Production method of semaglutide precursor

A technology of semaglutide and expression vector, which is applied in the field of genetic engineering and can solve the problems of low purity and low yield.

Active Publication Date: 2020-07-07
VONSUN PHARMATECH CO LTD +2
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] It can be seen from the above structural formula that the molecular formula of semaglutide is C 187 h 291 N 45 o 59 , with a molecular weight of 4113.57, one amino acid has been replaced in the natural GLP-1 molecular structure (the 34th lysine has been changed to arginine), the 8th alanine has been changed to an unnatural amino acid Aib, and in The 26-position lysine is connected to AEEA, glutamic acid and octadecanoic acid fatty chain, that is, the peptide GLP-1 (9-37), His-Aib and AEEA-AEEA-γ-Glu-Octadecanedioic Acid Mono-tert- Butylester consists of three parts. The synthetic method of the intermediate polypeptide GLP-1(9-37) in the prior art mainly adopts chemical synthesis to add, but there are disadvantages of low yield and low purity

Method used

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  • Production method of semaglutide precursor
  • Production method of semaglutide precursor
  • Production method of semaglutide precursor

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Construction of Recombinant Engineering Bacteria

[0033] The semaglutide precursor gene expressed in tandem was integrated with the plasmid pET-27b(+) using the whole genome synthesis technology to obtain sequence cDNA, the restriction enzyme cutting site is NdeI / XhoI, and the induced expression electrophoresis is as follows figure 1 As shown, the recombinant plasmid was transformed into the host Escherichia coli (Escherichia coli) BL21 (DE3) by chemical transformation to construct the recombinant engineering bacteria. After sequencing, the sequence of the engineered bacteria was consistent with the designed sequence.

[0034] Enzymes and Reagents:

[0035] The restriction endonucleases used in the design of molecular biology operations in the examples can be obtained from TAKARA company, and the corresponding operation steps are completely carried out in accordance with the relevant product instructions.

[0036] The agarose gel recovery kit and plasmid extraction k...

Embodiment 2

[0044] High-density fermentation and induced expression

[0045] Inoculate the positive recombinant engineered bacteria selected in the above Example 1 into 10mL LB liquid medium according to the inoculation amount of 1%, and cultivate them at 30°C and 200rpm to OD600=12~15. The inoculum was inoculated in 100mL LB liquid medium, shaken at 200rpm at 30°C, and the OD 600 In the process of about 4, it was inserted into a 2.5L fermentation medium for high-density culture. The initial fermentation temperature is 34°C, the stirring speed is 200rpm, the ventilation rate is 2L / min, and the pH is 6.7, and then the stirring speed and ventilation rate are continuously increased to 800rpm and 8L / min to maintain the dissolved oxygen above 30%. Density fermentation requires a large amount of foreign air. If the supply of foreign air is insufficient, it will not only keep the respiration of the bacteria, limit the production and reproduction of the bacteria, but also accumulate some metabol...

Embodiment 3

[0052] Renaturation of recombinant tandem expressed semaglutide precursor

[0053] Centrifuge the engineered bacterium culture medium after the induced expression in Example 2, take the thalline, add the breaking buffer according to the ratio of 1:10 (w:v), and use the ATS homogenizer 850bar, break the bacteria twice at a frequency of 40Hz, 8500rpm Centrifuge for 30 min to collect inclusion bodies. The obtained semaglutide pre-inclusion bodies expressed in series were stirred evenly with 0.5M Tris-HCl pH 8.5 buffer solution, and then 0.5% SDS and 1% Triton X-100 were added to obtain a solution ratio of 4% (w:v) Stir the protein mixture solution with a magnetic stirrer at room temperature for 3-4 hours to slowly dissolve the precipitate; centrifuge at 10,000 rpm for 10 min at 4°C and discard the precipitate. The obtained supernatant was diluted 10 times with deionized water, and then filtered with a 0.22 μm microporous membrane to obtain the soluble protein.

[0054] Various ...

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Abstract

The invention belongs to the technical field of genetic engineering, and particularly relates to a production method of a semaglutide precursor. According to the production method of the semaglutide precursor, a gene recombination technology is adopted, and compared with chemical synthesis, the production method of the semaglutide precursor has the advantages that impurities are reduced, and the purity and a yield are higher; and the production method of the semaglutide precursor comprises the main steps of introducing a plasmid with a tandem expression GLP-1(9-37) gene sequence into Escherichia coli BL21(DE3) by means of the gene recombination technology at first to construct recombinant engineering bacteria, conducting high-density fermentation and induction to obtain tandem expression protein which expresses the GLP-1(9-37), and carrying out the steps of degeneration, renaturation, digestion, purification and the like to obtain the semaglutide precursor GLP-1(9-37).

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, in particular to a preparation method of a semaglutide precursor. Background technique [0002] Semaglutide (Semaglutide) is a GLP-1 analogue produced by genetic recombination technology. It has high amino acid sequence homology with human GLP-1. The difference from natural GLP-1 is that Semaglutide In terms of pharmacokinetics and pharmacodynamics in humans, it is more suitable for a once-weekly dosing regimen, and it has higher drug stability. When the semaglutide drug is injected subcutaneously, it mainly prolongs the action time of the drug through the following mechanisms: first, it can slow down the absorption through the cross-linking of the drug protein; -IV and other enzymes have a relatively stable ability to prevent degradation, so that they can manage a longer plasma half-life. In patients with type 2 diabetes, its clinical administration showed that a single dose of sem...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/605C12N15/16C12N15/70C12N1/21C07K1/20C07K1/18C12R1/19
CPCC07K14/605C12N15/70
Inventor 文良柱韩宇鹏辛中帅赵珊珊平康康陈慧梅张明义王玉刚杨桦赵梅
Owner VONSUN PHARMATECH CO LTD
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