 30℃for 3.5h.

Wherein the purification is C8 reversed phase column purification.

Wherein, the parameter of the C8 reverse phase column is carbon content: 9%, specific surface area: 280m ² Particle size/g: 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 in SEQ ID NO. 1.

In order to solve the fourth technical problem, the invention discloses a gene for encoding the EDDIE (C69E, C168E) -Sumo combined tag, which comprises a nucleotide sequence shown as 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 tag.

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

The beneficial effects are that: compared with the prior art, the invention has the following advantages:

the invention relates to a linear polypeptide expression and purification method, fusing polypeptide with inclusion body protein EDDIE (C69E, C168E) and SUMO protein label, the fusion polypeptide can be expressed and purified in the form of inclusion body in colibacillus, and the target polypeptide is cut off by SUMO protease after dialysis renaturation, finally the small peptide is purified by C8 solid phase extraction reversed phase column, and the purity is 80% -90% by HPLC chromatography analysis; the method can effectively solve the problems of difficult renaturation of inclusion bodies and removal of labels.

Drawings

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

FIG. 1 shows the renaturation cleavage of EDDIE (C69E) -Sumo, in contrast to the non-renatured EDDIE (C69E) -Sumo protein, and 1-4 are renatured EDDIE (C69E) -Sumo proteins, respectively.

FIG. 2 shows the renaturation cleavage of EDDIE (C168E) -Sumo, the comparison is that of unrepeated EDDIE (C168E) -Sumo protein, and 1-4 are renatured EDDIE (C168E) -Sumo proteins, respectively.

FIG. 3 is a diagram of the detection of vector-constructed RCR amplification electrophoresis.

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

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

FIG. 6 is a diagram of a final lyophilized polypeptide product.

Detailed Description

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

Example 1:

(1) Construction of EDDIE (C69E, C168E) -Sumo Combined tag with amino acid sequence shown in SEQ ID NO.1

The primers shown in SEQ ID NO.3-24 are used for synthesizing the gene sequence shown in SEQ ID NO.1, and the expected length of the sequence is about 885bp respectively. The gene synthesis is carried out for 2 rounds: first round PCR reaction System (50. Mu.L): 10. Mu.L of Phusion Buffer (5X), 1. Mu.L of dNTP Mix (10 mmol/L), 2. Mu.L of primer Mix (SEQ ID NO.3 and SEQ ID NO. 24), 0.5. Mu.L of Phusion enzyme (5U/. Mu.L), 36.5. Mu. L H ₂ O. The first round of PCR reaction procedure was 98℃for 30sec of pre-denaturation; 18 cycles: 98℃for 10sec;58 ℃,20sec;72 ℃ for 1.5min; and at 72℃for 5min. Second round PCR reaction System (50. Mu.L): 10 μL of Phusion Buffer (5×), 1 μL of dNTP mix (10 mmol/L), EDDIE (C69 EC 168E) -Sumo-1: 1. Mu.L (10. Mu. Mol/L), EDDIE (C69 EC 168E) -Sumo-22: 1. Mu.L (10. Mu. Mol/L), first round template 1. Mu.L, 0.5. Mu.L Phusion enzyme (5U/. Mu.L), 35.5. Mu. L H ₂ O. The second round of PCR reaction procedure was 98℃pre-denatured for 30sec;25 cycles: 98℃for 10sec;58 ℃,20sec;72 ℃ for 1.5min;72℃for 5min.

The PCR products were detected by 1.5% agarose gel electrophoresis and recovered by hanging column PCR product purification kit using silica gel membrane to adsorb DNA: 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 mixture was transferred to an adsorption column 8000 Xg and centrifuged for 30sec.3. The liquid in the collection tube was poured off, 500. Mu.L of Wash Solution was added to the adsorption column, and the mixture was centrifuged at 9000 Xg for 30sec to pour the liquid in the collection tube. 4. The step 3 is repeated once. 5. The empty adsorbent column and collection tube were placed in a centrifuge and centrifuged at 9000 Xg for 1min.6. 40 mu LELUTION Buffer was added to the center of the adsorption membrane, and the mixture was allowed to stand still at room temperature for l-2min and centrifuged at 9000 Xg for 1min. The solution Buffer was 2.5mM Tris-HCI, pH 8.5. And then connecting the purified PCR product with pET-15bvector subjected to NcoI/XhoI double digestion, converting the PCR product into competent cells of escherichia coli TOP10, screening for ampicillin resistance, picking positive transformants, extracting plasmids, sending the plasmids to the company for sequencing, and displaying the correct sequence according to the sequencing result, wherein the nucleotide sequence is shown as SEQ ID NO. 25.

Cys at position 69 in the combined tag is mutated to Glu, cys at position 168 is mutated to Glu, and the conditions of renaturation and shearing of EDDIE (C69E) -Sumo and EDDIE (C168E) -Sumo are respectively shown in FIG. 1 and FIG. 2, and the EDDIE (C69E) -Sumo and the 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 for electrophoresis after overnight at 4 ℃ and the contrast is dialysis untreated. It was demonstrated that no self-cleavage reaction occurred after mutations at positions 69 and 168, which played a key role in self-cleavage, both sites were mutated according to the present invention.

(2) Fusion expression of the target polypeptide by the combined tag vector and the target polypeptide to obtain fusion protein

Amplification of EDDIE (C69E, C168E) -SUMO co-tagged vector (FIG. 3, lanes 1-2) gave 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 about 6497bp. PCR reaction System (50. Mu.L): 10. Mu.L of Phusion Buffer (5X), 1. Mu.L of dNTP mix (10 mmol/L), 1. Mu.L of upstream primer (10. Mu. Mol/L), 1. Mu.L of downstream primer (10. Mu. Mol/L), 1. Mu.L of PET15b-EDDIE (C69E, C E) -Sumo template, 0.5. Mu.L of LPhusion enzyme (5U/. Mu.L), 35.5. Mu. L H ₂ O. The PCR reaction procedure was 98℃for 30sec of pre-denaturation; 25 cycles: 98℃for 10sec;58 ℃,20sec;72 ℃ for 1.5min;72℃for 5min.

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

Respectively carrying out enzyme-linked (20 min at 50 ℃) on the gene fragments of the target polypeptides 1-4 and a linearization tag carrier, wherein an enzyme-linked system is a 20 mu L system: gibson recombinase: 5. Mu.L of the polypeptide gene fragment of interest: 10ng, linearized tag vector: 50ng, H ₂ O: the mixture was filled to 20. Mu.L. After expression identification, sending the obtained product to a company for sequencing, wherein a sequencing result shows that the sequence is correct; successfully constructs recombinant fusion polypeptide vector.

Inducible expression of the target protein by the fusion polypeptide vector in E.coli, 1) activation: the fusion polypeptide vector was plated on LB plates (Amp+), and incubated overnight at 37 ℃. The next morning one clone was picked into a tube containing 2ml of LB solution (Amp+) and shaken at 37℃and 200rpm to an OD600 of about 0.6. 2) 2ml of the bacterial liquid was added to 200ml of LB at 37℃and shaken at 200rpm to an OD600 of about 0.6. 3) Induction of expression: IPTG was added to a final concentration of 1mM and shaking was continued for 3-4 h. 4) Cell collection: centrifugation (5000 rpm,5min,4 ℃). 5) Resuspension washing: using a washing buffer:50mM Tris-HCl,5mM EDTA,1%Triton X-100, pH 8.0. 6) Cells were sonicated. 7) Collecting inclusion body sediment: the supernatant was removed by centrifugation (12,000 rpm,15min,4 ℃). 8) Washing inclusion bodies: washing buffer:50mM Tris-HCl,5mM EDTA,1%Triton X-100, pH 8.0. 9) Inclusion body dissolution: the fusion protein solution was obtained by solubilization with denaturation buffer (50 mM Tris-HCl,150mM NaCl,5mM imidazole, 8M urea, pH 8.0).

(3) Renaturation of fusion protein

4 different renaturation buffers were prepared, respectively: buffer1:1M Tris-HCl, pH 7.8,5% glycerol, 2mM Tcep; buffer2:500mM NaCL,20mM Tris-HCl, pH 9.5,2mM EDTA,5% glycerol, 2mM Tcep; buffer3:0.2M Arg,500mM NaCL,20mM Tris-HCl, pH 9.0,2mM EDTA,5% glycerol, 2mM Tcep,0.01%Tween-20; buffer4:0.2M Arg,500mM NaCl,20mM Tris-HCl, pH 9.0,2mM EDTA,5% glycerol, 2mM Tcep,0.01%Tween-20, 800mM urea.

Dialyzing and renaturating the 4 renaturation buffers with the fusion proteins respectively: 1) The 2.5K dialysis bag is cut into small sections of about 10cm in appropriate length. 2) The dialysis bag was placed in boiling water and boiled for 10 minutes. 3) Taking out the dialysis bag, draining water, sealing one end by using a dialysis clamp, adding the fusion protein solution obtained in the step (2) into the dialysis bag, draining bubbles, and uniformly sealing two tail ends by using the dialysis clamp. 4) And (3) placing the dialysis bag filled with different proteins in 5L renaturation buffer solution for dialysis, and replacing the dialysis solution every 30min until urea is dialyzed cleanly to obtain the renaturated fusion protein.

Among them, the present invention test found that the fusion protein (inclusion body protein of fusion polypeptide) was not easily precipitated under the conditions of high Tris (1M Tris) and high urea (800 mM urea), but urea affected the cleavage efficiency of SUMO protease, whereas high Tris (1M) did not affect the cleavage of SUMO protease. Thus Buffer1 is preferred: 1M Tris-HCl, pH 7.8,5% glycerol, 2mM Tcep. Therefore, the problems that EDDIE has more hydrophobic amino acids, is easy to precipitate after urea is removed by dialysis and is not easy to renaturate can be effectively solved.

(4) Cleavage of the SUMO tag in the fusion protein using SUMO protease:

taking the renatured fusion protein in the step (3) as a substrate, adding SUMO protease, controlling the substrate concentration at 1mg renatured fusion protein/mL renaturation buffer solution, and reacting at 30 ℃ for 3.5h to obtain an enzyme digestion product; meanwhile, the corresponding proteins which were not renatured were used as negative controls, respectively (FIG. 4).

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

(5) And (3) enzyme digestion product purification: purification of polypeptides by hydrophobic interactions of the polypeptides using C8 reverse column purification

And (3) 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 is not combined with the whole outflow of the C8 column, the polypeptide can be combined with the C8 column, washing the end of the column with 3mL of 5% acetonitrile after no liquid flows out, automatically flowing the washing liquid by gravity completely, and eluting the polypeptide by using 2mL of 95% acetonitrile to obtain the purified target polypeptide.

The purity of the eluted polypeptide is higher by HPLC detection, and the purity of B147-1 is as follows: 93%, glp-1 purity was 83% and the cherry50 purity was: 91, the purity of the cherry80 is: 86% (FIG. 5), the characteristic after lyophilization was white powder (FIG. 6). Therefore, the method provided by the invention is convenient to operate, and the purified polypeptide has better purity, can remove salt and has high universality.

Wherein, the parameter of the C8 reverse phase column is carbon content: 9%, specific surface area: 280m ² Particle size/g: average pore diameter of 40-75 μm:

in summary, the invention combines the advantages of EDDIE and SUMO protein, fuses the target polypeptide at the C end by combining EDDIE (C69E, C E) and SUMO, cuts off the tag by SUMO protease, and purifies the enzyme-cut product by adopting a solid-phase extraction small column C8 reverse phase column to obtain the polypeptide, thereby establishing an escherichia coli high-efficiency expression system, further obtaining the target polypeptide with high expression and high purity, being mainly used for biosynthesis of linear polypeptide, having the advantages of high yield, low cost and easy purification, and being a novel method for producing the polypeptide. The method solves the problem that small peptides are easy to degrade in the biological expression process and meets the requirement of expressing toxic peptides or antibacterial peptides.

The invention provides a method for improving the biological expression of linear polypeptide, and a method for realizing the technical scheme, wherein the method and the way are a plurality of methods, the method and the way are only the preferred embodiments of the invention, and it should be pointed out that a plurality of improvements and modifications can be made by one of ordinary skill in the art without departing from the principle of the invention, and the improvements and the modifications are also considered as the protection scope of the invention. The components not explicitly described in this embodiment can be implemented by using the prior art.

Sequence listing

<110> Hunan Zhongcheng full peptide Biochemical technology Co., ltd

<120> a method for increasing the biological expression of a linear polypeptide

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His Asp Arg Gly Glu Asp Asp Ile Glu Thr Thr Leu Arg Asp Leu Pro

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Arg Lys Gly Asp Glu Arg Ser Gly Asn His Leu Gly Pro Val Ser Gly

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Ile Tyr Ile Lys Pro Gly Pro Val Tyr Tyr Gln Asp Tyr Thr Gly Pro

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Val Tyr His Arg Ala Pro Leu Glu Phe Phe Asp Glu Thr Gln Phe Glu

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Glu Thr Thr Lys Arg Ile Gly Arg Val Thr Gly Ser Asp Gly Lys Leu

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Tyr His Ile Tyr Val Glu Val Asp Gly Glu Ile Leu Leu Lys Gln Ala

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Lys Arg Gly Thr Pro Arg Thr Leu Lys Trp Thr Arg Asn Thr Thr Asn

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<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 (8)

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

fusing EDDIE (C69E, C168E) -Sumo joint tag with a target polypeptide to express the target polypeptide;

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

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

fusion expression of EDDIE (C69E, C168E) -Sumo combined tag and target polypeptide to obtain fusion protein; renaturation of the fusion protein; removing Sumo tags in the renatured fusion protein by adopting specific protease which is suitable for the combined tag, thus obtaining the target polypeptide;

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

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

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

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

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

fusion expression of EDDIE (C69E, C168E) -Sumo combined tag and target polypeptide to obtain fusion protein; renaturation of the fusion protein; removing Sumo tags in the renaturated fusion protein by adopting specific protease which is suitable for the combined tags, and purifying to obtain the target polypeptide;

wherein the purification is performed by C8 inversePurifying a phase column; the parameters of the C8 reverse phase column are carbon content: 9%, specific surface area: 280m ² /g, particle size: 40-75 μm, average pore size:

7. a gene encoding an EDDIE (C69E, C168E) -Sumo co-tag, characterized by the nucleotide sequence shown in SEQ ID No. 2; wherein the EDDIE (C69E, C168E) -Sumo combined tag is an amino acid sequence shown in SEQ ID NO. 1.

8. A kit comprising the gene encoding the EDDIE (C69E, C168E) -Sumo combination tag of claim 7.

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