Method for direct secretory expressing of mature double-chain insulin glargine by using saccharomyces cerevisiae

A technology for insulin glargine and Saccharomyces cerevisiae, which is applied in the field of protein drug production, can solve the problems of wasting biological enzyme preparations, waste carbon source raw materials, etc., and achieve the effects of reducing the difficulty of the production process, simplifying the processing flow, and improving the efficiency of fermentation use.

Active Publication Date: 2019-10-15
SUN YAT SEN UNIV
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Problems solved by technology

However, the use of Saccharomyces cerevisiae still fails to avoid enzyme digestion in vitro and the formation of a large number of by-p

Method used

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  • Method for direct secretory expressing of mature double-chain insulin glargine by using saccharomyces cerevisiae
  • Method for direct secretory expressing of mature double-chain insulin glargine by using saccharomyces cerevisiae
  • Method for direct secretory expressing of mature double-chain insulin glargine by using saccharomyces cerevisiae

Examples

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Embodiment 1

[0062] Example 1 Construction of Kex2 protease recombinant expression system

[0063] The overall objective of the present invention is to directly synthesize and secrete mature insulin glargine from Saccharomyces cerevisiae cells. Among them, the intracellular enzyme cleavage of proinsulin glargine to form a double-chain structure must first be realized. In the molecular structure of insulin glargine, the end of the B chain contains a structure of double basic amino acids Arg-Arg, which can be recognized and cut by Kex2. Therefore, the way to achieve insulin cleavage can be the endogenous Kex2 naturally present in yeast. However, due to the insufficient expression of endogenous Kex2 in Saccharomyces cerevisiae cells, the cleavage of its own alpha factor is still incomplete. Therefore, in order to achieve recombinant expression of insulin glargine in Saccharomyces cerevisiae, it is necessary to co-express a higher level and higher efficiency of Kex2 to complete sufficient cl...

Embodiment 2

[0109] The direct synthesis and secretion of embodiment 2 insulin glargine

[0110] In this example, on the basis of the research in Example 1, the No. 4 strain constructed in Example 1 was selected. According to the same method, the fragment MBP-linker-eGFP in the recombinant plasmid pYES2-alpha-MBP-linker-eGFP The co-expression system of insulin glargine and Kex2 was constructed by replacing it with the optimized insulin glargine sequence (as shown in SEQ ID NO.3). The expressed protein was then immunologically characterized by ELISA, and it was confirmed that the co-expressed insulin was cleaved by Kex2. After purification by cation exchange chromatography, the molecular weight of the expressed protein was verified by MALDI-TOF, and its peptide fingerprint was further determined, and its disulfide bond position and Kex2 cleavage position were verified. In addition, the retention time was verified by liquid chromatography-mass spectrometry, and the amino acid sequence was d...

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Abstract

The invention discloses a method for direct secretory expressing mature double-chain insulin glargine by using saccharomyces cerevisiae. The method comprises the steps that firstly a chassis cell over-expressing Kex2 is constructed, then tandem signal peptide and codon-optimized insulin glargine DNA are transferred into a chassis cell over-expressing Kex2 to form a saccharomyces cerevisiae co-expression system for protein expression purification, and the insulin glargine is obtained. The method greatly reduces the difficulty of the production process of the insulin glargine, simplifies the downstream multi-step tedious and complicated processing flow of the traditional process, and enables yeast cells to directly perform the modification work in the cell. Meanwhile, the method reduces thecomplexity of purification, and the mature insulin protein can be directly extracted from a culture medium by relatively low-cost ion exchange purification. In addition, since the use of components such as trypsin is avoided, nearly 50% by-products in the original process are avoided, the fermentation use efficiency of nutrients such as carbon sources in the medium is improved, and the good application prospect is achieved.

Description

technical field [0001] The invention belongs to the technical field of protein medicine production. More specifically, it relates to a method for directly secreting and expressing mature double-chain insulin glargine by using Saccharomyces cerevisiae. Background technique [0002] Diabetes is a chronic disease with high incidence in today's society. Insulin products are the largest drug category in the diabetes market, accounting for about 53% of the market share, of which third-generation recombinant insulin is the main product. Insulin glargine belongs to the third generation of recombinant insulin. It is a long-acting insulin with no obvious peak and the risk of hypoglycemia and sudden death. Due to its safety and long-acting characteristics, insulin glargine Lantus has continued to become The product with the largest market share in the insulin market, accounting for more than 30% of the overall insulin market. [0003] Most of the existing insulin glargine production...

Claims

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Application Information

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IPC IPC(8): C12N15/81C12N15/62C12N1/19C12R1/865
CPCC12N15/81C12N15/62C12N9/60C07K14/62C07K2319/02C07K2319/21
Inventor 阿迪亚陆勇军
Owner SUN YAT SEN UNIV
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