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Fusion protein and method for preparing semeglutide intermediate polypeptide from fusion protein

A technology of fusion protein and fusion peptide, which is applied in the field of genetic engineering and polypeptide preparation, can solve problems such as the inability to effectively increase the expression of fusion proteins, the lack of industrial scale-up value, and the lack of industrial scale-up significance, so as to improve extraction and enzymatic digestion. Efficiency, reduced dissolution loss, improved yield and purity

Active Publication Date: 2022-04-08
NANJING HANXIN PHARMA TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Among them, the intracellular soluble expression (patent document with publication number CN104745597A, published in 2015) has a low expression level and does not have the value of industrial scale-up; the patent CN110498849A (published in 2019) related to inclusion body expression discloses a high A method for preparing the main peptide chain of semaglutide with high purity and high yield, but the preferred leader peptide sequence KPSTYI disclosed therein belongs to a short peptide sequence, which cannot effectively improve the expression of the fusion protein; in addition, in the patent publication number CN111378027A ( In the patent literature published in 2020), the intermediate polypeptide of semaglutide was tandemly expressed, and KexII protease cleavage site KR was used as the linker, which required two-step digestion with KexII enzyme and carboxypeptidase B to obtain semaglutide The intermediate polypeptide of meglutide has cumbersome steps and the KexII enzyme is expensive, which does not have the significance of industrial scale-up

Method used

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  • Fusion protein and method for preparing semeglutide intermediate polypeptide from fusion protein
  • Fusion protein and method for preparing semeglutide intermediate polypeptide from fusion protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] Example 1: Construction of recombinant engineering bacteria expressing semaglutide intermediate polypeptide fusion protein

[0055] A fusion protein sequence was designed for expression in E. coli: fusion peptide-DDDDK-Arg34GLP-1(9-37).

[0056] The amino acid sequence of the fusion peptide can firstly enhance the expression, and secondly can protect the intermediate polypeptide Arg34GLP-1(9-37) from being degraded by Escherichia coli's own protease. The amino acid sequence of the fusion peptide is MATKAVSVLKGDGPVQGIINFEQKESNGPVKVWGSIKGLTEGLHGFHVHKFVNQHLCGSHLVALYLV (SEQ ID NO: 3). The C-terminus of the fusion peptide sequence is connected to the intermediate polypeptide Arg34GLP-1(9-37) of semaglutide through DDDDK residues, so the complete amino acid sequence of the fusion protein is MATKAVSVLKGDGPVQGIINFEQKESNGPVKVWGSIKGLTEGLHGFHVHKFVNQHLCGSHLVALYLV DDDDK EGTFTSDVSSYLEGQAAKEFIAWLVRGRG (SEQ ID NO: 12), the isoelectric point of this sequence is 6.2, and the average ...

Embodiment 2

[0058] Example 2: Expression of sameglutide intermediate polypeptide fusion protein in shake flask system

[0059] The recombinant engineered bacterium S1 obtained in Example 1 was cultured in LB medium at 37° C. for 12 hours to obtain a seed solution, which was then inserted into TB medium according to the inoculum size of 0.2% (v / v) for cultivation. When the fermentation broth OD 600 When the value reached 6-8, IPTG with a final concentration of 0.1 mM was added for induction, and the fermentation was terminated after 16 hours of induction at 37° C., and the bacteria were collected by centrifugation.

Embodiment 3

[0060] Example 3: Expression and expression level detection of sameglutide intermediate polypeptide fusion protein in shake flask system

[0061] Wash the fermented cells obtained in Example 2, crush the cells with an ultrasonic breaker, and centrifuge the crushed suspension to collect inclusion bodies. SDS-PAGE was performed on the whole bacteria and inclusion bodies, and the electrophoretic purity of the target protein was detected by an optical densitometer. At the same time, the BCA kit was used to detect the total protein content of the whole bacteria and inclusion bodies. The expression amount of the intermediate polypeptide fusion protein was obtained by multiplying the total protein amount by the electrophoretic purity. After testing, recombinant engineering bacteria S1 was fermented and induced to express, and 1.56 g / L fusion protein was obtained, and 0.95 g / L inclusion bodies were obtained after crushing and washing.

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Abstract

The invention discloses a fusion protein and a method for preparing a semeglutide intermediate polypeptide from the fusion protein, and belongs to the technical field of gene engineering and polypeptide preparation. The fusion protein comprises a segment of fusion peptide, a protease restriction enzyme cutting site and a target main molecule sequence. By optimizing the sequence of the fusion peptide and changing the isoelectric point, hydrophilicity and other properties of the protein, the expression quantity of the fusion protein is effectively improved, and the highest expression quantity can reach 13.1 g / L; meanwhile, the character of the fusion protein is also improved, the development of subsequent extraction, enzyme digestion and purification processes is facilitated, and the amount of the intermediate polypeptide after enzyme digestion is 3.62 g / L. The production cost of the semeglutide intermediate polypeptide Arg34GLP-1 (9-37) is reduced from the source, and the method is beneficial to industrial amplification and suitable for industrial production.

Description

technical field [0001] The invention relates to a fusion protein and a method for preparing a semaglutide intermediate polypeptide, in particular to a fusion protein sequence capable of efficiently preparing a semaglutide intermediate polypeptide Arg34GLP-1 (9-37), which belongs to Technical fields of genetic engineering and polypeptide preparation. Background technique [0002] Type 2 diabetes mellitus (T2DM) is a chronic metabolic disorder syndrome caused by a variety of etiologies that result in impaired human islet β-cell function, insufficient insulin secretion, or increased insulin resistance in target tissues. , polyuria, weight loss and other symptoms, and there is a risk of complications such as sudden diabetic ketoacidosis and hyperosmolar coma. T2DM is a chronic metabolic disease that usually develops after the age of 35 to 40, accounting for more than 90% of diabetic patients. At present, the drugs for the clinical treatment of diabetes are mainly concentrated ...

Claims

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Application Information

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IPC IPC(8): C07K19/00C07K14/605C12N15/62C12N15/70C12N15/81C12N15/75C12N1/21C12N1/19C12R1/19C12R1/125C12R1/84
CPCC07K14/605C12R2001/19C12N15/81C12N15/70C12N15/62C12R2001/84C07K19/00C12R2001/125C12N15/74C12N15/75
Inventor 王璟汤传根张腾范晓阳陈松金波
Owner NANJING HANXIN PHARMA TECH CO LTD
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