CN114805610B - Recombinant genetic engineering bacterium for highly expressing insulin glargine precursor and construction method thereof - Google Patents
Recombinant genetic engineering bacterium for highly expressing insulin glargine precursor and construction method thereof Download PDFInfo
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- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
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- C07K14/62—Insulins
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
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- C07K—PEPTIDES
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- C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
- C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
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Abstract
The invention relates to the technical field of genetic engineering, in particular to a recombinant genetic engineering bacterium for efficiently expressing a insulin glargine precursor and a construction method thereof. The insulin glargine precursor is polypeptide obtained by sequentially connecting a leader peptide, an insulin glargine B chain and an insulin glargine A chain in series, wherein two arginines at the C end of the insulin glargine B chain are directly connected with the N end of the insulin glargine A chain, and the N end of the insulin glargine B chain is connected with the leader peptide. The design of the insulin glargine precursor simplifies the enzyme digestion step in the insulin glargine preparation process, and improves the enzyme digestion efficiency; meanwhile, the expression quantity of the recombinant strain in the recombinant gene engineering bacteria can be obviously improved.
Description
Technical Field
The invention relates to the technical field of genetic engineering, in particular to a recombinant genetic engineering bacterium for highly expressing insulin glargine precursor and a construction method thereof.
Background
Diabetes is a chronic disease that is highly prevalent in today's society. The insulin products are the first major drug variety in the diabetes market, and the third generation of recombinant insulin is the main drug variety. Insulin Glargine (insulin-arginine insulin, glargine for short) belongs to a long-acting insulin biological product obtained by a gene recombination technology. The insulin glargine has no obvious peak value and the risk of hypoglycemia, sudden death and the like caused by the peak value, and the insulin glargine is a product with the largest market share in the insulin market for years due to the characteristics of safety and long-acting property. The recombinant insulin glargine produced by Aventis is sold as Lantus (Lantus) abroad, and the recombinant insulin glargine injection produced by Prunus spinosa pharmaceutical industry Co., ltd is sold as ChanxiuLin.
The insulin glargine has the structural design as follows: the 21 st Asn of human insulin A chain is replaced by Gly, and 2 Arg is added after 30 th amino acid of B chain. The complete recombinant insulin glargine consists of 53 amino acids in both chain A and chain B, wherein the chain A contains 21 amino acids and the chain B contains 32 amino acids. Insulin glargine is usually prepared by expression in recombinant escherichia coli engineering bacteria in the form of inclusion bodies, the expression product is mostly recombinant insulin glargine precursor in the form of fusion protein, and the precursor structure is usually designed by adding C peptide (consisting of tens of amino acids) between a B chain and an A chain. The C peptide ensures the correct folding of the A and B chains and can be cleaved by trypsin and carboxypeptidase B. That is, most of the existing production methods of insulin glargine produce single-chain precursor of insulin glargine by recombinant escherichia coli engineering bacteria, and after in vitro renaturation, the mature double-chain structure is formed by in vitro hydrolysis and processing of proteases such as trypsin and carboxypeptidase, and then after impurities of mixed proteases and other derivatives are removed by a series of purification means, the insulin glargine is obtained by purification.
At present, recombinant genetically engineered bacteria can be used for expression preparation of insulin. The expression system comprises a prokaryotic gene expression system, a yeast expression system and an animal cell expression system. For prokaryotic gene expression systems, the most used is Escherichia coliEscherichiacoliAs a host bacterium, the protein expression system is the most widely applied protein expression system at present. The reason is that the research on genetic background and physiological characteristics of an escherichia coli expression system is clear, and a plurality of commercial engineering bacteria can be developed and used; and the escherichia coli is easy to culture and control, the transformation operation is simple, and the method has the characteristics of high expression level, low cost, short period and the like. For expressing foreign gene by using prokaryotic system, most studies utilize fusion proteinAnd (3) an expression mode, namely fusing various guide peptide sequences to a target gene to form a recombinant fusion protein. When expressed in E.coli, the leader peptide can secrete the target protein into the periplasm of cells or even outside the cells, and finally, the leader peptide is cleaved off by a protease or the like.
However, due to the structural particularity (double-chain structure, multiple pairs of intramolecular disulfide bonds and the like) of insulin glargine, technical defects that the expression level is low, the renaturation conditions of an inclusion body after expression are harsh, the enzyme digestion purification steps are complicated and the like generally exist in the expression process of the insulin glargine precursor at present. Therefore, there is still an urgent need to develop recombinant genetic engineering bacteria capable of efficiently expressing insulin glargine precursors, so as to improve fermentation efficiency, simplify the steps of enzyme digestion and purification, and effectively reduce production cost.
Disclosure of Invention
In order to solve the technical problems, the invention provides a novel insulin glargine precursor, a recombinant genetic engineering bacterium for expressing the insulin glargine precursor and a construction method thereof.
The concept is as follows:
insulin glargine: the 21 st Asn of human insulin A chain is replaced by Gly, and 2 Arg is added after 30 th amino acid of human insulin B chain, wherein, the sulfydryl in four cysteines of A7 (Cys) -B7 (Cys) and A20 (Cys) -B19 (Cys) form two disulfide bonds to connect the A chain and the B chain, and a pair of intra-A chain disulfide bonds A6 (Cys) -A11 (Cys) exist.
Insulin glargine polypeptide: refers to single-chain amino acid peptide segment of insulin glargine, specifically refers to polypeptide obtained by directly connecting insulin glargine B chain and insulin glargine A chain or connecting peptide (such as C peptide).
Insulin glargine precursor: the fusion protein is formed by connecting insulin glargine polypeptide with a guide peptide and the like through a restriction enzyme site sequence, and the guide peptide and the like can be cut off through protease to obtain the insulin glargine polypeptide. Specifically, the insulin glargine precursor is a fusion protein shown as SEQ ID No. 1-SEQ ID No. 10.
The invention provides a novel insulin glargine precursor, which is polypeptide obtained by sequentially connecting a leader peptide, an insulin glargine B chain and an insulin glargine A chain in series, wherein the C end of the insulin glargine B chain is directly connected with the N end of the A chain, and the N end of the insulin glargine B chain is connected with the leader peptide; the amino acid sequence of the insulin glargine precursor is selected from SEQ ID No. 1-SEQ ID No.
The C end of the insulin glargine B chain is provided with two arginines, the arginine is directly connected with glycine (G) at the N end of the insulin glargine A chain, so that the arginine can be directly cut by adopting an Arg-Arg recognition site of kex2 protease, the insulin glargine A chain and the insulin glargine B chain can be cut and separated by one-step cutting, the enzyme cutting efficiency is obviously improved, side reactions in the process of cutting C peptide by adopting trypsin or carboxypeptidase B are avoided, and related impurities are effectively controlled.
Aiming at the characteristics of direct-connected polypeptides of insulin glargine A chain and insulin glargine B chain, the leader peptide in the precursor is subjected to targeted research design and screened to obtain the leader peptide capable of realizing high expression of the insulin glargine precursor. Preferably, the amino acid sequence of the novel insulin glargine precursor of the present invention is selected from SEQ ID No. 1 or SEQ ID No. 10.
In a second aspect, the present invention provides a polynucleotide encoding the insulin glargine precursor as defined above. The correspondence between the polynucleotides and the amino acids of the insulin glargine precursor is shown in Table 1. Specifically, the sequence of the polynucleotide is selected from nucleotide sequences shown in SEQ ID No. 11 to SEQ ID No. 20. And preferably a nucleotide sequence shown as SEQ ID No. 11 or SEQ ID No. 20.
In a third aspect, the present invention provides a recombinant expression vector for expressing insulin glargine precursor comprising said polynucleotide encoding insulin glargine precursor. Specifically, the expression vector of the embodiment of the present invention is preferably a plasmid vector, and specifically, a plasmid pET-28a (+); and preferably byNdeI andXhoi cleavage sites are inserted into the polynucleotide of the present invention.
In a fourth aspect, the present invention provides a host bacterium comprising the above recombinant expression vector. The host bacteria are selected from prokaryotic cells, more preferably Escherichia coli, and specifically can be selected from Escherichia coli expression strain BL21 (DE 3). The recombinant gene engineering bacteria are prepared according to the following method:
(1) Synthesizing the above polynucleotide;
(2) Inserting the polynucleotide into an expression vector to construct a recombinant expression vector;
(3) And (3) introducing the recombinant expression vector into host bacteria to obtain recombinant genetic engineering bacteria for expressing the insulin glargine precursor.
The fifth aspect of the invention provides a construction method of recombinant genetic engineering bacteria for expressing insulin glargine precursors, which at least comprises the following steps:
s1, synthesizing and expressing a polynucleotide containing a insulin glargine precursor;
s2, inserting the polynucleotide into an expression vector to construct a recombinant expression vector;
and S3, introducing the recombinant expression vector into host bacteria to obtain recombinant genetic engineering bacteria for expressing the insulin glargine precursor.
Specifically, the polynucleotide sequence is selected from SEQ ID No. 11-SEQ ID No. 20; preferably SEQ ID No. 11-13 and SEQ ID No. 20; more preferably SEQ ID No. 11 or SEQ ID No.20, still more preferably SEQ ID No. 20.
Specifically, the expression vector is selected from a plasmid pET-28a (+), and the host cell is selected from an Escherichia coli expression strain BL21 (DE 3).
Compared with the prior art, the technical scheme provided by the invention has the following advantages:
according to the invention, through the design of the leader peptide and the design of directly connecting the B chain and the A chain of the insulin glargine, the recombinant genetic engineering bacterium with the obviously improved expression quantity is obtained, and the expression efficiency of the insulin glargine precursor is greatly improved. Moreover, the insulin glargine precursor obtained based on the method can be subjected to simplified enzyme digestion and purification steps to obtain the insulin glargine, and the enzyme digestion and purification efficiency is remarkably improved. Therefore, the recombinant gene engineering bacteria are more suitable for the commercial production of insulin glargine and have wider market prospect.
Drawings
FIG. 1 is a graph showing the results of inducible expression of 8 transformants obtained in example 1 of the present invention.
Detailed Description
In order that the above objects, features and advantages of the present invention may be more clearly understood, a solution of the present invention will be further described below. It should be noted that the embodiments of the present invention and features of the embodiments may be combined with each other without conflict.
In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention, but the present invention may be practiced in other ways than those described herein; it is to be understood that the embodiments described in this specification are only some embodiments of the invention, and not all embodiments.
The starting materials used in the following examples are all commercially available.
Examples materials used include:
(1) Strains and plasmids
The host bacterium Escherichia coli BL21 (DE 3) is a common tool strain for genetic engineering, and the recombinant plasmids pLA001-pLA010 are synthesized by Taobao biology Co.
(2) Culture medium
The LB liquid culture medium comprises the following formula: 10g/L of Tryptone (Tryptone), 5g/L of Yeast extract (Yeast extract) and 10g/L of sodium chloride (NaCl).
The LB solid culture medium has the formula: 10g/L Tryptone (Tryptone), 5g/L Yeast extract (Yeast extract), 10g/L sodium chloride (NaCl) and 20g/L Agar powder (Agar).
Experimental methods used in the examples:
coli transformation, SDS-PAGE is a routine procedure in the field of genetic engineering, see [ Michael r.green, joseph sambrook. Molecular cloning guide: fourth edition [ M ]. Haofing, et al, translation. Beijing: scientific Press, 2017, 124-125,1325-1330.
Example 1: construction and induced expression of recombinant engineering bacteria
1.1 construction of recombinant engineering bacteria
Designing a fusion protein comprising a leader peptide and a single-chain insulin glargine, i.e. an insulin glargine precursor,obtaining polynucleotide for coding fusion protein, synthesizing expression frame sequence of fusion protein, inserting expression frame into pET-28a (+) plasmidNdeIAndXhoIthe sites are named as recombinant plasmids pLA001-pLA 009. Respectively transforming the recombinant plasmids into escherichia coliEscherichia coliBL21 (DE 3), LB solid medium (containing 30. Mu.g/mL kanamycin) is coated with the transformation liquid, and the engineering bacteria containing the recombinant plasmid of the target gene are obtained by resistance selection.
The fusion proteins having the plasmid numbers inserted therein are shown in Table 1 below;
TABLE 1
| Recombinant plasmid number | Expression of fusion protein sequences | Polynucleotide sequences encoding fusion proteins |
| pLA001 | SEQ ID NO:1 | SEQ ID NO:11 |
| pLA002 | SEQ ID NO:2 | SEQ ID NO:12 |
| pLA003 | SEQ ID NO:3 | SEQ ID NO:13 |
| pLA004 | SEQ ID NO:4 | SEQ ID NO:14 |
| pLA005 | SEQ ID NO:5 | SEQ ID NO:15 |
| pLA006 | SEQ ID NO:6 | SEQ ID NO:16 |
| pLA007 | SEQ ID NO:7 | SEQ ID NO:17 |
| pLA008 | SEQ ID NO:8 | SEQ ID NO:18 |
| pLA009 | SEQ ID NO:9 | SEQ ID NO:19 |
1.2 inducible expression of recombinant engineering bacteria
Transformants were picked from the selection plates of the recombinant engineered bacteria, respectively, aseptically inoculated into LB liquid medium containing 30. Mu.g/mL kanamycin, and cultured overnight at 37 ℃ with shaking. According to the initial OD 600 Transfer to 20mL of LB liquid medium (containing 30. Mu.g/mL kanamycin) at a ratio of =0.05, and incubate at 37 ℃ with shaking to OD 600 And (4) adding IPTG (isopropyl thiogalactoside) with the final concentration of 1.0mmol/L to induce and express the target protein, wherein the concentration is 0.6 to 3.0. After 6 hours of induction, the cells were collected by centrifugation. SDS-PAGE (16% separation gel and 4% concentrated gel) was followed by gel imaging, and the expression results are shown in Table 2.
TABLE 2
| Number of plasmid contained in recombinant engineering bacterium | Expression level of recombinant engineered bacterium |
| pLA001 | + |
| pLA002 | + |
| pLA003 | + |
| pLA004 | - |
| pLA005 | - |
| pLA006 | - |
| pLA007 | - |
| pLA008 | - |
| pLA009 | - |
According to expression confirmation, only pLA001-pLA 003 can detect successful expression of the target fragment in the constructed expression system, and pLA 004-pLA 009 hardly detects the expression of the target fragment. However, the expression levels of pLA001 to pLA003 were only normal.
In an attempt to further improve the expression potential of the constructed recombinant engineered bacteria, the inventors attempted various design improvements of leader peptides. After a large amount of research and experiments, the method is finally obtained, and the expression quantity of the recombinant engineering bacteria can be remarkably improved by optimizing the amino acid at the tail end of the pLA001 guide peptide from PR to KR. The fusion proteins having the plasmid numbers inserted therein are shown in Table 3 below;
TABLE 3
| Recombinant plasmid number | Expression of fusion protein sequences | Polynucleotide sequences encoding fusion proteins |
| pLA010 | SEQ ID NO:10 | SEQ ID NO:20 |
Similarly, according to the methods 1.1 and 1.2, a recombinant engineering bacterium containing pLA010 was constructed, and the expression level thereof is compared with pLA001, as shown in table 4:
TABLE 4
| Sequence number of plasmid contained in recombinant engineering bacterium | Expression level of recombinant engineered bacterium |
| pLA001 | + |
| pLA010 | +++ |
The results of fermentation production of pLA001 and pLA010 show that the expression level of the latter is obviously improved, the expression level of pLA001 is about 3g/L through detection, the expression level of pLA010 can reach about 12g/L, and the expression level is obviously improved.
1.3 in the process of constructing recombinant engineering bacteria containing pLA010, 8 transformants picked from the screening plate thereof are induced and expressed according to the method shown in example 1.2, and the induced expression result is shown in figure 1.
As shown in FIG. 1, the recombinant engineering bacterium containing pLA010 constructed by the invention can realize high-efficiency expression of target protein, and has a good application prospect.
The above description is merely illustrative of particular embodiments of the invention that enable those skilled in the art to understand or practice the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Sequence listing
<110> Beijing-Hui-Heng Biotechnology Ltd
Jilin Huisheng biopharmaceutical Co.,Ltd.
<120> recombinant genetic engineering bacterium for high expression of insulin glargine precursor and construction method thereof
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<400> 17
atgaaaaaaa ccgctatcgc tatcgctgtt gctctggctg gtttcgctac cgttgctcag 60
gctaaacgtt tcgttaacca gcacctgtgc ggttctcacc tggttgaagc tctgtacctg 120
gtttgcggtg aacgtggttt cttctacacc ccgaaaaccc gtcgtggtat cgttgaacag 180
tgctgcacct ctatctgctc tctgtaccag ctggaaaact actgcggt 228
<210> 18
<211> 228
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 18
atgaaaaaat ctaccctggc tctggttgtt atgggtatcg ttgcttctgc ttctgttcag 60
gctaaacgtt tcgttaacca gcacctgtgc ggttctcacc tggttgaagc tctgtacctg 120
gtttgcggtg aacgtggttt cttctacacc ccgaaaaccc gtcgtggtat cgttgaacag 180
tgctgcacct ctatctgctc tctgtaccag ctggaaaact actgcggt 228
<210> 19
<211> 237
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 19
atgatcaccc tgcgtaaact gccgctggct gttgctgttg ctgctggtgt tatgtctgct 60
caggctatgg ctaaacgttt cgttaaccag cacctgtgcg gttctcacct ggttgaagct 120
ctgtacctgg tttgcggtga acgtggtttc ttctacaccc cgaaaacccg tcgtggtatc 180
gttgaacagt gctgcacctc tatctgctct ctgtaccagc tggaaaacta ctgcggt 237
<210> 20
<211> 351
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 20
atggctacca aagctgcttc tgttctgaaa ggtgacggtc cggttcaggg tatcatcaac 60
ttcgaacaga aagaatctaa cggtccggtt aaagtttggg gttctatcaa aggtctgacc 120
gaaggtctgc acggtttcca cgttcacgaa ttcggtgaca acaccgctgg ttctacctct 180
gctggtaaac gtttcgttaa ccagcacctg tgcggttctc acctggttga agctctgtac 240
ctggtttgcg gtgaacgtgg tttcttctac accccgaaaa cccgtcgtgg tatcgttgaa 300
cagtgctgca cctctatctg ctctctgtac cagctggaaa actactgcgg t 351
Claims (6)
1. The insulin glargine precursor is characterized by being polypeptide obtained by sequentially connecting a guide peptide, an insulin glargine B chain and an insulin glargine A chain in series, wherein the C end of the insulin glargine B chain is directly connected with the N end of the insulin glargine A chain, and the N end of the insulin glargine B chain is connected with the guide peptide;
the amino acid sequence of the insulin glargine precursor is SEQ ID No. 10.
2. A polynucleotide encoding the insulin glargine precursor according to claim 1, wherein the polynucleotide sequence is SEQ ID No. 20.
3. A recombinant expression vector for the expression of insulin glargine precursor, characterized in that said recombinant expression vector comprises the polynucleotide of claim 2, said expression vector is selected from the plasmid pET-28a (+).
4. The recombinant genetically engineered bacterium comprising the recombinant expression vector of claim 3, wherein the recombinant genetically engineered bacterium is obtained by introducing the recombinant expression vector into a host bacterium selected from escherichia coli BL21 (DE 3).
5. The recombinant genetically engineered bacterium of claim 4, wherein the recombinant genetically engineered bacterium is prepared by the following method:
(1) Synthesizing the polynucleotide of claim 2;
(2) Inserting the polynucleotide into an expression vector, wherein the expression vector is selected from a plasmid pET-28a (+), and constructing a recombinant expression vector;
(3) And (3) introducing the recombinant expression vector into host bacteria, wherein the host bacteria are selected from escherichia coli BL21 (DE 3), so as to obtain the recombinant genetic engineering bacteria for expressing the insulin glargine precursor.
6. A construction method of recombinant genetic engineering bacteria for expressing insulin glargine precursors is characterized by comprising the following steps:
(1) Synthesizing the polynucleotide of claim 2;
(2) Inserting the polynucleotide into an expression vector, wherein the expression vector is selected from a plasmid pET-28a (+), and constructing a recombinant expression vector;
(3) And (3) introducing the recombinant expression vector into host bacteria, wherein the host bacteria are selected from escherichia coli BL21 (DE 3), so as to obtain the recombinant genetic engineering bacteria for expressing the insulin glargine precursor.
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| CN106699872B (en) * | 2016-12-27 | 2019-12-24 | 江南大学 | Method for improving yield of insulin precursor |
| EP3807306A4 (en) * | 2018-06-18 | 2022-04-06 | Unichem Laboratories Limited | Leader sequence for higher expression of recombinant proteins |
| CN113773399B (en) * | 2020-06-09 | 2023-05-12 | 宁波鲲鹏生物科技有限公司 | Insulin glargine derivative and application thereof |
| CN113773392B (en) * | 2020-06-09 | 2023-04-07 | 宁波鲲鹏生物科技有限公司 | Preparation method of insulin glargine |
| CN114380903B (en) * | 2021-12-28 | 2023-07-25 | 上海仁会生物制药股份有限公司 | Insulin or analogue precursor thereof |
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