Recombinant engineering bacterium capable of efficiently expressing liraglutide precursor and application of recombinant engineering bacterium

A technology of recombinant engineering bacteria and liraglutide, applied in the field of genetic engineering, can solve the problems of unfavorable industrial development needs, protein cleavage problems, high cost, etc., achieve fast and convenient separation and purification methods, reduce subsequent processing steps, and reduce industrial production costs Effect

Active Publication Date: 2020-01-24
GAN&LEE PHARMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In terms of production technology, Liraglutide is currently produced by Novo Nordisk using Saccharomyces cerevisiae to express the liraglutide precursor molecule Arg34GLP-1(7-37) in an extracellular way, and after its Lys26 is linked to the fatty acid side chain Excessive amino acids are removed to obtain liraglutide molecules, which are further processed into injections and sold in China as imported drugs. The price is quite expensive and unaffordable for most patients
[0004] At present, prokaryotic expression and eukaryotic expression systems can be used to express foreign genes, but there are some defects in the eukaryotic expression system when expressing the target protein, for example: (1) When using yeast to express the target protein, the product protein will be inhomogeneous Degradation, incomplete signal peptide processing, multimer formation, etc.
Moreover, problems such as protein cleavage and low protein secretion efficiency may occur when expressing proteins in Saccharomyces cerevisiae
(2) The insect baculovirus expression system also has certain defects
In addition, although the baculovirus expression system can perform post-translational modification on the target protein, this modification has certain limitations; although the glycosylation site is the same as in mammalian cells, the properties of the oligosaccharide chain are different. different, unable to produce complex glycan side chains, which may be due to the inability of insect cells to process mature glycans into analogous forms in mammalian cells
(3) Mammalian expression system has relatively high cost and complex technology when expressing foreign genes, and there is potential contamination of animal viruses in the expression process, etc.
However, the amount of target protein produced by this protein expression method is very small, which is not conducive to the subsequent industrial development needs

Method used

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  • Recombinant engineering bacterium capable of efficiently expressing liraglutide precursor and application of recombinant engineering bacterium
  • Recombinant engineering bacterium capable of efficiently expressing liraglutide precursor and application of recombinant engineering bacterium
  • Recombinant engineering bacterium capable of efficiently expressing liraglutide precursor and application of recombinant engineering bacterium

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Example 1 Design of Liraglutide Precursor Molecule Containing Signal Peptide and Enterokinase Sequence and Construction of the Gene Expression Plasmid

[0040] 1. Construction of liraglutide precursor molecule

[0041] The liraglutide precursor molecule of the present invention has an A-B-C structure, wherein A is a signal peptide, B is a connecting peptide, and C is the sequence of Arg34GLP-1 (7-37); the signal peptide is MalE, PhoA, OmpF , PelB, LamB, Lpp or OmpA; the connecting peptide is enterokinase, thrombin, SUMO or Protease.

[0042] Add a His tag after the second amino acid at the N-terminal of the signal peptides MalE, PhoA, OmpF, and PelB respectively. The amino acid sequences are shown in SEQ ID NO.1-4, and the gene sequences are shown in SEQ ID NO.5-8. .

[0043] The amino acid sequence of Arg34GLP-1(7-37) is shown in SEQ ID NO.9.

[0044] The gene sequence of the liraglutide precursor molecule of the signal peptide and enterokinase sequence of the prese...

Embodiment 2

[0047] Example 2 Construction of Recombinant Engineering Bacteria Highly Expressing Liraglutide Precursor

[0048] The successfully constructed recombinant expression vector was transformed into BL21(DE3) host by chemical method for amplification, and the correct recombinant engineered bacteria were initially screened by colony PCR method, and then the plasmid was extracted for enzyme digestion identification. As a result of the identification, the correct recombinant engineered bacteria were obtained.

Embodiment 3

[0049] Example 3 The method of preparing liraglutide precursor by using recombinant engineered bacteria highly expressing liraglutide precursor

[0050] 1. Fermentation and inclusion body collection

[0051] Get the recombinant engineered bacterium constructed in Example 2, carry out high-density fermentation, obtain thalline after centrifugation, 10L fermented liquid can obtain 800g wet bacterium. During the fermentation process, recombinant fusion proteins were expressed with different signal peptides, SDS-polyacrylamide gel electrophoresis was performed, and the content of the target protein was determined by observing the band intensity. Among them, recombinant fusion proteins with LamB, Lpp, and OmpA as signal peptides The expression effect is not good, and the expressed recombinant protein band is very narrow or absent, see figure 2 ; The recombinant fusion protein with MalE, PhoA, OmpF, and PelB as signal peptides has a better expression effect, see image 3 , the fu...

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PUM

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Abstract

The invention provides a recombinant engineering bacterium for efficiently expressing a liraglutide precursor and an application of the recombinant engineering bacterium. According to the invention, asignal peptide and an enterokinase restriction enzyme cutting site are designed at the N end of a liraglutide precursor molecule Arg34G LP-1 (7-37), the C end is connected with termination codon, a target gene is formed, then, the target gene is inserted between two restriction enzyme cutting sites of an expression vector; the recombinant engineering bacterium for expressing the liraglutide precursor is constructed, the engineering bacterium is subjected to high-density fermentation culture and is expressed in the form of an inclusion body of a fusion protein, the expression quantity of the recombinant fusion protein is high, the recombinant fusion protein accounts for about 25%-35% of the total protein of the bacterium, and the expression quantity of the inclusion body of a target protein reaches 15-20g/L. The inclusion body is low in impurity protein content, beneficial to separation and purification, and high in purification efficiency and good in stability, the production cost isgreatly reduced, the production efficiency is improved, and the inclusion body has the good application prospect in the field of diabetes treatment medicine preparation.

Description

technical field [0001] The present invention relates to the technical field of genetic engineering, in particular to a recombinant engineering bacterium for highly expressing liraglutide and its application. Background technique [0002] In recent years, scientists have developed GLP based on the physiological mechanism of glucagon-like peptide 1 (GLP-1) to promote insulin secretion and regulate blood sugar. -1 receptor agonist. GLP-1 has great advantages in the treatment of type 2 diabetes mellitus (T2DM): ①Rely on the increase of human blood sugar concentration to regulate the body's blood sugar; ②Promote the proliferation and differentiation of islet β cells, inhibit their apoptosis, promote the secretion of insulin, and increase the body's insulin Sensitivity; ③Inhibit gastrointestinal emptying, increase satiety, reduce food intake, and reduce weight. But the only shortcoming is that GLP-1 is easily degraded by dipeptidyl peptidase in the human body, and its half-life ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/605C12N15/16C12N15/70C12N1/21C12P21/02C12R1/19
CPCC07K14/605C12N15/70
Inventor 杨柏成赵运星
Owner GAN&LEE PHARMA
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