CN117801125A - Fusion protein of exenatide precursor and application thereof - Google Patents

Abstract

The invention provides fusion protein of exenatide precursor and application thereof. Wherein the fusion protein comprises an exenatide precursor and a tag protein fused at the N end of the exenatide precursor; the exenatide precursor has an amino acid sequence shown as SEQ ID NO. 1; the tag protein is selected from any one of the following: fh8 tag protein, ffu209 tag protein, CBM tag protein, sumo tag protein, or Trx tag protein. The exenatide precursor is fused with the tag protein, has higher expression quantity, can be prepared into a large quantity of high-purity exenatide precursors, and can better perform industrial production and application of the exenatide.

Description

Fusion protein of exenatide precursor and application thereof

Technical Field

The invention relates to the technical field of protein expression, in particular to fusion protein of exenatide precursor and application thereof.

Background

Currently exenatide precursors are mainly obtained by chemical coupling synthesis and recombinant expression. The chemical coupling synthesis process is relatively complex, impurities are easy to generate in the synthesis process, chemical reagents are toxic, racemates can be possibly generated, and the purity and the recovery rate are relatively low; the synthesis period is longer, and the quality control of the product is more difficult. The recombinant expression adopts the technical means of genetic engineering to introduce a target gene expression element into a prokaryotic or eukaryotic organism, and can realize the efficient synthesis of target polypeptide through microbial fermentation, comprising two types of inclusion body expression and soluble expression, wherein a large amount of denaturants such as urea or guanidine hydrochloride are required in the denaturation and renaturation processes of the inclusion body expression, and the purification process is complex, so that the final yield is very low. The solubility expression has a certain influence on the solubility of the obtained fusion protein and the yield of the target peptide due to the different types of the introduced tag protein and the cleavage site connecting the tag protein and the target peptide.

Soluble expression as in patent CN 103911388B by fusing exenatide precursor with GB1 tag; introducing enterokinase cleavage site, and cutting by enterokinase to obtain exenatide precursor with fermentation yield of 500 mg/L. The patent CN 104894196A takes hirudin with smaller molecular weight as a fusion partner, the target polypeptide exenatide precursor or the derivative thereof is spliced at the downstream of the hirudin fusion partner for fusion expression, and TEV enzyme cutting sites are introduced, and the ratio of the small molecular target polypeptide in the fusion protein is effectively increased by fusing fusion tags with smaller molecular weight. However, the former has been to select a relatively smaller tag in order to pursue a higher exenatide precursor ratio, resulting in complicated purification process and use of expensive IgG packing, requiring ion exchange chromatography-gel filtration chromatography-sandwich chromatography (affinity chromatography, using expensive IgG packing-enzyme cleavage-affinity chromatography), and enterokinase having some non-specific drawbacks. The latter uses TEV protease for cleavage, but the recognition sequence of the enzyme is ENLYFQG (SEQ ID NO: 15)/ENLYFQGS (SEQ ID NO: 16), the patent uses ENLYFQH (SEQ ID NO: 17) as the recognition sequence, the optimal recognition sequence of the enzyme is not the optimal recognition sequence, the problem of the lower cleavage efficiency is caused, hirudin is a polypeptide drug, and if the purification effect is poor, the risk of polluting other drug polypeptides is caused.

Disclosure of Invention

The invention mainly aims to provide fusion protein of exenatide precursor and application thereof, so as to solve the problem of low efficiency of preparation of exenatide precursor in the prior art.

In order to achieve the above object, according to a first aspect of the present invention, there is provided a fusion protein of exenatide precursor, the fusion protein comprising exenatide precursor and a tag protein fused to the N-terminus of the exenatide precursor; the exenatide precursor has an amino acid sequence shown as SEQ ID NO. 1; the tag protein is selected from any one of the following: fh8 tag protein, ffu209 tag protein, CBM tag protein, sumo tag protein, or Trx tag protein.

Further, when the tag protein is selected from any one of the following, the exenatide precursor and the tag protein are linked by a linking peptide: fh8 tag protein, ffu209 tag protein, CBM tag protein or Trx tag protein; the connecting peptide is selected from (GGGS) nKR, and n is selected from any integer from 1 to 5.

Further, the above-mentioned connecting peptide is GGGSGGGSGGGSKR.

Further, the fusion protein has an amino acid sequence shown in SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 7, SEQ ID NO. 8 or SEQ ID NO. 9.

In order to achieve the above object, according to a second aspect of the present invention, there is provided a method for preparing exenatide precursor, comprising: preparing and obtaining the fusion protein; and cutting off the tag protein in the fusion protein by utilizing endoprotease to obtain the exenatide precursor.

Further, the endoprotease is selected from the group consisting of a Ulp1 enzyme and a KEX2 enzyme; the mass ratio of the Ulp1 enzyme to the fusion protein is 1:20-100; the mass ratio of KEX2 enzyme to fusion protein is 1:40-500.

Further, the method comprises the steps of utilizing endoprotease to cut off tag protein in the fusion protein, and further purifying a product after endoprotease treatment to obtain exenatide precursor; the purification comprises the following steps: regulating the pH value of the enzyme digestion system to the isoelectric point of the tag protein, adding an organic reagent, and centrifuging to obtain a supernatant, namely the exenatide precursor.

In order to achieve the above object, according to a third aspect of the present invention, there is provided a DNA molecule encoding the above fusion protein.

Further, the nucleotide sequence of the DNA molecule is a nucleotide sequence after codon optimization.

Further, the DNA molecule has a nucleotide sequence shown in SEQ ID NO. 4 or 5.

In order to achieve the above object, according to a fourth aspect of the present invention, there is provided a recombinant plasmid having the above DNA molecule attached thereto.

In order to achieve the above object, according to a fifth aspect of the present invention, there is provided a host cell into which the above recombinant plasmid is transformed.

Further, the host cell is a prokaryotic cell or a yeast cell.

In order to achieve the above object, according to a sixth aspect of the present invention, there is provided the use of the fusion protein described above or the exenatide precursor prepared by the method described above for preparing exenatide.

By applying the technical scheme of the invention, the exenatide precursor is fused with Sumo or Trx tag protein, and the exenatide precursor with the tag protein has higher expression quantity, can be prepared into a large amount of high-purity exenatide precursor, and can better perform industrial production and application of exenatide.

Drawings

The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the invention. In the drawings:

FIG. 1 shows an electrophoresis pattern of fusion expression of Fh8-exe, ffu209-exe, CBM-exe in E.coli in example 1 of the present invention;

FIG. 2 shows an electrophoresis pattern of fusion expression of Trx-exe in E.coli in example 1 of the present invention;

FIG. 3 shows an electrophoretogram of fusion expression of Sumo-exe in E.coli in example 1 of the present invention;

FIG. 4 shows an electrophoresis pattern of the fusion protein Trx-exe obtained by purification after expression in E.coli in example 2 of the present invention;

FIG. 5 shows an electrophoresis pattern of fusion proteins Sumo-exe obtained by purification after expression in E.coli in example 2 of the present invention;

FIG. 6 shows an electrophoretogram of exenatide precursor obtained by purification of the digested fusion protein Trx-exe of example 4 by acetonitrile precipitation;

FIG. 7 shows an electrophoretogram of exenatide precursor obtained by purification of the digested fusion protein Sumo-exe of example 4 by acetonitrile precipitation;

FIG. 8 shows a schematic mass spectrometry detection of exenatide precursor after cleavage and purification of the fusion protein of example 5 of the present invention.

Detailed Description

It should be noted that, in the case of no conflict, the embodiments and features in the embodiments may be combined with each other. The invention will be described in detail below with reference to the drawings in connection with embodiments.

As mentioned in the background art, the existing method for synthesizing exenatide precursor cannot efficiently synthesize a large amount of exenatide precursor products, and has the problems of complex purification operation, more impurities, higher cost and lower cleavage specificity. The technical scheme of simple purification technology and high exenatide precursor yield is provided, a fusion tag and a KEX2 enzyme recognition site KR with high specificity and a Sumo tag tertiary structure with high specificity are utilized to separate the fusion tag from a target polypeptide, and the characteristic that the polypeptide can be dissolved by isoelectric point difference of the polypeptide and easy precipitation of the tag protein in an organic solvent is utilized, so that the technical scheme of efficiently carrying out soluble expression of the exenatide precursor is established.

In a first exemplary embodiment of the present invention, there is provided a fusion protein of exenatide, the fusion protein comprising an exenatide precursor and a tag protein fused to the N-terminus of the exenatide precursor; the exenatide precursor has an amino acid sequence shown as SEQ ID NO. 1; the tag protein is selected from any one of the following: fh8 tag protein, ffu209 tag protein, CBM tag protein, sumo tag protein, or Trx tag protein.

SEQ ID NO. 1: (amino acid sequence of exenatide precursor)

HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS。

The exenatide precursor with the amino acid sequence is fused with tag proteins Sumo and Trx with smaller molecular weight, and the tag proteins are fused at the N section of the exenatide precursor, so that the fusion protein can be efficiently and solublely expressed in host cells, and a foundation is provided for obtaining a large amount of exenatide for subsequent treatment.

In order to further allow sufficient spatial distance between the two molecules to be fused to maintain their respective spatial configurations and their biological activities, a reasonable selection from existing linker peptides may be made as needed to link the two molecules to be fused, in a preferred embodiment, when the tag protein is selected from the group consisting ofIn any of the following cases, the exenatide precursor and the tag protein are linked by a linking peptide: fh8 tag protein, ffu209 tag protein, CBM tag protein or Trx tag protein; preferably, the linking peptide is selected from (GGGS) _n KR, n is selected from any integer from 1 to 5, preferably n is 3; preferably, the connecting peptide is GGGSGGGSGGGSKR (SEQ ID NO: 12).

When n=1, the connecting peptide is SEQ ID No. 10: GGGSKR; when n=2, the connecting peptide is SEQ ID No. 11: GGGSGGGSKR; when n=3, the connecting peptide is SEQ ID No. 12: GGGSGGGSGGGSKR; when n=4, the connecting peptide is SEQ ID No. 13: GGGSGGGSGGGSGGGSKR; when n=5, the connecting peptide is SEQ ID No. 14: GGGSGGGSGGGSGGGSGGGSKR.

In a preferred embodiment, the fusion protein has the amino acid sequence shown in SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 7, SEQ ID NO. 8 or SEQ ID NO. 9. The fusion protein is an exenatide precursor with N segments fused with tag proteins, the underlined part in the following amino acid sequences is the sequence of the tag proteins, and the amino acid sequences at the 116 th to 129 th positions in SEQ ID NO. 3, the amino acid sequences at the 76 th to 89 th positions in SEQ ID NO. 7, the amino acid sequences at the 164 th to 177 th positions in SEQ ID NO. 8 and the amino acid sequences at the 170 th to 184 th positions in SEQ ID NO. 9 are connecting peptide sequences.

SEQ ID NO. 2: (amino acid sequence of Sumo-exenatide precursor)

MHHHHHHGSLQDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPLRRLMEAFAKRQGKE MDSLRFLYDGIRIQADQAPEDLDMEDNDIIEAHREQIGGHGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS。

SEQ ID NO:3：（Trx-(G ₃ S) ₃ -amino acid sequence of KR-exenatide precursor

MHHHHHHSDKIIHLTDDSFDTDVLKADGAILVDFWAEWCGPCKMIAPILDEIADEYQGKLTVAKLNID QNPGTAPKYGIRGIPTLLLFKNGEVAATKVGALSKGQLKEFLDANLAGGGSGGGSGGGSKRHGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS。

SEQ ID NO. 7: (amino acid sequence of Fh 8-exenatide precursor)

MHHHHHHPSVQEVEKLLHVLDRNGDGKVSAEELKAFADDSKCPLDSNKIKAFIKEHDKNKDGKLDLKE LVSILSSGGGSGGGSGGGSKRHGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS。

SEQ ID NO. 8: (amino acid sequence of Ffu 209-exenatide precursor)

MHHHHHHATEPVPGFPQPTEHTQKAYSPTDDFTSRWTRADAKQIKAMSDPNAGSRENSMPKEYTMPTV PQDFPDMSNEEVWVWDTWPLTDEHANQYSVNGQEIIFSLVADRDLGFDERHQYARIGYFYRPAGVPADERPEDGGW TYGGQVFDEGVTGKIFEDQGGGSGGGSGGGSKRHGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS。

SEQ ID NO. 9: (amino acid sequence of CBM-exenatide precursor)

MHHHHHHTTPFMSNMTGWTTVNGTWADTIEGKQGRSDGDSFILSSASGSDFTYESDITIKDGNGRGAG ALMFRSDKDAKNGYLANVDAKHDLVKFFKFENGAASVIAEYKTPIDVNKKYHLKTEAEGDRFKIYLDDRLVIDAHD SVFSEGQFGLNVWDATAVFQNVTKESGGGSGGGSGGGSKRHGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS。

In a second exemplary embodiment of the present invention, there is provided a method for preparing exenatide precursor, comprising: preparing and obtaining the fusion protein; and cutting off the tag protein in the fusion protein by utilizing endoprotease to obtain the exenatide precursor. The fusion protein can be expressed in a host cell, preferably escherichia coli, in a large quantity to obtain the exenatide precursor with the tag protein, the tag protein is required to be excised for obtaining the purified exenatide precursor, and the exenatide precursor can be obtained after the exenatide precursor is treated by endoprotease capable of specifically recognizing the specific tag protein, so that the purpose of producing the exenatide precursor in a large quantity and high efficiency is achieved.

Wherein, the method for preparing and obtaining the fusion protein comprises the following steps: and carrying out homologous recombination connection on the nucleotide sequence of the exenatide precursor with the tag protein subjected to codon optimization and a carrier capable of expressing the protein, and carrying out positive screening to obtain the recombinant plasmid with normal sequencing. Transferring the correct plasmid into a proper host cell for pre-screening, and selecting an expression strain with the optimal expression amount for subsequent protein purification culture. And (3) carrying out protein induction expression by using an expression strain with the optimal expression quantity, carrying out centrifugation bacterial collection on the culture bacterial liquid, carrying out crushing after re-suspension, and carrying out step-by-step elution and purification on the supernatant to obtain the fusion protein.

The types of endoproteases required for the different tag proteins are also different, in a preferred embodiment the endoprotease is selected from the group consisting of a Ulp1 enzyme or a KEX2 enzyme, wherein the Ulp1 enzyme is capable of specifically recognizing the tertiary structure of the Sumo tag, thereby completely cleaving the Sumo tag protein from the target protein at the junction GH of the Sumo tag with the target protein; KEX2 recognizes the KR site of the linker peptide and completely cleaves the Trx tag protein and the linker peptide from the target protein. To further excise the tag protein as clean as possible, in a preferred embodiment the mass ratio of Ulp1 enzyme to fusion protein is 1:20-100, including 1:20, 1:40, 1:50, 1:60, 1:80, 1:100; the mass ratio of KEX2 enzyme to fusion protein is 1:40-500, including 1:40, 1:80, 1:100, 1:120, 1:200, 1:300, 1:400, 1:500; preferably, the digestion time is 16-18 hours. Preferably, the mass ratio of Ulp1 enzyme to exenatide precursor fusion protein is 1:40, and the mass ratio of KEX2 enzyme to exenatide precursor fusion protein is 1:200, preferably, the enzyme digestion time is 16h.

In order to further obtain exenatide precursor with higher purity, the product after endoprotease treatment is subjected to protein purification treatment, and in a preferred embodiment, after the label protein in the fusion protein is excised by endoprotease, the product after endoprotease treatment is further purified to obtain exenatide precursor.

Various methods of performing protein purification, including isoelectric point precipitation, salting out, column chromatography, membrane filtration, and the like, are suitable for use in the present application, any method of purifying a protein capable of further separating exenatide precursor from impurities, and in a preferred embodiment the purification comprises: regulating the pH value of the enzyme digestion system to the isoelectric point of the tag protein, adding an organic reagent, and centrifuging to obtain a supernatant, namely the exenatide precursor. Preferably, the organic reagent comprises methanol or acetonitrile. Wherein His-Sumo has an isoelectric point of 5.6. And centrifuging the labeled protein impurities separated out by isoelectric points, removing the impurities by precipitation, and finally obtaining a purified target product exenatide precursor in supernatant. Wherein, the exenatide precursor has smaller molecular weight, is more stable than protein, has good solubility in organic solvent and is not easy to be precipitated.

In a third exemplary embodiment of the invention, a DNA molecule is provided which encodes the fusion protein described above.

In the translation process, each host cell for protein expression has a certain preference for codons, and in order to obtain a protein product with higher expression level in a specific host cell, the nucleotide sequence for encoding the protein can be codon optimized for the specific host cell type. In a preferred embodiment, the nucleotide sequence of the DNA molecule is a codon optimized nucleotide sequence; preferably, the DNA molecule has the nucleotide sequence shown in SEQ ID NO. 4 or 5. The nucleotide sequence of the exenatide precursor with Sumo or Trx tag protein is subjected to codon optimization treatment with escherichia coli preference, wherein the underlined part in the following nucleotide sequence is the sequence of the tag protein, and the nucleotide sequence from 346 th to 387 th positions in SEQ ID NO. 5 is a connecting peptide sequence.

SEQ ID NO. 4: (nucleotide sequence of Sumo-exenatide precursor)

ATGCATCATCATCATCATCATGGCAGTCTGCAAGATAGCGAAGTGAATCAAGAAGCGAAGCCAGAAGT GAAACCGGAAGTTAAACCGGAGACCCACATCAATCTGAAGGTGAGCGACGGCAGCAGCGAGATCTTCTTCAAGATC AAGAAGACGACCCCGCTGCGTCGTCTGATGGAAGCCTTCGCCAAACGCCAAGGCAAAGAAATGGACAGTCTGCGCT TTCTGTACGATGGTATCCGCATCCAAGCCGATCAAGCCCCGGAAGATCTGGACATGGAGGACAACGACATCATCGA GGCGCATCGCGAACAGATCGGCGGCCATGGCGAAGGCACCTTTACCAGCGATCTGAGCAAACAGATGGAAGAAGAAGCGGTGCGCCTGTTTATTGAATGGCTGAAAAACGGCGGCCCGAGCAGCGGCGCGCCGCCGCCGAGCTAA。

SEQ ID NO. 5: (nucleotide sequence of Trx- (G3S) 3-KR-exenatide precursor)

ATGCATCATCATCATCATCATAGCGATAAAATTATTCACCTGACTGACGACAGTTTTGACACGGATGT ACTCAAAGCGGACGGGGCGATCCTCGTCGATTTCTGGGCAGAGTGGTGCGGTCCGTGCAAAATGATCGCCCCGATT CTGGATGAAATCGCTGACGAATATCAGGGCAAACTGACCGTTGCAAAACTGAACATCGATCAAAACCCTGGCACTG CGCCGAAATATGGCATCCGTGGTATCCCGACTCTGCTGCTGTTCAAAAACGGTGAAGTGGCGGCAACCAAAGTGGG TGCACTGTCTAAAGGTCAGTTGAAAGAGTTCCTCGACGCTAACCTGGCCGGTGGCGGTTCTGGCGGTGGCAGCGGCGGTGGCAGCAAACGTCATGGCGAAGGCACCTTTACCAGCGATCTGAGCAAACAGATGGAAGAAGAAGCGGTGCGCCTGTTTATTGAATGGCTGAAAAACGGCGGCCCGAGCAGCGGCGCGCCGCCGCCGAGCTAA。

In a fourth exemplary embodiment of the present invention, a recombinant plasmid is provided, to which the above-described DNA molecule is attached.

In a fifth exemplary embodiment of the present invention, a host cell transformed with the recombinant plasmid described above is provided. The host cell is a prokaryotic cell or a yeast cell, preferably E.coli.

By using the host cell, the recombinant plasmid can be replicated in the host cell, and DNA molecules carried on the recombinant plasmid can be transcribed and translated to obtain a large number of fusion proteins. By using the prior art, the host cells can be subjected to broken protein purification or other modes, so that fusion proteins can be obtained, and the subsequent preparation of exenatide by using exenatide precursors is facilitated.

In a sixth exemplary embodiment of the present invention, there is provided the use of the fusion protein described above or the exenatide precursor prepared by the method described above for preparing exenatide.

The present application is described in further detail below in conjunction with specific embodiments, which should not be construed as limiting the scope of the claims.

EXAMPLE 1 construction of pET-28a (+) expression vector

The nucleotide sequence of the E.coli-favored codon-optimized exenatide precursor was synthesized total-genetically from Jin Weizhi as follows: SEQ ID NO. 6: CATGGCGAAGGCACCTTTACCAGCGATCTGAGCAAACAGATGGAAGAAGAAGCGGTGCGCCTGTTTATTGAATGGCTGAAAAACGGCGGCCCGAGCAGCGGCGCGCCGCCGCCGAGCTAA.

The synthesized DNA fragments are respectively connected with pET-28a-Trx, pET-28a-Sumo, pET-28a-Fh8, pET-28a-Ffu209 and pET-28a-CBM vector frameworks through homologous recombination, BL21 (DE 3) competent cells are transformed by the connection products, a correct clone expression vector pET-28a-Tag-exe (exe is an abbreviation of Exenatide precursor) is obtained through monoclonal sequencing analysis, 3 correct clone activation is selected and then used as seeds to be inoculated in a 250 ml shake flask for pre-screening, and one optimal expression strain is selected. The tag proteins Fh8, ffu209, CBM and tag proteins Sumo and Trx all belong to tag proteins with smaller molecular weights (7.5-18 kDa), wherein Fh8 is a protein secreted by fasciola hepatica in early infection, the molecular weight is 7.5kDa, ffu209 is a protein derived from Gluconobacter salina, the molecular weight is 17.75kDa, CBM is a domain of beta-fructosidase, the molecular weight is 17.96kDa, sumo (a small molecule ubiquitin-like modified protein, the molecular weight is 11.4 kDa) and Trx (thioredoxin, the molecular weight is 11.7 kDa) are smaller and the soluble expression amount of the fusion protein is high.

BL21 (DE 3) strain 4 mL containing recombinant plasmid was inoculated into a 2L triangular flask containing 400 mL of LB medium, and when the culture was carried out at 37℃and 200 rpm with shaking until the OD600 became 1.0, IPTG was added at a final concentration of 0.2 mM, the induction was completed overnight at 25℃and the cells were collected by centrifugation. By ultrasonication, collecting the supernatant, SDS-PAGE detection of 17% separating gel, SDS-PAGE result graphs of fusion proteins with Fh8 tag, ffu209 tag, CBM tag, sumo tag and Trx tag are shown in FIGS. 1-3, it can be seen that the fusion proteins with CBM tag are mainly expressed as inclusion bodies in the pellet, the other fusion proteins are all expressed in a soluble state, and the expression amount of the fusion proteins with Fh8 tag and Ffu209 tag is significantly lower than that of the fusion proteins with Sumo tag (shown in FIG. 3) and Trx tag (shown in FIG. 2).

EXAMPLE 2 Sumo-exe and Trx-exe purification

Bacterial sludge expressing Fh8-exe, ffu209-exe, CBM-exe, sumo-exe and Trx-exe was resuspended in 20% bacterial concentrate, sonicated (5 s sonicated, 5 s intervals, 30% power), centrifuged, and the supernatant was filtered through a 0.45 μm filter to obtain a crude enzyme solution, which was then purified using affinity chromatography (AKTA System was equipped with 5 ml HisTrap HP). The specific flow is as follows: the filter membrane filtration samples were loaded at a flow rate of 5 ml/min, then washed with binding buffer (50 mM Tris-HCl,200 mM NaCl,pH 8.0) until unbound protein was completely eluted, then eluted with buffer 50 mM Tris-HCl,200 mM NaCl,50 mM imidazole, pH 8.0 for 4 column volumes of the hybrid protein, and finally eluted with elution buffer 50 mM Tris-HCl,200 mM NaCl,300 mM imidazole, pH 8.0 for the target protein. 1 g wet cells can obtain purified Sumo-exe fusion protein 13.7 mg (shown in FIG. 5), trx-exe fusion protein 15.5mg (shown in FIG. 4), and the expression level of the protein is improved greatly compared with Fh 8-lixix, ffu 209-lixix and CBM-lixix.

EXAMPLE 3 cleavage

The Ulp1 cleavage reaction is carried out at 30 ℃, and the specific process is as follows: 50 mM Tris-HCl,200 mM NaCl,300 mM imidazole, pH 8.0 purified Sumo-exe and Ulp1 with a mass ratio of 40:1 (mg/mg), enzyme digestion for 16h, SDS-PAGE detection cleavage efficiency can reach more than 90%. In addition, when the mass ratio of exenatide precursor fusion protein to be digested to Ulp1 is 100:1, the cleavage efficiency can reach 83%, and when the mass ratio is 20:1, the cleavage efficiency can reach 95%, but the cleavage efficiency cannot be further improved by increasing Ulp 1.

The KEX2 enzyme digestion reaction is carried out at 25 ℃, and the specific process is as follows: 50 mM Tris-HCl,200 mM NaCl,300 mM imidazole, pH 8.0 purified Trx-exe and KEX2 mass ratio 200:1 (mg/mg), and the cutting efficiency of SDS-PAGE detection can reach more than 90%. In addition, when the mass ratio of the exenatide precursor fusion protein to be digested to KEX2 is 50:1, the cleavage efficiency can reach more than 95%, but when the cleavage time exceeds 24 hours, non-specific digestion can be generated, and the mass ratio is 400:1, the enzyme cutting time is required to be prolonged to 48 hours, and the cutting efficiency can reach more than 95 percent.

EXAMPLE 4 purification of target polypeptide by acetonitrile precipitation

The product after completion of the cleavage in example 3 was adjusted to pH 5.6 (His-sumo isoelectric point) or 5.7 (His-Trx isoelectric point), then 60% acetonitrile was added to the reaction system, and after mixing, 2 h was subjected to shaking treatment at 30℃and after which the supernatant and precipitate were centrifuged at 12000 rpm, the purity of the purified product was checked by 17% SDS-PAGE, and the formation of the target polypeptide was checked (the purified product obtained by the cleavage product in example 3 is shown in FIGS. 6 to 7), and the purity of the treated exe purified product was shown in the following table:

EXAMPLE 5 Mass Spectrometry detection of polypeptide molecular weight

The molecular weight of the purified product obtained in example 4 was analyzed by LC-MS.

The method comprises the following steps: samples were first separated by HPLC column: agilent ZORBAX Edipse Plus C18,4.6 x 100mm, 3.5 μm, mobile phase a:0.1% trifluoroacetic acid, mobile phase B: 0.1% acetonitrile solution of trifluoroacetic acid, gradient elution mode: 0 min 10%B,9 min 95%B,12 min 100%B,12.1 min 10%B,15 min 10%B, column temperature 50 ℃, UV detector 210 nm, flow rate 0.3 ml/mi. The components separated by HPLC are subjected to Q exact HF combined quadrupole Orbitrap mass spectrometer, electrospray ion source (Dual AJS ESI) is adopted, positive ion mode detection is carried out, the sheath air flow rate is 35 arb, the auxiliary air flow rate is 8 arb, the spray voltage is 3800V, the temperature of an ion transmission tube is 320 ℃, the scanning range is 200-3000 m/z, mass spectrum data are processed by BioPharma Finder software, the theoretical molecular weight of exenatide precursor is 4187.6 Da, and the molecular weight of exenatide precursor analyzed by mass spectrum is 4186.9 (shown in figure 8).

In summary, it can be demonstrated that the exenatide precursor fused with Sumo or Trx tag protein in the present application, the product obtained after expression purification and subsequent cleavage of tag protein is consistent with the molecular weight of standard exenatide precursor, and efficient production of exenatide precursor can be performed by using the fusion protein in the present application.

From the above description, it can be seen that the above embodiments of the present invention achieve the following technical effects: according to the preparation method, through recombinant expression of Sumo or Trx tag proteins and exenatide precursors, a large number of fusion proteins which can be used for preparing the subsequent exenatide are obtained, and after enzyme digestion of the tag proteins and purification, purer exenatide precursors are obtained, so that efficient synthesis of the exenatide precursors is realized.

The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims (14)

1. A fusion protein of exenatide precursor, which is characterized by comprising exenatide precursor and a tag protein fused at the N end of the exenatide precursor;

the exenatide precursor has an amino acid sequence shown as SEQ ID NO. 1;

the tag protein is selected from any one of the following: fh8 tag protein, ffu209 tag protein, CBM tag protein, sumo tag protein, or Trx tag protein.

2. The fusion protein of claim 1, wherein when the tag protein is selected from any one of the following, the exenatide precursor and the tag protein are linked by a linking peptide: fh8 tag protein, ffu209 tag protein, CBM tag protein or Trx tag protein;

the connecting peptide is selected from (GGGS) _n KR and n are selected from any integer from 1 to 5.

3. The fusion protein of claim 2, wherein the linker peptide is GGGSGGGSGGGSKR.

4. The fusion protein of claim 1, wherein the fusion protein has the amino acid sequence shown as SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 7, SEQ ID NO. 8 or SEQ ID NO. 9.

5. A method of preparing an exenatide precursor, comprising:

preparing to obtain the fusion protein of any one of claims 1 to 4;

and cutting off the tag protein in the fusion protein by utilizing endoprotease to obtain the exenatide precursor.

6. The method according to claim 5, wherein the endoprotease is selected from the group consisting of a Ulp1 enzyme and a KEX2 enzyme;

the mass ratio of the Ulp1 enzyme to the fusion protein is 1:20-100;

the mass ratio of the KEX2 enzyme to the fusion protein is 1:40-500.

7. The method of claim 5, wherein said cleaving the tagged protein in said fusion protein with an endoprotease further comprises further purifying the endoprotease treated product to obtain said exenatide precursor;

the purification includes: regulating the pH value of the enzyme digestion system to the isoelectric point of the tag protein, adding an organic reagent, and centrifuging to obtain a supernatant, namely the exenatide precursor.

8. A DNA molecule encoding the fusion protein of any one of claims 1 to 4.

9. The DNA molecule of claim 8, wherein the nucleotide sequence of the DNA molecule is a codon optimized nucleotide sequence.

10. The DNA molecule of claim 9, wherein said DNA molecule has a nucleotide sequence as set forth in SEQ ID No. 4 or 5.

11. A recombinant plasmid, characterized in that it has linked thereto a DNA molecule according to any one of claims 8 to 10.

12. A host cell transformed with the recombinant plasmid of claim 11.

13. The host cell of claim 12, wherein the host cell is a prokaryotic cell or a yeast cell.

14. Use of the fusion protein of any one of claims 1-4 or the exenatide precursor prepared by the method of any one of claims 5-7 for preparing exenatide.