Method for preparing semaglutide by biochemical method

A biochemical and chemical technology, applied in the field of recombinant polypeptide preparation, can solve the problems of low yield, unfriendly environment and high cost

Pending Publication Date: 2021-08-20
NANJING FORESTRY UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Since the synthesis of the polypeptide main chain mainly adopts the solid-phase method in the preparation process, a large amount of organic solvents are required, which is no

Method used

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  • Method for preparing semaglutide by biochemical method
  • Method for preparing semaglutide by biochemical method
  • Method for preparing semaglutide by biochemical method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Construction of embodiment 1 fusion protein expression plasmid

[0050] Design the expression vector sequence (full sequence as shown in SEQ ID No.2), and send out the DNA sequence synthesis of the plasmid, which contains the coding gene, which contains the TrxA gene, TEV restriction site sequence and M II chain Sequence; After the plasmid was synthesized, PCR was used to confirm that the target gene of the fusion protein was synthesized correctly, and the upstream and downstream primers were designed F: 5'-TAATACGACTCACTATAGGG-3'R: 5'-GCTAGTTATTGCTCAGCGG-3'. The PCR conditions are: 98°C for 5min, 98°C for 30s, 56°C for 90s, 72°C for 90s, 36 cycles; PCR amplification system: template 1.5 μL, upstream and downstream primers 1.5 μL, sterilized double distilled water 20.5 μL, PrimerSTAR Mix 25 μL; After the PCR product is confirmed to run on the gel, send it to the detection sequence to confirm that the DNA sequence of the fusion protein is correct, and then construct the ...

Embodiment 2

[0051] Embodiment 2 Expression Engineering Bacteria Preparation and Positive Verification

[0052] Transform the constructed vector into the expression cell E.coli BL21(DE3) by electric shock for 1S, spread it on the LB plate containing kanamycin, put it in a 37°C incubator overnight, and grow a single colony Plasmid extraction and sequencing were carried out, and finally the recombinant engineering bacteria containing the esterase gene were confirmed to be positive engineering bacteria.

Embodiment 3

[0053] Expression and purification of embodiment 3 fusion protein

[0054] Transfer the seed liquid of the recombinant positive engineering strain of Example 2 to LB medium according to the inoculum size of 2%, cultivate it at 37°C until OD600=0.6, add 50 μl of 0.5mol / L IPTG, induce at 15°C for 16 hours, and collect by centrifugation For each 1 g of wet bacteria, resuspend in 10 mL of lysis buffer (50 mM Tris-HCl, 50 mM NaCl, 5% glycerol, pH 8.0, 17 μl (10 mg / mL PMSF)), ultrasonicate and centrifuge to obtain supernatant and precipitate. Collect the supernatant, 1:1 with nickel column adsorption buffer (10mM imidazole, 50mM NaCl, 50mM Tris-HCl pH8.0) (mixed), nickel column adsorption (prepacked column, commercially available), twice the column volume adsorption Buffer washing, 20mM imidazole buffer (20mM imidazole, 50mM NaCl, 50mM Tris-HCl pH8.0) was washed again to remove impurities. The elution buffer was used for elution of the target fusion protein (250mM imidazole, 50mM N...

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Abstract

The invention belongs to the field of preparation of recombinant polypeptides, particularly relates to the technical field of preparation of the recombinant polypeptides by a chemistry and biology combined method, and more particularly relates to a method for preparing semaglutide by a biochemical method. By creatively splitting 31 amino acids of a main peptide chain of the semaglutide are into a 3-site amino acid polypeptide chain (MI chain) and a 28-site amino acid polypeptide chain (MII chain), the possibility of efficiently and quickly preparing the semaglutide under an escherichia coli preparation system is realized. After the technical scheme disclosed by the invention is adopted, a semaglutide product can be rapidly, efficiently and highly specifically obtained, and meanwhile, a large number of chemical reagents are not needed, so that the method is a green and environment-friendly preparation method.

Description

technical field [0001] The invention belongs to the field of preparation of recombinant polypeptides, and in particular relates to the technical field of preparing recombinant polypeptides by combining chemical and biological methods, and more specifically relates to a method for preparing semaglutide by a biochemical method. Background technique [0002] Semaglutide is a GLP-1 analog with a molecular weight of about 4113.57754 (CAS#910463-68-2). It consists of a polypeptide chain containing 31 amino acids and a chemical side chain, and its chemical structure is as follows: [0003] [0004] GLP-1 is an active polypeptide with glucose-dependent hypoglycemic effect secreted by the intestinal cells of the body. It consists of 37 amino acids. There are two biologically active forms of GLP-1 secreted by L cells. -37) and GLP-1(7-36) amide, which differ in only one amino acid sequence. The two types of polypeptides exert the same activity by acting on the same receptor. Due...

Claims

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Application Information

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IPC IPC(8): C07K14/605C07K1/36C07K1/34C07K1/22C12N15/16C12N15/70C12N1/21C12R1/19
CPCC07K14/605C12N15/70
Inventor 叶建仁何伟陈本顺石利平江涛徐春涛邱磊潘声成尹斌何义
Owner NANJING FORESTRY UNIV
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