CN109628479A - A kind of recombinant expression method of glucagon-like-peptide-1 - Google Patents
A kind of recombinant expression method of glucagon-like-peptide-1 Download PDFInfo
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- CN109628479A CN109628479A CN201811587926.XA CN201811587926A CN109628479A CN 109628479 A CN109628479 A CN 109628479A CN 201811587926 A CN201811587926 A CN 201811587926A CN 109628479 A CN109628479 A CN 109628479A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- 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
- C07K14/575—Hormones
- C07K14/605—Glucagons
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/62—DNA sequences coding for fusion proteins
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
- C12P21/06—Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/95—Fusion polypeptide containing a motif/fusion for degradation (ubiquitin fusions, PEST sequence)
Abstract
A kind of recombinant expression method of glucagon-like-peptide-1 includes the following steps the fusion SUMO-rolGLP-1 for 1) constructing a kind of DNA sequence dna containing coding small ubiquitin-related modifier proteins and the DNA sequence dna for encoding Gluca Gen sample polypeptide -1；2) colibacillus engineering of a kind of coli expression carrier including fusion SUMO-rolGLP-1 and high expression fusion SUMO-rolGLP-1 is constructed；3) inducing expression is carried out to colibacillus engineering strain, obtains fusion protein；4) fusion protein is isolated and purified using nickel-Ago-Gel；5) albumen by step 4) purifying uses protease digestion, obtains the mixture of SUMO and rolGLP-1 albumen；6) after the mixture that step 5) obtains is using Ni-FF absorption SUMO albumen, rolGLP-1 albumen is collected by centrifugation in super filter tube.
Description
Technical field
The present invention relates to the recombinant expression methods of glucagon-like-peptide-1.
Background technique
According to the pathogenesis of diabetes, three types can be largely divided into: type 1 diabetes --- also known as insulin according to
Relying property diabetes, diabetes B --- also known as adult-onset diabetes, gestational diabetes mellitus, wherein diabetes B is most normal
See, accounts for about the ninety percent of whole diabetics.
In the enteron aisle of mammal, there is the pancreas hyperglycemia of about 3kD that L cell is secreted under the stimulation absorbed nourishment a kind of
Plain sample peptide -1 (GLP-1) has the function of promoting insulin secretion.GLP-1 by with throughout in pancreas, brain, gastrointestinal tract, the heart
GLP-1 receptor (GLP-1R) in the tissue such as dirty, lung is associated and shows the physiological action of blood glucose-control, but GLP-1R is in pancreas
In abundance to be much higher than its hetero-organization [3].GLP-1R secretion determines that GLP-1 is complicated and extensive in the characteristics of multiple tissue
Physiological function.In terms of physiology, GLP-1 major function includes: 1. to stimulate insulin secretion.GLP-1 is concentration of glucose
Dependent form hormone can just play when blood sugar concentration is higher than normal level and promote insulin secretion effect, mortality is not occurring
Blood sugar reducing function is played under the premise of the risk of hypoglycemia.2. GLP-1 can contain glucagon secretion in conjunction with alpha Cell of islet
[8].3. inhibiting β Apoptosis.Farilla et al. discovery, GLP-1 can stablize the three-dimensional structure of pancreas islet, and islet cells is inhibited to subtract
It is few；Hui [10] et al. discovery, GLP-1 can inhibit apoptotic proteins active, promote the expression of anti-apoptotic proteins, inhibit β to play
The effect of Apoptosis.4. GLP-1 can promote to transcribe insulin gene, it is proliferated islet cells and promotes pancreatic stem cell differentiation
β cell is proliferated for β cell.5. inhibiting bowel movement and gastric emptying, appetite is reduced, promotes liver, fat, musculature glycogen
Synthesis.
China is most one of the country of diabetes B number of patients, and disease incidence is up to 11.6%, and diabetes have become prestige
The serious public health problem of people's the level of physical diathesis is coerced, it is heavy that annual health care costs are brought to diabetic and country
The burden of weight, it is extremely urgent to develop the new treatment diabetes medicament having no toxic side effect.Since GLP-1 makees without gastrointestinal tract pair
With it is anti-mainly to develop three classes GLP-1 class in the market for protection beta Cell of islet, the safe hypoglycemic effect for promoting insulin secretion
Diabetes medicament: DDP-4 inhibitor, GLP-1R agonist, GLP-1 analog have shown good application prospect at this stage,
And part has been used for clinic.
Small ubiquitin-related modifier proteins (SUMO) are a kind of height for being distributed widely in about 15kD in various eukaryotic cells
Conservative little albumen, its structure and reactive mode is similar to ubiquitin, but has the function of different.SUMOization modification and ubiquitin
The common ground for changing modification is lysine residue that binding site is destination protein, cohesive process be required to activating enzymes, desmoenzyme,
The participation of ligase, but the activating enzymes of ubiquitination and ligase huge number, and both enzymes of SUMOization modification only have
One kind, this is likely to be the reason of protein stability after SUMO is modified significantly improves.Stability in addition to improving albumen,
SUMO can also inhibit protein degradation because the binding site of SUMO and ubiquitin is lysine, the two can it is emulative with
Binding Capacity, to play antiprotease hydrolysis function.In addition, SUMOization modification can also keep the steady of antigen-4 fusion protein gene group
It is qualitative, it folds polypeptide normally, improves the solubility of fusion protein.
Glucagon-like-peptide-1 is easily degraded as a kind of small molecular protein and is difficult to purify, and how to improve existing
GLP-1 recombinant expression method improves its yield and simplifies purification step as urgent problem to be solved in the prior art.
Summary of the invention
To solve aforementioned technical problem, the sequence of GLP-1 is recombinated to obtain rolGLP-1 by the present invention, then by SUMO with
It, is then cloned on the pET 22b expression vector in escherichia expression system, constructs a weight by rolGLP-1 fusion
Group SUMO-rolGLP-1 prokaryotic expression carrier, makes it in E. coli, isolates and purifies SUMO- using Ni-FF
RolGLP-1, then the SUMO-rolGLP-1 albumen after SUMO protease digestion is adsorbed into SUMO albumen, super filter tube using Ni-FF
RolGLP-1 is collected by centrifugation.
The technical solution adopted by the present invention is to provide a kind of recombinant expression method of glucagon-like-peptide-1, feature
It is that the described method comprises the following steps
1) it constructs a kind of containing the DNA sequence dna for encoding small ubiquitin-related modifier proteins (SUMO) and coding Gluca Gen
The fusion SUMO-rolGLP-1 of the DNA sequence dna of sample polypeptide -1 (rolGLP-1)；The fusion SUMO-rolGLP-1
In, in the DNA sequence dna of the coding Gluca Gen sample polypeptide -1 (rolGLP-1), dipeptidyl peptidase Ⅳ and trypsase are known
Other site has been removed, and the 8th Ala is replaced by Ser, and the 26th and 34 Lys is replaced by Gln and Asp respectively；It is described to melt
The DNA sequence dna of gene SUMO-rolGLP-1 is closed as shown in sequence 1.
In the step 1), the method for obtaining the fusion SUMO-rolGLP-1 is will by the method for recombinant PCR
The DNA sequence dna for encoding SUMO, which merge with the DNA sequence dna of coding rolGLP-1, obtains the fusion SUMO-rolGLP-
1；Detailed process includes: to be merged SUMO gene with rolGLP-1 gene by the method for recombinant PCR, by SUMO-GLP-1
Fusion gene cloning obtains pMD18T-Simple-SUMO-GLP-1, the carrier is i.e. such as sequence 1 to pMD18T-Simple carrier
Shown in fusion SUMO-GLP-1 gene order.
2) a kind of coli expression carrier pNK-EC-SUMO- including fusion SUMO-rolGLP-1 is constructed
The colibacillus engineering NK-SUMO-rolGLP-1 of rolGLP-1 and high expression fusion SUMO-rolGLP-1, it is specific
Step includes
2.1) the fusion SUMO-rolGLP-1 that step 1) obtains is cloned into pMD18T-Simple carrier, is wrapped
The cloning vector pMD18T-Simple-SUMO-rolGLP-1 of the SUMO-rolGLP-1 containing fusion；
2.2) coli expression carrier pNK-EC-SUMO-rolGLP-1, the structure of the coli expression carrier are provided
It is as follows to build process:
The fusion SUMO- in cloning vector pMD18T-Simple-SUMO-rolGLP-1 that step 2.1) is obtained
RolGLP-1 carries out digestion with BamH I and Nde I restriction endonuclease, and digestion fusion SUMO-rolGLP-1 after the recovery is connected into
PET-22b (+) carrier, building obtain coli expression carrier pNK-EC-SUMO-rolGLP-1；
2.3) Escherichia coli are converted with the coli expression carrier pNK-EC-SUMO-rolGLP-1 that step 2.2) obtains
BL21 (DE3) screens the bacterial strain of wherein highly expressed fusion SUMO-rolGLP-1 as colibacillus engineering NK-
SUMO-rolGLP-1。
3) inducing expression is carried out to colibacillus engineering strain NK-SUMO-rolGLP-1, obtains SUMO-rolGLP-1 and melts
Hop protein.
4) nickel-Ago-Gel (Ni-FF) purification procedures 3 are used) obtained SUMO-rolGLP-1 fusion protein,
5) the SUMO-rolGLP-1 albumen by step 4) purifying uses SUMO protease digestion SUMO-rolGLP-1, obtains
The mixture of SUMO and rolGLP-1 polypeptide
6) after the mixture for the SUMO and rolGLP-1 polypeptide that step 5) obtains is using Ni-FF absorption SUMO albumen, ultrafiltration
RolGLP-1 albumen is collected by centrifugation in pipe.
A kind of recombinant expression method of glucagon-like-peptide-1 provided by the invention is obtained with gene manipulation techniques
SUMO-rolGLP-1 fusion is cloned into coli expression carrier and obtains pNK-EC-SUMO-rolGLP-1, and
Colibacillus engineering strain NK-SUMO-rolGLP- is constructed based on coli expression carrier pNK-EC-SUMO-rolGLP-1
1, by the inducing expression to colibacillus engineering strain, expression product is isolated and purified using affinity chromatography, be can get
SUMO-rolGLP-1 albumen is carried out digestion using SUMO protease, produced after digestion by high-purity SUMO-rolGLP-1 albumen
Object is using the rolGLP-1 albumen that can be obtained high-purity after purification.
Detailed description of the invention
Fig. 1 is coli expression carrier pNK-EC-SUMO-rolGLP-1 described in step 2.2) in specific embodiment
Building process schematic diagram；
Fig. 2 is the gel for the fusion SUMO-rolGLP-1 that step 1) is obtained by recombinant PCR in specific embodiment
Electrophoretogram；
Fig. 3 carries out NK-SUMO-rolGLP-1 with colibacillus engineering strain for step 3) in specific embodiment and induces table
Up to the inducing expression and soluble analysis gel electrophoresis figure for obtaining SUMO-rolGLP-1 fusion protein.
Fig. 4 carries out NK-SUMO-rolGLP-1 with colibacillus engineering strain for step 3) in specific embodiment and induces table
Up to the SDS-PAGE electrophoresis detection and Western blot testing result figure for obtaining SUMO-rolGLP-1 fusion protein
Fig. 5 is to purify SUMO-rolGLP-1 albumen and digestion products in step 4) and step 5) in specific embodiment
Gel electrophoresis figure；
Fig. 6 is the gel electrophoresis figure of the rolGLP-1 albumen of step 6) after purification in specific embodiment.
Specific embodiment
1) is melted the DNA sequence dna of the DNA sequence dna for encoding SUMO and coding rolGLP-1 by the method for recombinant PCR
It closes and obtains the fusion SUMO-rolGLP-1.
PCR primer used in the recombinant PCR is the DNA sequencing fragment and coding rolGLP-1 according to coding SUMO
DNA sequencing fragment design, comprising:
Primer 1, sequence are SEQ ID NO:2
Primer 2, sequence are SEQ ID NO:3
Primer 3, sequence are SEQ ID NO:4
Primer 4, sequence are SEQ ID NO:5
The specific method of the recombinant PCR be using primer 1 and primer 2 as upstream and downstream, be to encode the DNA sequence dna of SUMO
Template expands SUMO-Linker gene, PCR reaction condition are as follows: 94 DEG C of initial denaturation 5min, 94 DEG C of denaturation 30s, 60 DEG C are annealed
1min 72 DEG C, extends 1min, recycles 20 times, 72 DEG C of extension 5min；
It is expanded using primer 3 and primer 4 as upstream and downstream primer using the DNA sequence dna for encoding rolGLP-1 as template
Linker-rolGLP-1 gene, PCR reaction condition are 94 DEG C of initial denaturation 5min, 94 DEG C of denaturation 30s, 60 DEG C of annealing 1min, 72 DEG C
Extend 1min, recycles 20 times, 72 DEG C re-extend 5min.
It is respectively upstream and downstream primer with primer 1 and primer 4, with SUMO-Linker gene and Linker-rolGLP-1 base
Because template obtains SUMO-rolGLP-1 gene, PCR reaction condition: 94 DEG C of initial denaturation 5min, 94 DEG C of denaturation 30s, 60 DEG C are annealed
1min, 72 DEG C of extension 1min are recycled 20 times, and 72 DEG C re-extend 5min.Fusion SUMO-rolGLP-1 is obtained, sequence is such as
Shown in sequence 1, the gel electrophoresis figure of amplified production is as shown in Fig. 2, swimming lane 1 is 100bp DNA Ladder Marker, swimming in figure
Road 2~5 is the fusion SUMO-rolGLP-1 product that recombinant PCR expands.
The DNA sequence dna of the SUMO-Linker gene is as shown in SEQ ID NO:6, the Linker-rolGLP-1 gene
DNA sequence dna as shown in SEQ ID NO:7.
2) a kind of coli expression carrier pNK-EC-SUMO- including fusion SUMO-rolGLP-1 is constructed
The colibacillus engineering NK-SUMO-rolGLP-1 of rolGLP-1 and high expression fusion SUMO-rolGLP-1, it is specific
Step includes
2.1) the fusion SUMO-rolGLP-1 that step 1) obtains is cloned into pMD18T-Simple carrier, is wrapped
The cloning vector pMD18T-Simple-SUMO-rolGLP-1 of the SUMO-rolGLP-1 containing fusion；
2.2) coli expression carrier pNK-EC-SUMO-rolGLP-1, the structure of the coli expression carrier are provided
It is as follows to build process:
The fusion SUMO- in cloning vector pMD18T-Simple-SUMO-rolGLP-1 that step 2.1) is obtained
RolGLP-1 carries out digestion with BamH I and Nde I restriction endonuclease, and digestion fusion SUMO-rolGLP-1 after the recovery is connected into
PET-22b (+) carrier, building obtain coli expression carrier pNK-EC-SUMO-rolGLP-1；The Bacillus coli expression
Carrier pNK-EC-SUMO-rolGLP-1 building process schematic diagram is as shown in Figure 1.
2.3) with step 2.2) obtain through correct coli expression carrier pNK-EC-SUMO-rolGLP-1 is sequenced
It converts e. coli bl21 (DE3), screens the bacterial strain of wherein highly expressed fusion SUMO-rolGLP-1 as Escherichia coli
Engineering bacteria NK-SUMO-rolGLP-1.
3) inducing expression is carried out to colibacillus engineering strain NK-SUMO-rolGLP-1, obtains SUMO-rolGLP-1 and melts
Hop protein
By colibacillus engineering strain NK-SUMO-rolGLP-1, in 37 DEG C, 180rpm shaking flask culture to OD600 be 0.6-
Final concentration of 1mmol/L inducer IPTG is added when 0.8, and 4h is induced under the conditions of 37 DEG C, centrifuging and taking thallus carries out ultrasonication,
Extract proteins, which carry out 15%SDS-PAGE electrophoresis detection, proves successful expression SUMO-GLP-1 fusion protein.Western blot
(immunoblotting) the result shows that, the antibody of SUMO-rolGLP-1 fusion protein and GLP-1 specificity can carry out specificity
In conjunction with.For the gel electrophoresis figure of inducing expression and soluble analysis as shown in figure 3, in figure, M is albumen marker, and swimming lane 1 is the positive
It compares (whole cell after induction), swimming lane 2 is to precipitate after induction is broken, and swimming lane 3 is supernatant after induction is broken, and swimming lane 4 is negative right
According to (before induction).The SDS-PAGE electrophoresis detection and Western blot of SUMO-rolGLP-1 fusion protein are detected such as Fig. 4 institute
Show, swimming lane M in Fig. 4 (A): standard Marker；Swimming lane 1: before induction；Swimming lane 2,3: supernatant after ultrasonication, Fig. 4 (B) are SUMO-
The Wester-blot result of rolGLP-1 fusion protein.
4) SUMO-rolGLP-1 is isolated and purified using Ni-FF
Supernatant after step 3) ultrasonic treatment is subjected to affinity chromatography (Ni-FF chromatographic column) purification process, is obtained high-purity
The SUMO-rolGLP-1 albumen of degree.It is concentrated using the super filter tube of 10kDa, desalination.Purifying
5) SUMO protease digestion SUMO-rolGLP-1 albumen is used, the mixture of SUMO and rolGLP-1 polypeptide is obtained
Digestion will be carried out with SUMO enzyme by the SUMO-rolGLP-1 albumen of concentration desalination in step 4), obtains SUMO and rolGLP-1
The mixture of albumen.The gel electrophoresis figure of SUMO-rolGLP-1 and digestion products are purified as shown in figure 5, M is ultra-low molecular in figure
Measure albumen marker, swimming lane 1SUMO-rolGLP-1 albumen；Swimming lane 2-6 is product after SUMO-rolGLP-1 proteolytic cleavage.
6) after by digestion products using Ni-FF absorption SUMO albumen, rolGLP-1 albumen is collected by centrifugation by step in super filter tube
5) mixture for the SUMO and rolGLP-1 albumen that digestion obtains carries out affinity chromatography (Ni-FF chromatographic column) purification process, SUMO
Albumen can be adsorbed by Ni-FF, then be centrifuged with the super filter tube of 3kDa, and collecting filtrate is rolGLP-1 albumen, the purifying
The liquid chromatogram content of rolGLP-1 albumen afterwards is 91.81%, gel electrophoresis figure such as Fig. 6 of rolGLP-1 albumen after purification
Shown, in figure, M is Ultra-low molecular weight albumen marker, and swimming lane 1, swimming lane 2 are rolGLP-1 after purification.
Claims (2)
1. a kind of recombinant expression method of glucagon-like-peptide-1, it is characterized in that the described method comprises the following steps
1) it constructs a kind of more containing the DNA sequence dna for encoding small ubiquitin-related modifier proteins (SUMO) and coding Gluca Gen sample
The fusion SUMO-rolGLP-1 of the DNA sequence dna of peptide -1 (rolGLP-1)；In the fusion SUMO-rolGLP-1, institute
In the DNA sequence dna for the coding Gluca Gen sample polypeptide -1 (rolGLP-1) stated, dipeptidyl peptidase Ⅳ and trypsase identify position
Point has been removed, and the 8th Ala is replaced by Ser, and the 26th and 34 Lys is replaced by Gln and Asp respectively；The fusion base
Because the DNA sequence dna of SUMO-rolGLP-1 is as shown in sequence 1；
In the step 1), the method for obtaining the fusion SUMO-rolGLP-1 is that will be encoded by the method for recombinant PCR
The DNA sequence dna of SUMO, which merge with the DNA sequence dna of coding rolGLP-1, obtains the fusion SUMO-rolGLP-1；Tool
Body process includes: to be merged SUMO gene with rolGLP-1 gene by the method for recombinant PCR, and SUMO-GLP-1 is merged
Gene cloning obtains pMD18 T-Simple-SUMO-GLP-1, the carrier is as shown in sequence 1 to pMD18 T-Simple carrier
Fusion SUMO-GLP-1 gene order；
2) a kind of coli expression carrier including fusion SUMO-rolGLP-1 is constructed
The colibacillus engineering NK-SUMO- of pNK-EC-SUMO-rolGLP-1 and high expression fusion SUMO-rolGLP-1
rolGLP-1；
3) inducing expression is carried out to colibacillus engineering strain NK-SUMO-rolGLP-1, obtains SUMO-rolGLP-1 and merges egg
It is white；
4) nickel-Ago-Gel (Ni-FF) purification procedures 3 are used) obtained SUMO-rolGLP-1 fusion protein；
5) the SUMO-rolGLP-1 albumen by step 4) purifying uses SUMO protease digestion SUMO-rolGLP-1, obtains SUMO
With the mixture of rolGLP-1 albumen；
6) mixture for the SUMO and rolGLP-1 albumen that step 5) obtains using Ni-FF absorption SUMO albumen after, super filter tube from
The heart collects rolGLP-1 albumen.
2. a kind of recombinant expression method of glucagon-like-peptide-1 as described in claim 1, it is characterized in that the step 2)
It specifically includes:
2.1) the fusion SUMO-rolGLP-1 that step 1) obtains is cloned into pMD18 T-Simple carrier, is included
The cloning vector pMD18 T-Simple-SUMO-rolGLP-1 of fusion SUMO-rolGLP-1；
2.2) coli expression carrier pNK-EC-SUMO-rolGLP-1, the building of the coli expression carrier are provided
Journey is as follows:
The fusion SUMO- in cloning vector pMD18 T-Simple-SUMO-rolGLP-1 that step 2.1) is obtained
RolGLP-1 carries out digestion with BamH I and Nde I restriction endonuclease, and digestion fusion SUMO-rolGLP-1 after the recovery is connected into
PET-22b (+) carrier, building obtain coli expression carrier pNK-EC-SUMO-rolGLP-1；
2.3) e. coli bl21 is converted with the coli expression carrier pNK-EC-SUMO-rolGLP-1 that step 2.2) obtains
(DE3), the bacterial strain of wherein highly expressed fusion SUMO-rolGLP-1 is screened as colibacillus engineering NK-SUMO-
rolGLP-1。
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| WO2021135765A1 (en) * | 2019-12-31 | 2021-07-08 | 翰宇药业（武汉）有限公司 | Salt conversion method for glp-1 analogue |
| WO2022032991A1 (en) * | 2020-08-10 | 2022-02-17 | 安徽新熙盟生物科技有限公司 | Method for efficiently expressing and purifying mannanase, homologues thereof and glp-1 recombinant fusion protein, and use thereof |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2021135765A1 (en) * | 2019-12-31 | 2021-07-08 | 翰宇药业（武汉）有限公司 | Salt conversion method for glp-1 analogue |
| CN113121675A (en) * | 2019-12-31 | 2021-07-16 | 翰宇药业（武汉）有限公司 | Salt conversion method of GLP-1 analogue |
| WO2022032991A1 (en) * | 2020-08-10 | 2022-02-17 | 安徽新熙盟生物科技有限公司 | Method for efficiently expressing and purifying mannanase, homologues thereof and glp-1 recombinant fusion protein, and use thereof |
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Application publication date: 20190416 |