CN115850385B - Expression-promoting peptide and application thereof - Google Patents
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Abstract
The application relates to the technical field of genetic engineering, in particular to an expression-promoting peptide suitable for recombinant expression of insulin peptide chains and GLP-1 related target agonist polypeptides and application thereof. The recombinant genetic engineering bacteria constructed based on the expression promoting peptide can obviously enhance the expression quantity of insulin precursor protein, GLP-1 and related multi-target excited polypeptide precursors with difficult expression or low expression quantity, effectively reduce the production cost and have wide commercialized prospect.
Description
The present application claims full priority from patent application No. 202210788609.4 filed on 7/4 of 2022. The entire contents of this application are incorporated herein by reference in their entirety.
Technical Field
The application relates to the technical field of genetic engineering, in particular to an expression-promoting peptide and application thereof.
Background
With the development of socioeconomic performance, the living standard of people is gradually improved, the dietary structure of people is greatly changed, the occurrence rate of obesity is increased, and the number of diabetics is further increased sharply. Statistics data show that the number of diabetics in China exceeds 1 hundred million, and simultaneously, more than 1.5 hundred million invisible pre-diabetics exist. Diabetes has become the third greatest chronic disease.
Diabetes is a complex chronic metabolic disease caused by long-term interaction of genetic and environmental factors, and is a disease characterized by hyperglycemia due to lack of insulin secretion, and is classified into type I diabetes and type II diabetes, wherein type II diabetes (T2 DM) accounts for more than 95% of diabetics in China.
The most important drugs for treating diabetes mellitus are insulin and derivatives thereof, glucagon-like peptide-1 (GLP-1) receptor agonists (GLP-1 RAs) and related multi-target co-agonists. The above medicines are protein or polypeptide medicines.
Known methods for obtaining insulin include animal tissue extraction and genetic engineering expression, and the use of genetic engineering to express human insulin and its analogues has long been the mainstream means of industry. Microorganisms expressing insulin in genetic engineering are mainly classified into 2 types, which are E.coli and yeast, respectively. For prokaryotic gene expression systems, the most commonly used is Escherichia coli as host bacteria, which is also the most widely used protein expression system at present. The reason is that the genetic background and physiological characteristic of the escherichia coli expression system are clearly researched, and various commercial engineering bacteria can be developed for use; and the escherichia coli is easy to culture and control, simple in transformation operation, and has the characteristics of high expression level, low cost, short period and the like. When a prokaryotic system is used for expressing exogenous genes, most of researches utilize a fusion protein expression mode to fuse various different leader peptide sequences to target genes to form recombinant fusion proteins. When expressed in E.coli, the leader peptide may secrete the target protein into the periplasm or even outside the cell, and finally, the leader peptide is cleaved off by a protease or the like. However, for polypeptides partially having a specific structure or amino acid sequence, such as insulin precursors, partial GLP-1 polypeptides, and GLP-1 related multi-target agonist polypeptides, it is often difficult to express or the expression level is low, and commercial use requirements cannot be satisfied. For this reason, it is sometimes necessary to add a fragment of an expression-promoting peptide which can significantly enhance the expression level of the fusion protein after being linked to a leader peptide, and finally obtain a target protein fragment after treatment such as cleavage. At present, relatively few researches are carried out on an expression promoting peptide capable of further enhancing the high expression of the protein or polypeptide, an expression vector constructed by using the expression promoting peptide and having high expression potential and recombinant engineering bacteria thereof.
Therefore, the method is suitable for obtaining a plurality of proteins or pro-expression peptides of polypeptides which are difficult to express or have low expression level, and constructs recombinant engineering bacteria with high expression capacity based on the pro-expression peptides, so that the expression level of the polypeptides or the proteins can be improved, the production cost is effectively reduced, and great demands still exist.
Disclosure of Invention
In order to solve the technical problems, the application provides an expression-promoting peptide, and recombinant engineering bacteria constructed based on the expression-promoting peptide can efficiently express insulin peptide chain precursors and GLP-1 related target agonist polypeptides.
As used herein, the term "protein of interest" refers to a product of interest or a precursor protein thereof that is desired to obtain high expression levels.
As used herein, the term "leader peptide" refers to a polypeptide sequence, also known as a "leader peptide," "leader peptide," or "secretory peptide," that is linked to a polypeptide or protein of interest, directs the soluble expression, secretion, or facilitates the correct folding of the polypeptide or protein of interest for soluble expression.
As used herein, the term "pro-expression peptide" refers to a polypeptide sequence that is linked after the leader peptide and before the protein or polypeptide of interest, further enhances the amount of expression of the protein or polypeptide of interest, or is capable of promoting expression of the protein or polypeptide of interest that is difficult to express by conventional leader peptides.
The term "peptide" refers to a molecule comprising amino acid sequences linked by peptide bonds, whether in length, post-translational modification or function.
As used herein, the term "insulin" includes natural insulin and insulin analogs.
As used herein, the term "natural insulin" refers to a hormone that is a 51 amino acid residue polypeptide (5808 daltons), which plays an important role in many critical cellular processes. The mature form of human insulin consists of 51 amino acids arranged into an A chain (GlyAl-AsnA 21) and a B chain (PheB 1-ThrB 30) with a total molecular weight of 5808 Da. The molecule is stabilized by two interchain chains (A20-B19, A7-B7) and one interchain disulfide bond (A6-A11). Insulin of the present application includes natural, synthetically provided, or genetically engineered (e.g., recombinant) sources, and in various embodiments of the present application, insulin may be human natural insulin or insulin analogs.
As used herein, the term "insulin analogue" refers to a modified form of insulin, which is a more rapid acting form or a longer acting form of insulin. Non-limiting examples of such analogs include Insulin lispro, insulin deglutition (Insulin deglutch), insulin Aspart (Insulin Aspart), and Insulin Glargine (Glargine Insulin). "lispro" insulin analogues are essentially identical in primary structure to human insulin, differing from human insulin by exchange of lysine at the B28 position and proline at the B29 position. It is a short acting insulin analogue. "glargine" insulin analogues differ from human insulin by the substitution of glycine with asparagine at a21, and the addition of two arginine residues at the C-terminus of the B chain. Insulin glargine solution was formulated and injected at pH 4.0. These modifications raise the isoelectric point to a more neutral pH, reduce solubility under physiological conditions, and cause precipitation of insulin glargine at the site of injection, thereby slowing absorption. Insulin glargine is a long-acting analogue that lasts for 20-24 hours.
As used herein, the term "insulin precursor" refers to a single chain molecule formed by linking insulin a and B chains of a natural insulin or insulin analog, specifically by linking insulin B and insulin a chains via a C peptide.
As used herein, the term "polynucleic acid fragment" refers to a nucleotide chain consisting of naturally occurring bases, sugars and inter-sugar (backbone) linkages. The nucleic acid fragments of the application may be deoxyribonucleic acid fragments (DNA) or ribonucleic acid fragments (RNA) and may include naturally occurring bases including adenine, guanine, cytosine, thymidine, and uracil. The nucleic acid fragment encoding insulin that may be used according to the methods provided herein may be any nucleic acid fragment encoding an insulin polypeptide or a precursor thereof (including proinsulin and preproinsulin).
As used herein, the term "coding sequence" refers to a polynucleotide sequence that is transcribed into mRNA when placed under the control of appropriate control sequences, and the mRNA is translated into a polypeptide. The boundaries of the coding sequence are typically determined by a start codon located at the beginning of the open reading frame at the 5 'end of the mRNA and a stop codon located at the 3' end of the open reading frame of the mRNA.
The term "GLP-1 polypeptide" generally refers to GLP-1 (7-37) polypeptides and analogs thereof. GLP-1 (7-37) polypeptide refers to polypeptide consisting of amino acids 7-37 of human natural GLP-1 polypeptide, and GLP-1 (7-37) polypeptide analogue refers to polypeptide obtained by modifying human natural GLP-1 (7-37) amino acid, wherein the modification comprises removing and/or replacing (replacing) and/or adding (extending) one or more amino acid residues, and the amino acid can be naturally occurring amino acid or artificial amino acid.
The term "GLP-1 related multi-target agonist polypeptide" refers to a polypeptide that possesses co-agonists that activate both the GLP-1 receptor and other diabetes related multi-target receptors, including but not limited to glucose-dependent insulinotropic polypeptide (GIP) receptor, glucagon (GCG) receptor. The co-agonist may be a GLP-1 receptor and GIP receptor dual-target co-agonist (GLP-1/GIP co-agonist), a GLP-1 receptor and GCG receptor dual-target co-agonist (GLP-1/GCG co-agonist), or a GLP-1 receptor, GIP receptor and GCG receptor tri-target co-agonist (GLP-1/GIP/GCG co-agonist).
The first aspect of the application provides the application of the polypeptide shown as SEQ ID NO. 1 as the expression promoting peptide for constructing recombinant escherichia coli engineering bacteria. The polypeptide shown as SEQ ID NO. 1 can be used as an expression promoting peptide, can efficiently express some target proteins or polypeptides which are difficult to express or obviously enhance the target proteins or polypeptides with low expression level, and is particularly suitable for insulin precursors, GLP-1 polypeptides, GLP-1 related multi-target agonist polypeptides and the like.
The second aspect of the application provides a fusion protein containing target protein, which is formed by sequentially connecting at least a guide peptide, an expression promoting peptide, an enzyme cutting site fragment and a target protein fragment, wherein the amino acid sequence of the expression promoting peptide is shown as SEQ ID NO. 1.
Wherein the amino acid sequence of the cleavage site is selected from K or DDDDK (SEQ ID NO: 2). The enzyme cutting site K can be hydrolyzed by Lys-C enzyme, and the DDDDK enzyme cutting site can be hydrolyzed by enterokinase.
As an improvement of the technical scheme of the application, the leader peptide can be a leader peptide sequence conventional in the art, preferably FKFEFKFE (SEQ ID NO: 7) or FEFKFEFK (SEQ ID NO: 8).
Wherein the target protein is selected from the group consisting of insulin precursors, GLP-1 polypeptides and GLP-1 related multi-target agonist polypeptides to increase the expression level thereof. As a specific embodiment of the present application, the amino acid sequence of the protein of interest is selected from the group consisting of: SEQ ID NO 3-SEQ ID NO 6.
The amino acid sequence of the protein or polypeptide of the present application is specifically shown in table 1:
TABLE 1
In a third aspect the application provides a polynucleotide fragment for encoding the fusion protein described above. As a specific embodiment of the application, the polynucleotide fragment has a sequence shown in any one of SEQ ID No. 9 to SEQ ID No. 16.
The fourth aspect of the application provides an expression vector comprising the polynucleotide fragment described above, wherein the expression vector is a recombinant pET-30a (+) expression vector.
The fifth aspect of the application provides a recombinant escherichia coli engineering bacterium, which comprises the expression vector, and escherichia coli is preferably BL21 (DE 3). Furthermore, the construction method of the recombinant escherichia coli engineering bacteria at least comprises the following steps:
(1) Synthesizing a polynucleotide fragment encoding the fusion protein of the application;
(2) Cloning the polynucleotide fragment into an expression plasmid, and constructing a recombinant expression vector;
(3) And (3) converting the expression vector into escherichia coli to obtain recombinant escherichia coli engineering bacteria.
Preferably, the polynucleotide sequence of the application is shown in any one of SEQ ID No. 9-SEQ ID No. 16; the plasmid of the application is preferably pET-30a (+); the E.coli of the application is preferably BL21 (DE 3).
The sixth aspect of the application provides a method for preparing the recombinant escherichia coli engineering bacteria, which comprises the following steps:
(1) Synthesizing a polynucleotide encoding the fusion protein of the application;
(2) Cloning the polynucleotide into an expression plasmid, and constructing a recombinant expression vector;
(3) And (3) converting the expression vector into escherichia coli to obtain recombinant escherichia coli engineering bacteria.
Preferably, the polynucleotide sequence of the application is shown in any one of SEQ ID No. 9-SEQ ID No. 16; the plasmid of the application is preferably pET-30a (+); the E.coli of the application is preferably BL21 (DE 3).
Compared with the prior art, the technical scheme provided by the embodiment of the application has the following advantages:
the application provides an expression promoting peptide and recombinant genetically engineered bacterium which can obviously improve the expression level of target proteins in escherichia coli, and can obviously enhance the expression level of insulin precursor proteins, GLP-1 and related multi-target co-excited polypeptide precursors with difficult expression or low expression level.
Drawings
Fig. 1 to 3 are electrophoresis diagrams of embodiments of the present application.
Detailed Description
In order that the above objects, features and advantages of the application will be more clearly understood, a further description of the application will be made. It should be noted that, without conflict, the embodiments of the present application and features in the embodiments may be combined with each other.
In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present application, but the present application may be practiced otherwise than as described herein; it will be apparent that the embodiments in the specification are only some, but not all, embodiments of the application.
EXAMPLE 1 preparation of Long-acting acylated insulin derivatives
(1) The fusion protein sequences were designed as shown in table 2:
TABLE 2
Where "/" indicates that the sequence is absent.
(2) Constructing a plasmid: the coding sequences of the fusion proteins shown in Table 2 were constructed, corresponding to the fusion proteins numbered 1 to 8 in Table 3, respectively, and specific coding polynucleotide fragments are shown in Table 3.
TABLE 3 Table 3
The constructed coding nucleotide fragments are inserted into a prokaryotic expression plasmid pET-30a through Nde1 and Xho1 sites respectively and are sequenced and verified to obtain a recombinant expression plasmid.
(3) Recombinant engineering bacteria construction: adding the constructed recombinant expression plasmid into 30 mu L BL21 (DE 3), carrying out ice bath for 30min, carrying out heat shock for 30 s-90 s at 42 ℃, adding 500 mu L LB culture medium after 2min of ice bath, carrying out shake culture for 1 hour at 37 ℃, coating onto an LB solid culture plate, standing overnight at 37 ℃, picking up a monoclonal, adding the monoclonal into 5mL LB liquid culture medium containing kanamycin, carrying out shake overnight at 37 ℃, taking 500 mu L of overnight bacterial liquid, adding 500 mu L of 50% glycerol bacteria, mixing uniformly, and obtaining glycerol frozen bacteria, and preserving at-80 ℃.
(4) Shake flask expression
The frozen strain is selected and added into 50mL of LB culture medium, shaking is carried out at 37 ℃ for overnight, the overnight bacterial liquid is added into the LB liquid culture medium according to the proportion of 1/100, and 0.5mM IPTG (isopropyl-beta-D-thiogalactoside) is added after 4 hours to induce expression. Shaking culture is carried out for 24 hours at the constant temperature of 37 ℃. The bacteria were harvested by centrifugation at 7000rpm for 20 minutes.
(5) SDS-PAGE electrophoresis detection of the expression results:
the obtained electrophoresis detection expression results are shown in fig. 1-3:
in FIG. 1, lane 1 shows the result of expression of the fusion protein of No. 2, and lane 2 shows the result of expression of the fusion protein of No. 1.
As can be seen from FIG. 1, in the recombinant engineering bacteria constructed by using the peptide-promoting fusion protein (No. 1) of the present application, the expression of the target protein can be clearly seen, but in the recombinant engineering bacteria constructed by the peptide-promoting fusion protein (No. 2) of the present application, the target protein is not substantially expressed, or the expression level is very low.
In FIG. 2, lane 1 shows the results of expression of the fusion protein numbered 3; lane 2 shows the expression result of the fusion protein numbered 4; lane 3 is the fusion protein No. 3 non-induced control (step 3 without IPTG-induced expression).
As can be seen from FIG. 2, after the recombinant engineering bacteria constructed by the fusion protein (No. 4) without the expression promoting peptide of the application are induced, the expression condition of the recombinant engineering bacteria is similar to that of the recombinant engineering bacteria constructed by the non-induced No. 3, and the expression of the target protein is basically absent or the expression quantity is very low; the recombinant engineering bacteria constructed by using the fusion protein (number 3) of the peptide expression promoting peptide can see the obvious expression of the target protein.
In FIG. 3, lane 1 is the result of expression of the fusion protein numbered 5, lane 2 is the result of expression of the fusion protein numbered 6, and lane 3 is the uninduced expression of the fusion protein numbered 5 (i.e., the step of IPTG induction expression is not performed in step 3); lane 4 shows the result of expression of the fusion protein numbered 7, lane 5 shows the result of expression of the fusion protein numbered 8, and lane 6 shows the uninduced expression of the fusion protein numbered 7 (i.e., the step of not performing IPTG-induced expression in step 3).
As can be seen from the figure 3, the recombinant engineering bacteria constructed by the fusion protein without the expression promoting peptide of the application have basically no expression or very low expression level of the target protein after induction; the recombinant engineering bacteria constructed by using the fusion protein of the peptide expression promoting peptide can be used for obviously expressing the target protein. As described above, the recombinant genetically engineered bacteria constructed by using the expression-promoting peptide sequence of the present application can observe the expression of the target protein after induction, but the recombinant genetically engineered bacteria constructed without using the expression-promoting peptide sequence of the present application and the recombinant genetically engineered bacteria constructed without using the expression-promoting peptide sequence of the present application are substantially free from the expression of the target protein or have very low expression level. The effect of the peptide expression promotion is obvious.
The foregoing is only a specific embodiment of the application to enable those skilled in the art to understand or practice the application. 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 application. Thus, the present application is not intended to be limited to the embodiments shown and described herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Claims (8)
1. The polypeptide shown as SEQ ID NO. 1 is used for constructing the expression promoting peptide in recombinant escherichia coli engineering bacteria, and the amino acid sequence of the target protein expressed by the recombinant escherichia coli engineering bacteria is selected from SEQ ID NO. 3-SEQ ID NO. 6.
2. The fusion protein containing the target protein is characterized by being formed by sequentially connecting at least a guide peptide, an expression promoting peptide, an enzyme cleavage site fragment and a target protein fragment, wherein the amino acid sequence of the expression promoting peptide is shown as SEQ ID NO. 1;
the amino acid sequence of the cleavage site is selected from K, DDDDK;
the amino acid sequence of the target protein is selected from SEQ ID NO 3-SEQ ID NO 6;
the amino acid sequence of the leader peptide is shown as SEQ ID NO. 7 or SEQ ID NO. 8.
3. A polynucleotide fragment encoding the fusion protein of claim 2.
4. An expression vector comprising the polynucleotide fragment of claim 3.
5. The expression vector of claim 4, wherein the expression vector is a recombinant pET-30a (+) expression vector.
6. A recombinant escherichia coli engineering bacterium, characterized in that the recombinant escherichia coli engineering bacterium comprises the expression vector of claim 4 or 5, and the escherichia coli is selected from BL21 (DE 3).
7. The recombinant escherichia coli engineering bacterium according to claim 6, wherein the construction method of the recombinant escherichia coli engineering bacterium at least comprises the following steps:
(1) Synthesizing the polynucleotide fragment of claim 3;
(2) Cloning the polynucleotide fragment into a plasmid pET-30a (+) to construct an expression vector;
(3) And (3) transforming the expression vector into escherichia coli BL21 (DE 3) to obtain the recombinant escherichia coli engineering bacteria.
8. A method for preparing the recombinant escherichia coli engineering bacteria of claim 7, comprising the steps of:
(1) Synthesizing the polynucleotide fragment of claim 3;
(2) Cloning the polynucleotide fragment to plasmid pET-30a (+) to construct an expression vector;
(3) And (3) transforming the expression vector into escherichia coli BL21 (DE 3) to obtain the recombinant escherichia coli engineering bacteria.
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| CN116693652B (en) * | 2023-08-02 | 2024-01-05 | 北京惠之衡生物科技有限公司 | GLP-1/GIP receptor dual agonist derivative and preparation method and application thereof |
| CN117756914B (en) * | 2023-12-15 | 2024-09-03 | 瀚晖制药有限公司 | Preparation method of lithocarpic insulin |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2016161983A1 (en) * | 2015-04-10 | 2016-10-13 | 中国医学科学院药物研究所 | Fusion carrier protein and application thereof in promoting target protein or polypeptide expression |
| WO2017215619A1 (en) * | 2016-06-15 | 2017-12-21 | 中国科学院上海生命科学研究院 | Fusion protein producing point mutation in cell, and preparation and use thereof |
| CN110128552A (en) * | 2019-03-14 | 2019-08-16 | 美药星（南京）制药有限公司 | A kind of fusion protein and its method for preparing Liraglutide intermediate polypeptide |
| CN110724187A (en) * | 2018-07-16 | 2020-01-24 | 甘李药业股份有限公司 | Recombinant engineering bacterium for efficiently expressing liraglutide precursor and application thereof |
| CN111072783A (en) * | 2019-12-27 | 2020-04-28 | 万新医药科技(苏州)有限公司 | Method for preparing GLP-1 or analog polypeptide thereof by adopting escherichia coli expression tandem sequence |
| CN113502296A (en) * | 2021-09-10 | 2021-10-15 | 北京惠之衡生物科技有限公司 | High-expression semaglutide precursor recombinant engineering bacterium and construction method thereof |
Family Cites Families (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| SG11201600684XA (en) * | 2013-07-31 | 2016-02-26 | Biogenomics Ltd | Process for production of insulin and insulin analogues |
| EP3303380B1 (en) * | 2015-06-02 | 2020-01-15 | Novo Nordisk A/S | Insulins with polar recombinant extensions |
| CN105254763B (en) * | 2015-06-30 | 2019-10-11 | 成都谨信恒生物技术有限公司 | A kind of Exendin-4 fusion protein, preparation method and applications |
| CN106434717A (en) * | 2015-11-05 | 2017-02-22 | 杭州九源基因工程有限公司 | Method for biosynthesis preparation of human GLP-1 polypeptide or analogue thereof |
| EP3606560A2 (en) * | 2017-04-05 | 2020-02-12 | Novo Nordisk A/S | Oligomer extended insulin-fc conjugates |
| US11649269B2 (en) * | 2018-10-05 | 2023-05-16 | Novo Nordisk A/S | Bifunctional compounds comprising insulin peptides and EGF(A) peptides |
| CN109295067B (en) * | 2018-10-26 | 2020-12-29 | 吉林惠升生物制药有限公司 | Codon-optimized degummed insulin precursor gene and expression method thereof |
| CA3122637A1 (en) * | 2018-12-11 | 2020-06-18 | Sanofi | Peptide binder |
| CN115867308A (en) * | 2020-05-15 | 2023-03-28 | 伊莱利利公司 | Time-acting acylated insulin compounds |
| CN116410297A (en) * | 2021-07-19 | 2023-07-11 | 内蒙古博睿精创科技有限公司 | Long-acting GLP-1 polypeptide analogue, and preparation method and application thereof |
| CN114874314B (en) * | 2021-12-28 | 2022-11-11 | 北京惠之衡生物科技有限公司 | Recombinant engineering bacterium for highly expressing GLP-1 analogue and construction method thereof |
-
2022
- 2022-09-07 CN CN202211089977.6A patent/CN115850385B/en active Active
- 2022-09-07 CN CN202211090263.7A patent/CN115947822B/en active Active
- 2022-09-13 CN CN202211111660.8A patent/CN115716876B/en active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2016161983A1 (en) * | 2015-04-10 | 2016-10-13 | 中国医学科学院药物研究所 | Fusion carrier protein and application thereof in promoting target protein or polypeptide expression |
| WO2017215619A1 (en) * | 2016-06-15 | 2017-12-21 | 中国科学院上海生命科学研究院 | Fusion protein producing point mutation in cell, and preparation and use thereof |
| CN110724187A (en) * | 2018-07-16 | 2020-01-24 | 甘李药业股份有限公司 | Recombinant engineering bacterium for efficiently expressing liraglutide precursor and application thereof |
| CN110128552A (en) * | 2019-03-14 | 2019-08-16 | 美药星（南京）制药有限公司 | A kind of fusion protein and its method for preparing Liraglutide intermediate polypeptide |
| CN111072783A (en) * | 2019-12-27 | 2020-04-28 | 万新医药科技(苏州)有限公司 | Method for preparing GLP-1 or analog polypeptide thereof by adopting escherichia coli expression tandem sequence |
| CN113502296A (en) * | 2021-09-10 | 2021-10-15 | 北京惠之衡生物科技有限公司 | High-expression semaglutide precursor recombinant engineering bacterium and construction method thereof |
Non-Patent Citations (1)
| Title |
|---|
| 李朋彦.利拉鲁肽前体肽GLP-1(7-37)K34R在大肠埃希菌中的表达与优化.医药导报.2020,(2021年第6期),参见全文. * |
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| CN115716876B (en) | 2023-06-23 |
| CN115850385A (en) | 2023-03-28 |
| CN115716876A (en) | 2023-02-28 |
| CN115947822A (en) | 2023-04-11 |
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