Main peptide chain of semaglutide and preparation method thereof

A semaglutide and peptide chain technology, applied in the field of semaglutide main peptide chain and its preparation, can solve the lack of high-purity and high-yield semaglutide main peptide preparation method, inclusion body dissolution and modification. Long renaturation time, unsuitable for large-scale production, etc., to reduce the renaturation process, reduce the cost of chemical reagents, and reduce impurities

Inactive Publication Date: 2019-11-26
南京迪维奥医药科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The above-mentioned recombinant protein obtained high-yield and high-purity polypeptide Arg34GLP-1 (9-37) through operations such as dissolution, renaturation, enzyme digestion, and separation, which solved the problems of many impurities, low yield, and use in the prior art. A large number of organic reagents are not friendly to the environment, soluble expression in cells leads to low expression, inclusion body dissolution and renaturation time are long, and low protein concentration leads to too large renaturation volume, which is not suitable for large-scale production and limits the possibility of increasing production capacity. question
[0012] At present, there is a lack of a high-purity and high-yield semaglutide main peptide chain and its preparation method

Method used

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  • Main peptide chain of semaglutide and preparation method thereof
  • Main peptide chain of semaglutide and preparation method thereof
  • Main peptide chain of semaglutide and preparation method thereof

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Experimental program
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Effect test

Embodiment 1

[0045] The recombinant protein of a semaglutide intermediate polypeptide of the present invention is characterized in that: the recombinant protein of the semaglutide intermediate polypeptide is a leader peptide-DDDDK-GLP-1(9-37), and the recombinant protein of the semaglutide intermediate polypeptide is The amino acid sequence of the recombinant protein of the marutide intermediate polypeptide is shown in SEQ ID NO.1;

[0046] In the preparation of semaglutide intermediates, the following leader peptides can be used:

[0047] (1) MFLKGDGYVQGIINFEGLHHLVALGLV; (2) KSI; (3) TrxA; (4) DsbA, (5) DsbC; (6) Sumo; (7) GST; (8) Intein; (9) KPSTYI.

[0048] A gene recombinant expression plasmid of the present invention contains the coding gene encoding the recombinant protein described in claim 1.

[0049] A kind of recombinant engineering bacterium comprising the gene recombination expression plasmid of the present invention adopts the gene recombination expression plasmid to transfe...

Embodiment 2

[0061] The difference between embodiment 2 and embodiment 1 is:

[0062] A kind of preparation method of semaglutide main peptide chain of the present invention comprises the following steps:

[0063] In step (6), the bacterium is subjected to high-pressure homogenization, the inclusion bodies are collected, and then the inclusion bodies are washed and denatured; the washed inclusion bodies are subjected to an alkaline condition with a pH of 14, according to the protein concentration of 30g / L inclusion body dissolution buffer was added to dissolve and denature inclusion bodies.

[0064] In step (7), the intermediate polypeptide GLP-1 (9-37) is obtained by enzymatic conversion, separation and purification; the specific method of the enzyme conversion, separation and purification is: the recombinant protein after step (6) renaturation The pH range of the solution described by enterokinase is 11.0; the recombinant protein after step (6) denaturation and renaturation is hydrolyze...

Embodiment 3

[0067] The difference between embodiment 3 and embodiment 1 is:

[0068] A kind of preparation method of semaglutide main peptide chain of the present invention comprises the following steps:

[0069] In step (6), the bacterium is subjected to high-pressure homogenization, the inclusion bodies are collected, and then the inclusion bodies are washed and refolded; the washed inclusion bodies are subjected to an alkaline condition with a pH of 12, according to the protein concentration of 5g / L inclusion body dissolution buffer was added to dissolve and denature inclusion bodies.

[0070] In step (7), the intermediate polypeptide GLP-1 (9-37) is obtained by enzymatic conversion, separation and purification; the specific method of the enzyme conversion, separation and purification is: the recombinant protein after step (6) renaturation The pH range of the solution described by enterokinase is 10.5; the recombinant protein after step (6) denaturation and renaturation can be obtaine...

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Abstract

The invention discloses a main peptide chain of semaglutide and a preparation method thereof; and more specifically, the invention discloses recombinant protein of intermediate polypeptide of somaluptide. The recombinant protein of the intermediate polypeptide of the somaluptide is leader peptide-DDDDK-GLP-1 (9-37), and has an amino acid sequence as shown in SEQ ID NO. 1. When the intermediate polypeptide of the somalutide is being prepared, the following leader peptides can be adopted: (1) MFLKGDGYVQGIINFEGLHHLVALGLV; (2) KSI; (3) TrxA; (4) DsbA, (5) DsbC; (6) Sumo; (7) GST; (8) Intein; and (9) KPSTYI. The intermediate polypeptide of the somalutide prepared by the preparation method is high in polypeptide purity that the polypeptide purity can be up to 89% or above; and moreover, yield ofthe intermediate polypeptide can be higher than 82%. By adopting ion-exchange separation and purification, the preparation method has the characteristics of being high in degree of separation, good in purification effects, few in impurities and easy to operate.

Description

technical field [0001] The invention relates to the technical field of genetic engineering and polypeptide preparation methods, in particular to a semaglutide main peptide chain and a preparation method thereof. Background technique [0002] Diabetes is a group of metabolic diseases characterized by hyperglycemia. Hyperglycemia is caused by defective insulin secretion or impaired biological action, or both. The long-term high blood sugar in diabetes leads to chronic damage and dysfunction of various tissues, especially the eyes, kidneys, heart, blood vessels, and nerves. [0003] Type 1 diabetes, formerly known as insulin-dependent diabetes, mostly occurs in children and adolescents, and can also occur in various ages. The onset is relatively sharp, and there is absolutely insufficient insulin in the body, and ketoacidosis is prone to occur. Insulin therapy must be used to obtain satisfactory curative effect, otherwise it will be life-threatening. Among them, type 1 diabe...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/605C12N15/70C12N1/21C12R1/19
CPCC07K14/605C12N15/70
Inventor 许驰名
Owner 南京迪维奥医药科技有限公司
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