Recombinant engineering bacterium for efficiently expressing liraglutide precursor and application thereof
阅读说明：本技术 一种高效表达利拉鲁肽前体的重组工程菌及其应用 (Recombinant engineering bacterium for efficiently expressing liraglutide precursor and application thereof ) 是由 杨柏成 赵运星 于 2018-07-16 设计创作，主要内容包括：本发明提供了一种高效表达利拉鲁肽前体的重组工程菌及其应用。本发明在利拉鲁肽前体分子Arg34G LP-1(7-37)的N端设计信号肽和肠激酶酶切位点与之紧邻,C端连接终止密码子,三者构成目的基因,然后插入到表达载体两个酶切位点之间,构建表达利拉鲁肽前体的重组工程菌,将工程菌进行高密度发酵培养,以融合蛋白的包涵体形式表达,重组融合蛋白表达量高,重组融合蛋白约占菌体总蛋白的25％-35％,目的蛋白包涵体表达量达15-20g/L。包涵体杂蛋白含量低,利于分离纯化,纯化效率高,且稳定性好,极大地降低了生产成本,提高了生产效率,在糖尿病治疗药物制备领域具有良好的应用前景。(The invention provides a recombinant engineering bacterium for efficiently expressing liraglutide precursor and application thereof. The invention designs signal peptide and enterokinase enzyme cutting site adjacent to the enzyme cutting site of liraglutide precursor molecule Arg34G LP-1(7-37) at the N end, the C end is connected with a stop codon, the three form a target gene, then the target gene is inserted between two enzyme cutting sites of an expression vector, recombinant engineering bacteria for expressing liraglutide precursor is constructed, the engineering bacteria are subjected to high-density fermentation culture and expressed in the form of inclusion bodies of fusion protein, the expression amount of the recombinant fusion protein is high, the recombinant fusion protein accounts for about 25-35% of the total protein of the bacteria, and the expression amount of the inclusion bodies of the target protein reaches 15-20 g/L. The inclusion body has low content of foreign protein, is beneficial to separation and purification, has high purification efficiency and good stability, greatly reduces the production cost, improves the production efficiency and has good application prospect in the field of preparation of diabetes treatment drugs.)
1. A liraglutide precursor molecule comprising the structure of a-B-C, wherein a is a signal peptide, B is a linker peptide, and C is a sequence of Arg34GLP-1 (7-37); the signal peptide is selected from MalE, PhoA, OmpF and PelB or an amino acid sequence which is formed by replacing, deleting and/or adding one or more amino acid residues and has the same function with MalE, PhoA, OmpF and PelB; the connecting peptide is selected from enzyme cutting sites of enterokinase, thrombin, SUMO or Protease.
2. The liraglutide precursor molecule according to claim 1, wherein the signal peptide is optionally followed by a purification tag at its N-or C-or N-terminal first or second amino acid, preferably the tag is a His-tag, further preferably followed by a His-tag at the N-terminal second amino acid of the signal peptide;
the amino acid sequence of Arg34GLP-1(7-37) is shown in SEQ ID NO. 9.
3. The encoding gene of the liraglutide precursor molecule according to claim 1 or 2, wherein the nucleotide sequence is represented by SEQ ID No.10, SEQ ID No.11, SEQ ID No.12 or SEQ ID No. 13.
4. Biomaterial containing the encoding gene for a liraglutide precursor molecule according to claim 1 or 2, which is an expression vector, an expression cassette, a recombinant bacterium or a transgenic cell line.
5. Use of the liraglutide precursor molecule or the gene encoding the same as defined in claim 1 or 2 or the biomaterial as defined in claim 4 for the preparation of liraglutide.
6. The recombinant engineering bacterium is characterized by being constructed by the following method: connecting the encoding gene of the liraglutide precursor molecule of claim 1 or 2 to an expression vector, constructing to obtain a recombinant expression vector, and transforming the recombinant expression vector into escherichia coli.
7. The recombinant engineered bacterium of claim 6, wherein the expression vector is selected from the group consisting of a pET series vector, a pASK series vector, and a pBad series vector.
8. A microbial agent comprising the recombinant engineered bacterium according to claim 6 or 7.
9. Use of the recombinant engineered bacterium of claim 6 or 7 or the microbial inoculum of claim 8 in the preparation of liraglutide.
10. A method for preparing a liraglutide precursor, comprising the steps of:
(1) seed activation: selecting the recombinant engineering bacteria of claim 6 or 7 to inoculate a culture medium;
(2) liquid seed activation: selecting lawn from the culture medium in the step (1) to a liquid seed culture medium, and performing activated culture;
(3) fermentation culture: inoculating the liquid seeds obtained in the step (2) to a liquid fermentation culture medium, and culturing;
(4) collecting thalli, crushing and washing the inclusion body;
(5) and (3) denaturation and renaturation, and enzyme digestion and purification.
Technical Field
The invention relates to the technical field of genetic engineering, in particular to a recombinant engineering bacterium for efficiently expressing liraglutide and application thereof.
Background
In recent years, scientists develop a GLP-1 receptor agonist according to the physiological mechanism that glucagon-like peptide 1(GLP-1) has the functions of promoting insulin secretion and regulating blood sugar, and based on the pathological mechanism that a patient with
Liraglutide (trade name: victoza), an amidated long-acting GLP-1 analog developed by norathanol corporation for once daily subcutaneous injection, is structurally characterized by replacing Lys34 of GLP-1(7-37) with Arg, and connecting a derivative obtained by connecting a 16-carbon palmitic fatty acid (N-epsilon- (γ -Glu (N- α -hexadecanoyl)) to the side chain of Lys26, and has 97% homology with natural GLP-1 in terms of production process, liraglutide is currently produced by norathanol corporation using saccharomyces cerevisiae to express the precursor molecule of liraglutide, Arg34GLP-1(7-37), extracellularly, and after connecting the fatty acid side chain to Lys26, an excess amino acid is cleaved to obtain a molecule of liraglutide, which is further processed to prepare an injection solution and sold in china in an imported pharmaceutical form, the price is quite expensive and most patients are hard to bear.
At present, prokaryotic expression and eukaryotic expression systems can be adopted for the expression of exogenous genes, but the eukaryotic expression systems have some defects in the expression of target proteins, such as: (1) when yeast is used for expressing target protein, the problems of nonuniform degradation of product protein, incomplete processing of signal peptide, formation of multimer and the like can be caused. Moreover, when the saccharomyces cerevisiae expresses proteins, problems of protein cleavage, low protein secretion efficiency and the like occur. (2) Insect baculovirus expression systems also have certain drawbacks. For example, the heterologous protein is not continuously expressed as the polyhedrosis virus infecting the host dies. Each round of new protein synthesis requires re-infection of the host cell. In addition, although baculovirus expression systems are capable of post-translational modification of proteins of interest, there are limits to such modifications; although their glycosylation sites are the same as in mammalian cells, the properties of oligosaccharide chains differ and complex glycosyl side chains cannot be generated, probably because insect cells cannot process mature sugar chains into a similar form in mammalian cells. (3) The mammalian expression system has relatively high cost and complex technology when expressing exogenous genes, and has potential animal virus pollution in the expression process.
In the aspect of prokaryotic expression systems, an escherichia coli expression system is the most deeply researched and rapidly developed expression system, has clear genetic background and gene expression regulation and control mechanism, is frequently used for expressing polypeptide and protein due to various expression vectors and host strains, and is the currently preferred exogenous expression system. The escherichia coli expression system consists of an expression vector, an exogenous gene and host bacteria, wherein the expression vector is the core part of the expression vector, and the general commonalities are as follows: the copy number and expression quantity of the recombinant plasmid are high; the application range is wide; the expression product is easy to purify; has good stability in the thalli. At present, known and widely applied escherichia coli expression vectors are mainly divided into non-fusion expression vectors, secretory expression vectors, surface expression vectors and the like, and the problems of low expression quantity of recombinant foreign proteins, high downstream separation and processing difficulty and the like are solved to a great extent. Wherein the expression pattern of the fusion protein expression technology is as follows: prokaryotic promoter → SD sequence → initiation codon → prokaryotic structural gene fragment → target gene sequence → termination codon, is a relatively common expression vector, is particularly suitable for the expression of small molecular polypeptides and proteins, and can significantly improve the expression success rate and the expression quantity of recombinant proteins.
For the expression of exogenous gene by using prokaryotic system, most studies utilize the expression mode of fusion protein to fuse various signal peptide sequences onto target gene to form recombinant protein, and when the recombinant protein is expressed in escherichia coli, the signal peptide can secrete the target protein to the periplasm of cell and even outside the cell, for example, chinese patent application 201610753093.4. However, the expression mode of the protein generates little target protein, which is not beneficial to the subsequent industrial development demand. If the recombinant genetic engineering strain capable of efficiently expressing the liraglutide precursor can be obtained, the yield of the liraglutide precursor can be greatly improved, the production cost is reduced, and the price of the liraglutide medicament is reduced, so that the liraglutide is beneficial to the livelihood.
Disclosure of Invention
The invention aims to provide a recombinant engineering bacterium for efficiently expressing a liraglutide precursor.
The invention also aims to provide application of the recombinant engineering bacteria in preparation of liraglutide medicaments.
In order to achieve the purpose, the invention designs a signal peptide and a connecting peptide which are adjacent to the N end of a liraglutide precursor molecule Arg34GLP-1(7-37), the C end is connected with a stop codon to form a target gene, and then the target gene, the signal peptide and the connecting peptide are inserted between two enzyme cutting sites of an expression vector to construct a recombinant engineering bacterium for expressing the liraglutide precursor. The liraglutide precursor molecule has an amino acid sequence with an A-B-C structure, wherein A is a signal peptide, B is a connecting peptide, and C is a sequence of Arg34GLP-1 (7-37); the signal peptide is selected from MalE, PhoA, OmpF and PelB or an amino acid sequence which is formed by replacing, deleting and/or adding one or more amino acid residues and has the same function with MalE, PhoA, OmpF and PelB; the connecting peptide is selected from enzyme cutting sites of enterokinase, thrombin, SUMO or Protease.
The signal peptide is optionally added with a purification tag after the first or second amino acid at the N-terminal or C-terminal or N-terminal, preferably, the tag is a His tag, further preferably, the second amino acid at the N-terminal of the signal peptide is added with a His tag, the amino acid sequences are respectively shown in SEQ ID NO.1-4, and the nucleotide sequences are respectively shown in SEQ ID NO. 5-8.
The amino acid sequence of Arg34GLP-1(7-37) is shown in SEQ ID NO. 9.
The invention provides a gene for coding the liraglutide precursor molecule, wherein the nucleotide sequence of the liraglutide precursor molecule gene of which the signal peptide is selected from MalE, PhoA, OmpF and PelB is respectively shown as SEQ ID NO.10, SEQ ID NO.11, SEQ ID NO.12 or SEQ ID NO. 13.
Furthermore, the invention provides a biological material containing the gene, wherein the biological material is an expression vector, an expression cassette, a recombinant bacterium or a transgenic cell line.
The invention provides application of the liraglutide precursor molecule containing the signal peptide and the enterokinase sequence in preparation of liraglutide.
The invention provides application of a gene for encoding the liraglutide precursor molecule containing the signal peptide and the enterokinase sequence in preparation of liraglutide.
The invention provides an application of the biological material in preparation of liraglutide.
The invention provides a recombinant engineering bacterium for efficiently expressing liraglutide precursors, which is constructed by the following method: and (2) connecting genes (respectively shown as SEQ ID NO.10-13 or specific sequences thereof) for coding the liraglutide precursor molecules containing the signal peptide (respectively MalE, PhoA, OmpF and PelB) and the enterokinase sequence to an expression vector to construct a recombinant expression vector, and then transforming the recombinant expression vector into escherichia coli to obtain the recombinant engineering bacteria for efficiently expressing the liraglutide precursor. The amino acid sequences of the liraglutide precursor are respectively shown as SEQ ID NO.14, SEQ ID NO.15, SEQ ID NO.16 or SEQ ID NO. 17.
The expression vector is selected from pET series vectors, pASK series vectors or pBad series vectors. The pET series of vectors is preferred.
In the examples of the present invention, pET series vectors were selected as pET-30a (+) or pET-40b (+). One skilled in the art can select a plasmid containing a strong promoter as the expression vector plasmid. The recombinant expression vector carrying the gene can be transformed into cells or tissues by a conventional biological method such as Ti plasmid, Ri plasmid, plant virus vector, direct DNA transformation, microinjection, conductance, Agrobacterium mediation and the like.
In the examples of the present invention, the target gene and pET-30a (+), pET-40b (+) were digested simultaneously with restriction enzymes NdeI and HindIII, respectively, and the target fragments were recovered and ligated with T4DNA ligase to obtain expression plasmids. Transforming the recombinant protein into escherichia coli DH5 alpha for amplification, primarily screening correct recombinant engineering bacteria by using a colony PCR method, and sequencing, wherein the result shows that the target gene sequence is a sequence shown by SEQ ID NO.10-13 and encodes amino acid shown by SEQ ID NO. 14-17.
The microbial inoculum containing the recombinant engineering bacteria also belongs to the protection scope of the invention.
The invention provides application of the recombinant engineering bacteria or a microbial inoculum containing the recombinant engineering bacteria in preparation of liraglutide.
The invention provides a preparation method for preparing a liraglutide precursor, which comprises the following steps:
(1) seed activation: selecting the recombinant engineering bacteria of the invention to inoculate to a culture medium;
(2) liquid seed activation: selecting lawn from the culture medium in the step (1) to a liquid seed culture medium, and performing activated culture;
(3) fermentation culture: inoculating the liquid seeds obtained in the step (2) to a liquid fermentation culture medium, and culturing;
(4) collecting thalli, crushing and washing the inclusion body;
(5) and (3) denaturation and renaturation, and enzyme digestion and purification.
The invention utilizes an escherichia coli prokaryotic expression system, a signal peptide and an enterokinase enzyme cutting site are designed in front of the N end of a liraglutide precursor molecule Arg34GLP-1(7-37) and are adjacent to the enzyme cutting site, the C end is connected with a stop codon to form a construct, and then the construct is inserted between two enzyme cutting sites of an expression vector to express in an inclusion body form of fusion proteins MalE-Arg34GLP-1(7-37), PhoA-Arg34GLP-1(7-37), OmpF-Arg34GLP-1(7-37) or PelB-Arg34GLP-1(7-37), thereby realizing the high-efficiency expression of the recombinant fusion protein, wherein the recombinant fusion protein accounts for about 25-35% of the total protein of thalli, the expression quantity of the inclusion body of the target protein (total mass collected by the inclusion body/mass of the thalli multiplied by 100%) reaches 13.12-17.49%, the expression level of the target protein inclusion body (the expression level of the inclusion body is the total mass of the inclusion body collected/the total volume of the thallus collected) reaches 15-20 g/L.
And finally, cutting the fusion protein by using high-specificity enterokinase to obtain the liraglutide precursor molecule Arg34GLP-1(7-37) with the right size. The invention has the advantages of simple operation, short period for protein expression, low cost for production and the like, reduces the industrial production cost and can achieve the required purpose in a short time. Further, (1) a rapid and convenient separation and purification mode; (2) the protein stability is high; (3) effectively prevent degradation by intracellular proteases; (4) the purity of the target protein in inclusion bodies is high, even up to 90%, so that the subsequent processing steps can be reduced.
Drawings
FIGS. 1A-1D are recombinant expression vector maps constructed by using encoding genes of liraglutide precursor molecules of different signal peptides. FIG. 1A shows MalE signal peptide, vector pET-40B (+), FIG. 1B shows PhoA signal peptide, vector pET-40B (+), FIG. 1C shows OmpF signal peptide, vector pET-30a (+), FIG. 1D shows PelB signal peptide, and vector pET-30a (+).
FIG. 2 is an expression diagram of recombinant fusion proteins constructed by signal peptides other than MalE, PhoA, OmpF, PelB, in which lane 1: uninduced mycoprotein, lane 2: LamB, lane 3: lpp, lane 4: OmpA.
FIG. 3 is a SDS-PAGE purity detection spectrum of the cells, lane 1: marker, lane 2: MalE signal peptide, vector pET-40b (+), lane 3: PhoA signal peptide, vector pET-40b (+), lane 4: OmpF signal peptide, vector pET-30a (+), lane 5: PelB signal peptide, vector pET-30a (+).
FIG. 4 is a HPLC-detected renaturation liquid map, FIG. 4A shows MalE signal peptide, vector pET-40B (+), FIG. 4B shows PhoA signal peptide, vector pET-40B (+), FIG. 4C shows OmpF signal peptide, vector pET-30a (+), FIG. 4D shows PelB signal peptide, and vector pET-30a (+).
FIG. 5 is a SDS-PAGE profile of the enzyme digestion, lane 1: marker, lane 2: MalE signal peptide, vector pET-40b (+), lane 3: PhoA signal peptide, vector pET-40b (+), lane 4: OmpF signal peptide, vector pET-30a (+), lane 5: PelB signal peptide, vector pET-30a (+).
FIG. 6 is a map of the HPCL assay after purification, FIG. 6A: MalE signal peptide, vector pET-40B (+), FIG. 6B: PhoA signal peptide, vector pET-40B (+), FIG. 6C: OmpF signal peptide, vector pET-30a (+), FIG. 6D: PelB signal peptide, vector pET-30a (+).
Detailed Description
The following examples are intended to further illustrate the present invention but should not be construed as limiting the invention. Modifications or substitutions to methods, procedures, or conditions of the invention may be made without departing from the spirit and scope of the invention.
Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art.
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