CN114457099B - Biological fermentation preparation method of cable Ma Lutai core peptide chain - Google Patents

Biological fermentation preparation method of cable Ma Lutai core peptide chain Download PDF

Info

Publication number
CN114457099B
CN114457099B CN202111556110.2A CN202111556110A CN114457099B CN 114457099 B CN114457099 B CN 114457099B CN 202111556110 A CN202111556110 A CN 202111556110A CN 114457099 B CN114457099 B CN 114457099B
Authority
CN
China
Prior art keywords
peptide chain
fusion protein
core peptide
lutai
expression
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Active
Application number
CN202111556110.2A
Other languages
Chinese (zh)
Other versions
CN114457099A (en
Inventor
陈本顺
李大伟
徐春涛
何伟
尹强
张维冰
邱磊
江涛
尹斌
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Jiangsu Alpha Pharmaceutical Co ltd
Original Assignee
Jiangsu Alpha Pharmaceutical Co ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Jiangsu Alpha Pharmaceutical Co ltd filed Critical Jiangsu Alpha Pharmaceutical Co ltd
Priority to CN202111556110.2A priority Critical patent/CN114457099B/en
Publication of CN114457099A publication Critical patent/CN114457099A/en
Application granted granted Critical
Publication of CN114457099B publication Critical patent/CN114457099B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • 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
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
    • C07K2319/21—Fusion polypeptide containing a tag with affinity for a non-protein ligand containing a His-tag
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/50—Fusion polypeptide containing protease site

Abstract

The application relates to the technical field of bioengineering, in particular to a biological fermentation preparation method of a core peptide chain of a cable Ma Lutai, wherein the core peptide chain is Lys 26 Arg 34 GLP‑1(9‑37)、Lys 26 Arg 34 GLP-1 (10-37) or Lys 26 Arg 34 GLP-1 (11-37); the specific method comprises the steps of coupling a core peptide chain with endonuclease or mutant thereof required by fusion protein to obtain a fusion protein DNA sequence, constructing an expression vector of the fusion protein, loading the expression vector into an expression cell, obtaining inclusion bodies after expression of the fusion protein, and carrying out enzyme digestion after renaturation and purification to obtain a product. Compared with the method that enzyme digestion and renaturation are carried out simultaneously, the method has the advantages of no need of adding additional endonuclease, low cost, economy, high efficiency and environmental friendliness.

Description

Biological fermentation preparation method of cable Ma Lutai core peptide chain
Technical Field
The application relates to the technical field of bioengineering, in particular to a preparation method of a cable Ma Lutai core peptide chain.
Background
Diabetes is a metabolic disease characterized by chronic elevated blood glucose, a progressive disease caused by reduced islet beta cell function and insulin resistance. Insulin secretion defects in islet beta cells and inappropriate secretion of alpha cell glucagon are important causes of deregulation of the insulin and glucagon ratio in type 2 diabetics.
Cord Ma Lutai the original research company is Norand Nord, as the most interesting diabetes drug, cord Ma Lutai is the global seventh approved GLP-1 receptor agonist, and the effect of reducing blood glucose and weight after type 2 diabetics receive 1-week injection of cord Ma Lutai is significantly better than that of placebo, sitagliptin, insulin glargine U100 and other diabetes-related drugs according to a post-hoc analysis of SUSTAIN 1-5 study published on the society of European diabetes research annual meeting (EASD 2017) at 9 and 12.
The biochemical combination preparation method of the cable Ma Lutai has few reports, and in the preparation method of the original research patent (US 9732137), lys in a main peptide chain 26 Arg 34 GLP-1(11-37)/Lys 26 Arg 34 GLP-1 (9-37) is prepared by yeast fermentation, redundant amino acid is cut off by exonuclease, and a target peptide chain is obtained.
The inventor research team reported in patent CN113278061A a method for preparing cable Ma Lutai by combining biological method and chemical method, which designs a route of 3 peptide+28 peptide, wherein MII peptide chain is Lys 26 Arg 34 GLP-1 (10-37) peptide chain, expressed by a fusion protein, the DNA sequence of which comprises a TrxA DNA sequence containing His-tag gene sequence and an MII chain DNA sequence. The method is novel, simple in reaction operation, high in yield and less in impurity. However, the method requires additional commercial endonuclease to carry out enzyme digestion to obtain the target polypeptide Lys 26 Arg 34 GLP-1 (10-37), the market price of the current endonucleases is very expensive, leading to higher cost of the preparation route.
Therefore, how to find a technical route with low cost, high yield and few impurities becomes one of the problems to be solved in the art.
Disclosure of Invention
The application aims to provide a biological fermentation preparation method of a core peptide chain of a cable Ma Lutai, which aims to solve the problems in the prior art.
In order to achieve the above purpose, the present application provides the following technical solutions:
biological fermentation preparation method of cable Ma Lutai core peptide chain, wherein the core peptide chain is Lys 26 Arg 34 GLP-1(9-37)、 Lys 26 Arg 34 GLP-1 (10-37) or Lys 26 Arg 34 GLP-1 (11-37); the specific method comprises the steps of coupling a core peptide chain with endonuclease or mutant thereof required by fusion protein to obtain a fusion protein DNA sequence, constructing an expression vector of the fusion protein, loading the expression vector into an expression cell, obtaining inclusion bodies after expression of the fusion protein, and carrying out enzyme digestion after renaturation and purification to obtain a product.
Wherein the structure of the fusion protein DNA sequence is any one of the following: (1) 6xHis-TEVp mutant-TEV site-core peptide chain; (2) TEV site-core peptide chain-TEV site-6xHis-TEVp or TEVp mutant.
Further, the fusion protein DNA sequence is SEQ ID NO: 1-3.
Further, the operon in the expression vector of the fusion protein is lac operon, and Km antibiotic labeling is used.
Further, the expression cell is Escherichia coli.
Further, the expression cell is E.coli BL21 (DE 3).
Specifically, the biological fermentation preparation method of the cable Ma Lutai core peptide chain comprises the following steps:
1) Synthesizing a coding gene, wherein the coding gene comprises an enzyme cutting site sequence and a core peptide chain sequence, and also comprises a TEVp mutant sequence or a TEVp sequence;
2) Constructing an expression vector of the fusion protein, and loading an expression cell, wherein the expression cell is escherichia coli;
3) After positive bacteria are determined, escherichia coli fermentation is carried out through a fermentation medium, and expression of fusion proteins is promoted;
4) Obtaining inclusion bodies by using cell disruption methods such as ultrasonic disruption or high-pressure homogenizer, and washing and denaturing the inclusion bodies;
5) And renaturation of the fusion protein is carried out, self-digestion reaction is carried out, and the obtained mixed polypeptide is separated and purified to obtain a required cord Ma Lutai core peptide chain sample.
Compared with the prior art, the application has the beneficial effects that:
compared with a chemical method, the biological fermentation preparation method of the cable Ma Lutai core peptide chain has the advantages of mild reaction, convenience in operation and relatively fewer impurities. Compared with the method in the patent CN113278061A, the method skillfully utilizes the insoluble characteristic of TEVp, redesigns the fusion protein structure, replaces the common fusion protein expressed by the KSI in a sinking way, expresses the polypeptide chain in an inclusion body mode, facilitates purification, obtains the target polypeptide by directly carrying out enzyme digestion on the fusion protein after renaturation, is convenient to operate, reduces the additional enzyme digestion step of adding the TEVp or other fusion proteins after renaturation, reduces the residue of exogenous proteins while greatly reducing the cost, reduces the difficulty of later purification and ensures the product yield.
Drawings
FIG. 1 is a schematic diagram of the vector construction of the present application;
FIG. 2 shows Lys 26 Arg 34 GLP-1 (10-37) is exemplified by the first structural schematic of the fusion protein of the application (corresponding to fusion protein A1 in the example and fusion protein A2 in the comparative example);
FIG. 3 shows Lys 26 Arg 34 GLP-1 (10-37) is exemplified, and the second structural schematic of the fusion protein of the application (corresponding to fusion protein B1 and fusion protein B2 in examples).
Detailed Description
The following description of the embodiments of the present application will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present application, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the application without making any inventive effort, are intended to be within the scope of the application.
EXAMPLE 1 DNA sequence Synthesis of fusion protein A1 and construction of plasmid
Total synthesis of TEVp238 delta-mutant-Lys by Shanghai JieRui biology 26 Arg 34 GLP-1 (10-37) DNA sequence, his tag is added to the N end of the sequence, TGA is added to the C end of the sequence, and a primer F is designed:CATATGCATCATCATCATCATCATGGCGAATCTCTGT，R：CTCGAGTTAGCCCCACGACCACGCA (SEQ ID NO: 6-7) and ligating the sequence of interest into a vector, the fusion protein sequence being detailed in SEQ ID NO:1, the structure of which is shown in figure 2, is constructed to express His tag-TEVp 238 delta-mutant-TEV protease site-Lys 26 Arg 34 Recombinant fusion expression vector of GLP-1 (10-37) as shown in FIG. 1. Wherein TEVp238 delta-variant is one of the TEVp mutants.
Example 2 preparation of expression engineering bacteria and Positive verification
The recombinant expression vector in example 1 was subjected to gel running verification and then transformed into E.coli BL21 competence, positive colonies were screened out by Km antibiotics, full positive colonies were selected, DNA sequencing verification was performed after amplification, and glycerol bacteria preservation was performed after confirmation.
Example 3 expression of fusion protein A1
Fermenting the preserved recombinant strain with 15L fermenter, and culturing at 37deg.C to OD 600 At 20, IPTG was added at 0.2mM, and the OD of the strain was detected after 0h of induction 600 Induced to OD 600 And (5) placing the cells in a tank, centrifuging at 6000rpm for 10min, and collecting the cells.
EXAMPLE 4 acquisition of inclusion bodies
The cells obtained in example 3 were homogenized and crushed under high pressure, and the inclusion body pellet was collected by centrifugation. Washing with buffer solution (Tris-HCl 8.0) for 3 times, dissolving in buffer solution, adding PMSF, ultrasonic crushing (300W power, running for 5s at intervals of 5s for 10 min), centrifuging (10000 rpm,8 min) to obtain inclusion body and impurity precipitate, washing with detergent to purify the precipitate, centrifuging, and collecting inclusion body.
EXAMPLE 5 preliminary purification of fusion protein A1 (TEVp 238 delta)
Adding the inclusion body obtained in the example 4 into a denaturation buffer containing urea with a final concentration of 8M, wherein the denaturation buffer is prepared by adding 500mM NaCl,20mM Tris-HCl,5mM imidazole, 8M urea and ddH2O to 500mL, the pH is 7.9, ice compress is carried out for 30-60 min to dissolve the inclusion body, loading the inclusion body on a nickel column (balanced by double column volume buffer), loading the inclusion body on the nickel column for 2-4 times repeatedly, and preparing an elution buffer: 500mM NaCl,20mM Tris-HCl,0.5M imidazole, 8M urea, ddH 2 O is supplemented to 500mL, and the fusion protein after preliminary purification is obtained by eluting with a column volume elution buffer solution with pH of 7.9,2-5.
EXAMPLE 6 preparation of target peptide Lys from cleavage reaction of fusion protein A1 (TEVp 238. DELTA.) 26 Arg 34 GLP-1 (10-37)
Diluting the initially purified denatured fusion protein solution obtained in example 5 to a concentration of 2mg/mL, placing into a dialysis bag, and performing renaturation digestion buffer replacement at 4 ℃ with a buffer ratio of 50mM Tris-HCl,0.5 mM EDTA,5% glycerol, ddH 2 The O is supplemented to 1L, and the pH value is 8.0-8.5. After 16h, the cleavage reaction is stopped, the conversion rate is detected, and purification is carried out by preparing a liquid phase or an akta purification system, so that the target peptide chain A (the amino acid sequence is shown in SEQ ID NO: 5) is obtained, and the conversion rate of the fusion protease cleavage reaction after renaturation is about 48% after liquid phase detection.
EXAMPLE 7 preparation of the peptide Lys of interest by fusion protein B1 (TEVp) 26 Arg 34 GLP-1 (11-37) or peptide Lys 26 Arg 34 GLP-1 (10-37) or peptide Lys 26 Arg 34 GLP-1(9-37)
With Lys 26 Arg 34 GLP-1 (10-37) as an example, reference examples 1 to 5 were used to prepare denatured and initially purified fusion protein B1 (see FIG. 3 for details of structure, TEV site-Lys 26 Arg 34 GLP-1 (10-37) -TEV site-6xHis-TEVp, the sequence of which is shown in SEQ ID NO: 2) Diluting the obtained primary purified denatured fusion protein solution to a concentration of 2mg/mL, and loading into a low molecular weight cut-offIn a dialysis bag, the renaturation enzyme digestion buffer solution is replaced at 4 ℃ and the buffer solution is prepared from 50mM Tris-HCl,0.5 mM EDTA,5% glycerol and ddH 2 The O is supplemented to 1L, and the pH value is 8.0-8.5. After 24h, the cleavage reaction is stopped, the conversion is detected and purified by preparing a liquid phase or akta purification system to obtain the target peptide Lys 26 Arg 34 GLP-1 (10-37) has about 71% conversion rate of fusion protease cleavage reaction after renaturation is successful through liquid phase detection.
Example 8 preparation of target peptide Lys by fusion protein B2 (TEVp 238. DELTA.) 26 Arg 34 GLP-1 (11-37) or peptide Lys 26 Arg 34 GLP-1 (10-37) or peptide Lys 26 Arg 34 GLP-1(9-37)
With Lys 26 Arg 34 GLP-1 (10-37) as an example, reference examples 1 to 5 were used to prepare denatured and initially purified fusion protein B2 (see FIG. 3 for details of structure, TEV site-Lys 26 Arg 34 GLP-1 (10-37) -TEV site-6xHis-TEVp238 delta-variant, SEQ ID NO: 3) Diluting the obtained primary purified denatured fusion protein solution to a concentration of 2mg/mL, loading into a low molecular weight cutoff dialysis bag, and performing renaturation enzyme digestion buffer replacement at 4 ℃ with 50mM Tris-HCl,0.5 mM EDTA,5% glycerol, and ddH 2 The O is supplemented to 1L, and the pH value is 8.0-8.5. After 24h, the cleavage reaction is stopped, the conversion is detected and purified by preparing a liquid phase or akta purification system to obtain the target peptide Lys 26 Arg 34 GLP-1 (10-37) has about 74 percent of conversion rate of fusion protease cleavage reaction after renaturation is successful through liquid phase detection.
To highlight the beneficial effects of the fusion protein structure of the present application, the following comparative experiments are exemplified.
Comparative example preparation of the peptide Lys of interest by fusion protein A2 (TEVp) 26 Arg 34 GLP-1(10-37)
With Lys 26 Arg 34 GLP-1 (10-37) as an example, reference examples 1-5 were used to prepare denatured and initially purified fusion protein A2 (endonucleases TEVp, structure details are shown in FIG. 2, 6XHis-TEVp-TEV site-Lys 26 Arg 34 GLP-1 (10-37), the sequence of which is shown in SEQ ID NO: 4) Denaturation after preliminary purification obtainedDiluting the fusion protein solution to a concentration of 2mg/mL, placing into a dialysis bag, and performing renaturation enzyme digestion buffer solution replacement at 4 ℃ with a buffer solution ratio of 50mM Tris-HCl,0.5 mM EDTA,5% glycerol and ddH 2 The O is supplemented to 1L, and the pH value is 8.0-8.5. After 16h, the cleavage reaction was stopped, the conversion was detected and purified by a preparative liquid phase or akta purification system to obtain the target peptide chain A, which was subjected to liquid phase detection and renaturation to give a fusion protein cleavage reaction conversion of about 39%.
As can be seen from the above examples and comparative examples, the application does not need to add additional endonuclease, and has low cost, economy and high efficiency; in addition, the conversion rate of the fusion protease digestion reaction of the three structures is better than that of the fusion protease digestion reaction of the structure of the 6xHis-TEVp-TEV site-core peptide chain, the structure of the 6xHis-TEVp mutant-TEV site-core peptide chain-TEV site-6xHis-TEVp mutant-TEV site.
Although embodiments of the present application have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made therein without departing from the principles and spirit of the application, the scope of which is defined in the appended claims and their equivalents.
Sequence listing
<110> Jiangsu alpha pharmaceutical Co., ltd
<120> biological fermentation preparation method of core peptide chain of Soxhlet Ma Lutai
<160> 7
<170> SIPOSequenceListing 1.0
<210> 1
<211> 885
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 1
atgcatcatc atcatcatca tggcgaatct ctgtttaaag gtccgcgtga ttataatccg 60
attagttcta ccatttgtca tctgaccaat gaatcagatg gccataccac ctcactgtat 120
ggcattggct ttggcccgtt tattattacc aataaacatc tgtttcgtcg taataatggc 180
accctgttag ttcagtcact gcatggtgtg tttaaagtta aaaataccac caccttacag 240
cagcatctga ttgatggtcg cgatatgatt attattcgta tgccgaaaga ttttccgccg 300
tttccgcaga aactgaaatt tcgcgaaccg cagcgtgaag aacgcatttg tctggtgacc 360
accaattttc agaccaaatc tatgtcttct atggtgagtg atacctcttg tacctttccg 420
agtagcgatg gtattttttg gaaacattgg attcagacca aagatggcca ggcaggctct 480
ccgttagtgt caacccgcga tggctttatt gtgggcattc atagcgcaag taattttacc 540
aataccaata attattttac ctcagtgccg aaaaatttta tggaactgct gaccaatcag 600
gaagcacagc agtgggtgag cggttggcgc ctgaatgccg atagcgtgct gtggggcggc 660
cataaagtgt ttatggttaa accggaagaa ccgtttcagc cggttaaaga agcaacccag 720
ctgatgaatg aactggaagc ggcggcgaaa gaagcggcag cgaaagaggc tgcggctaaa 780
gaaaacctgt atttccaagg tacctttacc agcgatgtta gcagctacct ggagggtcaa 840
gcggcgaagg agttcattgc gtggctggtg cgtggtcgtg gctaa 885
<210> 2
<211> 918
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 2
atggaaaacc tgtatttcca aggtaccttt accagcgatg ttagcagcta cctggagggt 60
caagcggcga aggagttcat tgcgtggctg gtgcgtggtc gtggcgaaaa cctgtatttc 120
caaggtcatc atcatcatca tcatgaagcg gcggcgaaag aagcggcagc gaaagaggct 180
gcggctaaag gcgaatctct gtttaaaggt ccgcgtgatt ataatccgat tagttctacc 240
atttgtcatc tgaccaatga atcagatggc cataccacct cactgtatgg cattggcttt 300
ggcccgttta ttattaccaa taaacatctg tttcgtcgta ataatggcac cctgttagtt 360
cagtcactgc atggtgtgtt taaagttaaa aataccacca ccttacagca gcatctgatt 420
gatggtcgcg atatgattat tattcgtatg ccgaaagatt ttccgccgtt tccgcagaaa 480
ctgaaatttc gcgaaccgca gcgtgaagaa cgcatttgtc tggtgaccac caattttcag 540
accaaatcta tgtcttctat ggtgagtgat acctcttgta cctttccgag tagcgatggt 600
attttttgga aacattggat tcagaccaaa gatggccagg caggctctcc gttagtgtca 660
acccgcgatg gctttattgt gggcattcat agcgcaagta attttaccaa taccaataat 720
tattttacct cagtgccgaa aaattttatg gaactgctga ccaatcagga agcacagcag 780
tgggtgagcg gttggcgcct gaatgccgat agcgtgctgt ggggcggcca taaagtgttt 840
atggttaaac cggaagaacc gtttcagccg gttaaagaag caacccagct gatgaatgaa 900
ctggtgtata gccagtaa 918
<210> 3
<211> 906
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 3
atggaaaacc tgtatttcca aggtaccttt accagcgatg ttagcagcta cctggagggt 60
caagcggcga aggagttcat tgcgtggctg gtgcgtggtc gtggcgaaaa cctgtatttc 120
caaggtcatc atcatcatca tcatgaagcg gcggcgaaag aagcggcagc gaaagaggct 180
gcggctaaag gcgaatctct gtttaaaggt ccgcgtgatt ataatccgat tagttctacc 240
atttgtcatc tgaccaatga atcagatggc cataccacct cactgtatgg cattggcttt 300
ggcccgttta ttattaccaa taaacatctg tttcgtcgta ataatggcac cctgttagtt 360
cagtcactgc atggtgtgtt taaagttaaa aataccacca ccttacagca gcatctgatt 420
gatggtcgcg atatgattat tattcgtatg ccgaaagatt ttccgccgtt tccgcagaaa 480
ctgaaatttc gcgaaccgca gcgtgaagaa cgcatttgtc tggtgaccac caattttcag 540
accaaatcta tgtcttctat ggtgagtgat acctcttgta cctttccgag tagcgatggt 600
attttttgga aacattggat tcagaccaaa gatggccagg caggctctcc gttagtgtca 660
acccgcgatg gctttattgt gggcattcat agcgcaagta attttaccaa taccaataat 720
tattttacct cagtgccgaa aaattttatg gaactgctga ccaatcagga agcacagcag 780
tgggtgagcg gttggcgcct gaatgccgat agcgtgctgt ggggcggcca taaagtgttt 840
atggttaaac cggaagaacc gtttcagccg gttaaagaag caacccagct gatgaatgaa 900
ctgtga 906
<210> 4
<211> 897
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 4
atgcatcatc atcatcatca tggcgaatct ctgtttaaag gtccgcgtga ttataatccg 60
attagttcta ccatttgtca tctgaccaat gaatcagatg gccataccac ctcactgtat 120
ggcattggct ttggcccgtt tattattacc aataaacatc tgtttcgtcg taataatggc 180
accctgttag ttcagtcact gcatggtgtg tttaaagtta aaaataccac caccttacag 240
cagcatctga ttgatggtcg cgatatgatt attattcgta tgccgaaaga ttttccgccg 300
tttccgcaga aactgaaatt tcgcgaaccg cagcgtgaag aacgcatttg tctggtgacc 360
accaattttc agaccaaatc tatgtcttct atggtgagtg atacctcttg tacctttccg 420
agtagcgatg gtattttttg gaaacattgg attcagacca aagatggcca ggcaggctct 480
ccgttagtgt caacccgcga tggctttatt gtgggcattc atagcgcaag taattttacc 540
aataccaata attattttac ctcagtgccg aaaaatttta tggaactgct gaccaatcag 600
gaagcacagc agtgggtgag cggttggcgc ctgaatgccg atagcgtgct gtggggcggc 660
cataaagtgt ttatgaataa accggaagaa ccgtttcagc cggttaaaga agcaacccag 720
ctgatgaatg aactggtgta tagccaggaa gcggcggcga aagaagcggc agcgaaagag 780
gctgcggcta aagaaaacct gtatttccaa ggtaccttta ccagcgatgt tagcagctac 840
ctggagggtc aagcggcgaa ggagttcatt gcgtggctgg tgcgtggtcg tggctaa 897
<210> 5
<211> 28
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 5
Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala
1 5 10 15
Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly
20 25
<210> 6
<211> 37
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 6
catatgcatc atcatcatca tcatggcgaa tctctgt 37
<210> 7
<211> 25
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 7
ctcgagttag ccccacgacc acgca 25

Claims (6)

1. A biological fermentation preparation method of a cable Ma Lutai core peptide chain is characterized by comprising the following steps of: the core peptide chain is Lys 26 Arg 34 GLP-1 (10-37); the specific method comprises the steps of coupling a core peptide chain with endonuclease or mutant thereof required by fusion protein to obtain a fusion protein DNA sequence, constructing an expression vector of the fusion protein, loading the expression vector into an expression cell, obtaining inclusion bodies after expression of the fusion protein, and carrying out enzyme digestion after renaturation and purification to obtain a product; the fusion protein DNA sequence is SEQ ID NO: 1-3.
2. The method for the biological fermentation production of a core peptide chain of cord Ma Lutai as claimed in claim 1, wherein: the structure of the fusion protein DNA sequence is any one of the following: (1) 6xHis-TEVp mutant-TEV site-core peptide chain; (2) TEV site-core peptide chain-TEV site-6xHis-TEVp; (3) TEV site-core peptide chain-TEV site-6xHis-TEVp mutant.
3. The method for the biological fermentation production of a core peptide chain of cord Ma Lutai as claimed in claim 2, wherein: the operon in the expression vector of the fusion protein was the lac operon, and Km antibiotic labeling was used.
4. A process for the biological fermentation preparation of the core peptide chain of cord Ma Lutai as claimed in claim 3, wherein: the expression cell is Escherichia coli.
5. The method for the biological fermentation production of a core peptide chain of cord Ma Lutai as claimed in claim 4, wherein: the expression cell is E.coli BL21 (DE 3).
6. The method for the biological fermentation production of the core peptide chain of cord Ma Lutai as claimed in any one of claims 1 to 5, comprising the steps of:
1) Synthesizing a coding gene, wherein the coding gene comprises an enzyme cutting site sequence and a core peptide chain sequence, and also comprises a TEVp mutant sequence or a TEVp sequence;
2) Constructing an expression vector of the fusion protein, and loading an expression cell, wherein the expression cell is escherichia coli;
3) After positive bacteria are determined, escherichia coli fermentation is carried out through a fermentation medium, and expression of fusion proteins is promoted;
4) Obtaining inclusion bodies by using cell disruption methods such as ultrasonic disruption or high-pressure homogenizer, and washing and denaturing the inclusion bodies;
5) And renaturation of the fusion protein is carried out, self-digestion reaction is carried out, and the obtained mixed polypeptide is separated and purified to obtain a required cord Ma Lutai core peptide chain sample.
CN202111556110.2A 2021-12-18 2021-12-18 Biological fermentation preparation method of cable Ma Lutai core peptide chain Active CN114457099B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202111556110.2A CN114457099B (en) 2021-12-18 2021-12-18 Biological fermentation preparation method of cable Ma Lutai core peptide chain

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202111556110.2A CN114457099B (en) 2021-12-18 2021-12-18 Biological fermentation preparation method of cable Ma Lutai core peptide chain

Publications (2)

Publication Number Publication Date
CN114457099A CN114457099A (en) 2022-05-10
CN114457099B true CN114457099B (en) 2023-12-15

Family

ID=81406092

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202111556110.2A Active CN114457099B (en) 2021-12-18 2021-12-18 Biological fermentation preparation method of cable Ma Lutai core peptide chain

Country Status (1)

Country Link
CN (1) CN114457099B (en)

Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108912221A (en) * 2018-07-31 2018-11-30 成都英普博集生物科技有限公司 For producing auxilin, encoding gene, recombination fusion protein, recombinant expression carrier and the preparation method of recombination fusion protein
CN109879970A (en) * 2019-03-14 2019-06-14 美药星（南京）制药有限公司 A kind of fusion protein and its method for preparing Liraglutide intermediate polypeptide
CN110498849A (en) * 2019-09-16 2019-11-26 南京迪维奥医药科技有限公司 A kind of main peptide chain of Suo Malu peptide and preparation method thereof
CN111019926A (en) * 2018-10-10 2020-04-17 上饶市康可得生物科技有限公司 TEV protease variants, fusion proteins thereof, and methods of making and using
WO2020190757A1 (en) * 2019-03-15 2020-09-24 Novetide Ltd. Improved processes for the preparation of semaglutide
CN113278061A (en) * 2021-06-22 2021-08-20 南京林业大学 Method for preparing Somalutide by biochemical method
WO2021249564A1 (en) * 2020-06-11 2021-12-16 宁波鲲鹏生物科技有限公司 Semaglutide derivative, and preparation method therefor and application thereof
CN113801234A (en) * 2020-06-11 2021-12-17 宁波鲲鹏生物科技有限公司 Somaltulipide derivative and application thereof

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GR20140100479A (en) * 2014-09-23 2016-05-05 Novetide, Ltd., Synthesis of liraglutide

Patent Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108912221A (en) * 2018-07-31 2018-11-30 成都英普博集生物科技有限公司 For producing auxilin, encoding gene, recombination fusion protein, recombinant expression carrier and the preparation method of recombination fusion protein
CN111019926A (en) * 2018-10-10 2020-04-17 上饶市康可得生物科技有限公司 TEV protease variants, fusion proteins thereof, and methods of making and using
CN109879970A (en) * 2019-03-14 2019-06-14 美药星（南京）制药有限公司 A kind of fusion protein and its method for preparing Liraglutide intermediate polypeptide
WO2020190757A1 (en) * 2019-03-15 2020-09-24 Novetide Ltd. Improved processes for the preparation of semaglutide
CN110498849A (en) * 2019-09-16 2019-11-26 南京迪维奥医药科技有限公司 A kind of main peptide chain of Suo Malu peptide and preparation method thereof
WO2021249564A1 (en) * 2020-06-11 2021-12-16 宁波鲲鹏生物科技有限公司 Semaglutide derivative, and preparation method therefor and application thereof
CN113801234A (en) * 2020-06-11 2021-12-17 宁波鲲鹏生物科技有限公司 Somaltulipide derivative and application thereof
CN113278061A (en) * 2021-06-22 2021-08-20 南京林业大学 Method for preparing Somalutide by biochemical method

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
胰高血糖素样肽-1融合蛋白在原核系统中的表达;桑延霞;张军军;;生物技术(04);第35-39页 *

Also Published As

Publication number Publication date
CN114457099A (en) 2022-05-10

Similar Documents

Publication Publication Date Title
WO2020182229A1 (en) Fusion protein and method of preparing liraglutide intermediate polypeptide thereof
CN111072783B (en) Method for preparing GLP-1 or analog polypeptide thereof by adopting escherichia coli expression tandem sequence
JP4504014B2 (en) Methods for producing insulinotropic GLP-1 (7-36) polypeptides and / or GLP-1 analogs
CN110724187B (en) Recombinant engineering bacterium for efficiently expressing liraglutide precursor and application thereof
CN106434717A (en) Method for biosynthesis preparation of human GLP-1 polypeptide or analogue thereof
US8877895B2 (en) Prokaryotic expression construct
CN113105536B (en) New proinsulin glargine and method for preparing insulin glargine by using same
CN112979781A (en) Sebastes schlegeli IL-1 beta recombinant protein and preparation method and application thereof
CN114790474B (en) Preparation method of Somalutide
CN114292338B (en) Fusion protein and method for preparing semaglutide intermediate polypeptide by using same
Kim et al. Large-scale refolding and enzyme reaction of human preproinsulin for production of human insulin
CN111548392A (en) Dissolution promoting label and application thereof
CN114457099B (en) Biological fermentation preparation method of cable Ma Lutai core peptide chain
CN110938151B (en) Fusion protein for expressing parathyroid hormone PTH, recombinant plasmid and recombinant engineering bacteria
CN107629129B (en) Method for producing and purifying polypeptides
CN114380903B (en) Insulin or analogue precursor thereof
EP3741774A1 (en) N-terminal fusion partner for producing recombinant polypeptide, and method for producing recombinant polypeptide using same
CN111705032B (en) Prokaryotic expression cell and preparation method and application thereof
CN114807205B (en) Recombinant engineering bacterium for expressing liraglutide precursor and construction method and application thereof
CN114686504A (en) Application of Lpp or mutant thereof as molecular chaperone in secretory expression of recombinant protein in escherichia coli
CN102277327B (en) Colon bacillus for over-expressing RimL and application on preparing N-extrasin alpha acetylate
CN106754945B (en) Production method of toxin Tx4(6-1) tag-free recombinant protein
WO2024087761A1 (en) Method for producing polypeptide from recombinant fusion protein and use thereof
CN114057897B (en) Sebastes schlegeli IL-6 recombinant protein, soluble expression method and application
CN114774397B (en) Bovine enterokinase light chain protein mutant and recombinant fusion protein

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant