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Engineering bacterium for gene recombination tandem expression of linaclotide

A technology of linaclotide and tandem expression is applied in recombinant engineering bacteria, the fusion protein of gene recombination and tandem expression of linaclotide and the field of its preparation, which can solve the problem of high production cost and environmental protection cost, high cost and lack of industrialization significance. Large and other problems, to achieve the effect of low cost

Active Publication Date: 2022-04-15
修实生物医药(南通)有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The original research and the domestically reported preparation methods all use solid-phase synthesis of polypeptides. Chinese patent CN103626849A discloses a synthesis method. Although the total yield is recorded as high as 69.60%, the method will form a disulfide bond in the later stage of cyclization It needs to be divided into three steps, the operation is very complicated, and the industrialization is of little significance; Chinese patents CN104163853A, CN104231051A, CN102875655A, and CN104844693A respectively disclose the synthesis method of linaclotide, and the highest total yield is recorded as 27%-43.5%. The synthesis needs to use expensive modified amino acids, resins and other raw materials, the cost is still high, and the production process needs to use N,N-dimethylformamide (DMF), N,N-diisopropylethylamine (DIPEA), A large number of organic solvents such as N,N-diisopropylcarbodiimide (DIC), trifluoroacetic acid (TFA), dimethyl sulfoxide (DMSO), anhydrous ether, and acetonitrile have high production costs and environmental protection costs.

Method used

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  • Engineering bacterium for gene recombination tandem expression of linaclotide
  • Engineering bacterium for gene recombination tandem expression of linaclotide
  • Engineering bacterium for gene recombination tandem expression of linaclotide

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0184] The invention relates to a method for expressing linaclotide by gene recombination and serially expressing linaclotide engineering bacteria.

[0185] 1. Recombinant protein design

[0186] Design 3: TrxA fusion tag, four linaclotide sequences in series, the nucleotide and protein sequences of which are shown in SEQ ID NO:9 and SEQ ID NO:10, respectively.

[0187] 2. Construction of engineering bacteria

[0188] Entrust BGI to synthesize gene fragments. The synthesized gene fragment was double-digested with restriction endonucleases Nco I and Xho I, connected to the same double-digested pET28a plasmid, and verified by sequencing; CaCl 2 Prepare Escherichia coli BL21(DE3) competent cells by treatment method, heat shock at 42°C to transfer the recombinant plasmid into the cells, culture at 37°C overnight, and pick monoclonal strains for product expression verification.

[0189] 3. Engineering bacteria shake flask fermentation

[0190] The verified strains were inoculat...

Embodiment 2

[0226] The invention relates to a method for expressing linaclotide by gene recombination and serially expressing linaclotide engineering bacteria.

[0227] 1. Recombinant protein design

[0228] Design 4: TrxA fusion tag, 6 linaclotide sequences in series, the nucleotide and protein sequences of which are shown in SEQ ID NO:11 and SEQ ID NO:12, respectively.

[0229] 2. Construction of engineering bacteria

[0230] Entrust BGI to synthesize gene fragments. The synthesized gene fragment was double-digested with restriction endonucleases Nco I and Xho I, connected to the same double-digested pET28a plasmid, and verified by sequencing; CaCl 2 Prepare Escherichia coli BL21(DE3) competent cells by treatment method, heat shock at 42°C to transfer the recombinant plasmid into the cells, culture at 37°C overnight, and pick monoclonal strains for product expression verification.

[0231] The engineering bacteria were preserved. The preservation date is November 15, 2021. The preser...

Embodiment 3

[0263] The invention relates to a method for expressing linaclotide by gene recombination and serially expressing linaclotide engineering bacteria.

[0264] 1. Recombinant protein design

[0265] Design 6: TrxA fusion tag, tandem 10 linaclotide sequences, the nucleotide and protein sequences of which are respectively shown in SEQ ID NO:15 and SEQ ID NO:16.

[0266] 2. Construction of engineering bacteria

[0267] Entrust BGI to synthesize gene fragments. The synthesized gene fragment was double-digested with restriction endonucleases Nco I and Xho I, connected to the same double-digested pET28a plasmid, and verified by sequencing; CaCl 2 Prepare Escherichia coli BL21(DE3) competent cells by treatment method, heat shock at 42°C to transfer the recombinant plasmid into the cells, culture at 37°C overnight, and pick monoclonal strains for product expression verification.

[0268] 3. Engineering bacteria shake flask fermentation

[0269]The verified strains were inoculated in ...

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Abstract

The invention relates to an engineering bacterium for high expression of linaclotide, a specific amount of linaclotide and a recombinant tag are connected in series to obtain a fusion protein, the fusion protein is used for expression production of linaclotide, and the yield of linaclotide is greatly improved.

Description

technical field [0001] The invention belongs to the field of medicinal chemistry and relates to a recombinant engineering bacterium, in particular to a fusion protein expressing linaclotide in series through genetic recombination and a preparation method thereof. Background technique [0002] Linaclotide is an innovative drug developed by Ironwood for the treatment of irritable bowel syndrome (IBS-C) and adult chronic idiopathic constipation (CIC). Pound Bomb drug, which was approved for marketing in China in 2019 (product name: Lingzeshu). The drug can bind to guanylate cyclase C on the surface of intestinal cells, promote the increase of intracellular and extracellular cGMP concentration, thereby stimulating the secretion of intestinal juice, promoting intestinal activity and increasing the frequency of defecation, and at the same time, it can relieve visceral pain. [0003] The original research and the domestically reported preparation methods all use solid-phase synthe...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/70C07K19/00C12N15/62C07K7/08C12R1/19
CPCY02A50/30
Inventor 吴寅嵩谭莹莹万民熙金锋杨晓爽
Owner 修实生物医药(南通)有限公司