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Preparation method of liraglutide intermediate polypeptide

A technology of expressing vectors and recombinant vectors, applied in the field of biomedicine

Inactive Publication Date: 2015-05-06
JIANGSU WANBANG BIOPHARMLS +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] According to the above structural formula, the molecular formula of liraglutide is C172H265N43O51, with a molecular weight of 3751.20, is a replacement of one amino acid in the molecular structure of natural GLP-1 (lysine at position 34 is changed to arginine), and at position 26 A 16-carbon palmitoyl fatty acid test chain is added, which is composed of polypeptide GLP-1 (7-37) and Nα-alkanoyl-Glu(ONsu)-OtBu. In the prior art The synthesis method of the intermediate polypeptide GLP-1 (7-37) mainly adopts chemical synthesis, which has the problems of many impurities and low yield

Method used

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  • Preparation method of liraglutide intermediate polypeptide
  • Preparation method of liraglutide intermediate polypeptide
  • Preparation method of liraglutide intermediate polypeptide

Examples

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Embodiment 1

[0037] Example 1 Construction of Recombinant Engineering Bacteria

[0038] Synthesize the gene of hI-GLP-1 fusion protein by conventional chemical synthesis method, insert the obtained sequence cDNA into the corresponding restriction site of plasmid pET-27b(+) through NdeI / XhoI restriction site, and construct the Recombinant plasmids such as figure 1 As shown, the electrophoresis after digestion of the recombinant plasmid is as follows figure 2 As shown, the recombinant plasmid inserted into the gene encoding hI-GLP-1 fusion protein was transformed into the host Escherichia coli by conventional chemical transformation method to construct recombinant engineering bacteria. like Figure 3-4 As shown, after sequencing, the sequence in the engineered bacteria was consistent with the design.

Embodiment 2

[0039] Example 2 High-density fermentation

[0040] Inoculate the positive clone of the recombinant engineered bacteria obtained in Example 1 in 10 mL of LB medium, 30 ° C, 250 rpm for overnight culture, then inoculate in 250 mL of LB medium at a ratio of about 0.4%, and shake to OD 600When the value reached 2, it was used as a seed liquid, and was inserted into 12L of fermentation medium for high-density cultivation. The initial fermentation temperature is 30°C, the stirring speed is 300rpm, the ventilation rate is 30L / min, and the pH is 6.7. Afterwards, the stirring speed and ventilation rate are continuously increased to 1000rpm and 80L / min respectively to maintain the dissolved oxygen above 30%. Because of high-density fermentation A large amount of oxygen is needed. If the oxygen supply is insufficient, it will not only inhibit the respiration of the bacteria, limit the growth and reproduction of the bacteria, but also accumulate harmful substances that will poison the ba...

Embodiment 3

[0046] Embodiment 3 Purification of polypeptide intermediate

[0047] After leaving the tank, the cells obtained in Example 2 were collected by centrifugation, and the crushing buffer was added at a weight-to-volume ratio of 1:10. The high-pressure homogenizer was crushed twice under a pressure of 700 bar, and the inclusion body precipitate was collected by centrifugation. The wet weight of the inclusion body was 30 g / L fermentation broth. The precipitate was added to the washing buffer at a weight-to-volume ratio of 1:10, stirred magnetically at room temperature for 1 hour, and the precipitate collected by centrifugation was washed twice with the washing buffer. The inclusion body dissolving buffer was then dissolved overnight at a weight-to-volume ratio of 1:10. After the dissolved inclusion bodies are centrifuged and ultrafiltered to remove impurities, such as Figure 9 As shown, dilute to a protein concentration of 0.2 mg / ml, refold overnight at 4°C in refolding buffer, ...

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Abstract

The invention belongs to the technical field of polypeptide preparation and relates to a preparation method of a liraglutide intermediate GLP-1(7-37). The preparation method utilizes a genetic recombination technology. Compared with chemical synthesis, the preparation method reduces impurities and improves purity and a yield. The preparation method comprises the following steps of introducing plasmids with hI-GLP-1(7-37) gene sequences into cells of escherichia coli by a gene engineering method to construct recombinant engineering bacteria, carrying out fermentation induction to obtain a hI-GLP-1(7-37) expression fusion protein, and carrying out renaturation, digestion conversion and separation purification to obtain GLP-1(7-37).

Description

technical field [0001] The invention relates to the field of biomedicine, in particular to a preparation method of a liraglutide intermediate polypeptide. Background technique [0002] Diabetes mellitus (Diatetes mellitus) is a group of metabolic diseases characterized by elevated plasma glucose (blood sugar) levels caused by defective insulin secretion or insufficient insulin action. Diabetes has become a major international medical problem due to its complexity, many complications, and low cure rate, and has been called an immortal cancer by the World Health Organization. In recent years, with the improvement of living standards, changes in dietary structure, increasingly tense pace of life, aging population, less active and more sedentary lifestyle and many other factors, the global incidence of diabetes has increased rapidly, and the age of onset is becoming younger Diabetes has become the third chronic disease that seriously threatens human health after tumors and card...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/605C12N15/16C12N15/70C12N1/21C12R1/19
CPCC07K14/605
Inventor 文良柱朱亮赵梅
Owner JIANGSU WANBANG BIOPHARMLS
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