Method for preparation of GLP-1 polypeptide or analogue thereof through MFH fusion protein and application of GLP-1 polypeptide or analogue thereof

A GLP-1 and fusion protein technology, applied in the biological field, can solve the problems of low yield, harsh production conditions, and single source, and achieve the effect of simple purification steps, high recovery rate, and mild conditions

Active Publication Date: 2014-10-15
HUBEI UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Moreover, enzymatic hydrolysis requires a specific amino acid sequence
Enzymatic hydrolysis industrial production requires a large amount of protease enzymes, which directly affects production costs
Specific proteases currently widely used, such as thrombin, enterokinase, and Factor Xa, have a single source, harsh production conditions, and low yields, which cannot meet industrial applications.
Although TEV protease can be prepared by large-scale fermentation, like many proteases, the hydrolyzate still has excess amino acids

Method used

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  • Method for preparation of GLP-1 polypeptide or analogue thereof through MFH fusion protein and application of GLP-1 polypeptide or analogue thereof
  • Method for preparation of GLP-1 polypeptide or analogue thereof through MFH fusion protein and application of GLP-1 polypeptide or analogue thereof
  • Method for preparation of GLP-1 polypeptide or analogue thereof through MFH fusion protein and application of GLP-1 polypeptide or analogue thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] Embodiment 1 fusion protein MFH-DP-H6-E7-GLP-1 and MFH-DP-H6-E7-R34 vector construction and expression

[0058] The codons preferred by Escherichia coli were used to design primers, and the overlap extension PCR method was used to plasmid pCMFH-GLP template (HJ Li, CX Zhou and JZ Su, Chemical ligation and cleavage on solid support facilitate recombinant peptide purification. Protein Expression and Purification50,238 –246,2006), amplified and constructed DNA sequences encoding DP-H6-E7-GLP-1 and DP-H6-E7-R34 fusion proteins respectively, and then, the DNA sequences of the two GLP-1 derivative peptides were passed After double digestion with EcoR I and BamH I, insert into the high-efficiency expression vector pMFH-MCS (HJ Li, CX Zhou and JZ Su, Protein Expression and Purification50,238–246,2006) that was cut with EcoR I and BamH I , constructed into prokaryotic expression recombinant plasmids of GLP-1 and its derivative peptides: pMFH-GLP-1 ( figure 1 ) and pMFH-R34 ( ...

Embodiment 2

[0064] Embodiment 2 Utilizes the fermentation method that lactose induces engineered bacteria to produce fusion protein

[0065] The present invention also determines the use of lactose as an inducer to induce the expression of the fusion protein. Because lactose itself cannot induce the initiation of the Lac promoter, it needs to be converted into allolactose through the action of β-galactosidase before it acts as an inducer. The invention enables engineering bacteria to express target fusion protein through seed cultivation and design of fermentation medium conditions. Specific steps are as follows:

[0066] (1) BL21 expressing fusion protein MFH-DP-H6-E7-GLP-1, MFH-DP-H6-E7-R34 was picked from the plate to 5 ml (containing ampicillin) LB medium, 200 rpm / min, 37°C, shake the flask for 12-16 hours. The composition of LB medium is as follows: yeast extract 5g / L, casein hydrolyzate 10g / L, sodium chloride 10g / L, initial pH 6.5-8.0.

[0067] (2) After cultivation, take 1 mil...

Embodiment 3

[0074] Example 3 Fusion protein ethanol precipitation purification

[0075] The present invention confirms that fusion proteins can be efficiently purified by ethanol precipitation. Collect the bacterial cells expressed by the lactose-induced fusion protein by centrifugation at 5000-6000 rpm for 15-30 minutes, resuspend the bacterial cells in PBS (containing 6M urea) at room temperature, and stir for 30 minutes. Minutes, then the broken cells were processed by a high-pressure homogenizer, the cell broken liquid was centrifuged at 12000 rpm for 20-30 minutes, and the total protein supernatant was obtained, and the supernatant was precipitated by ethanol for 2-8 hours (1:1 volume ratio; -20°C), high-speed centrifugation at 12,000 rpm to remove the precipitate (-20°C), and the secondary supernatant was subjected to ethanol precipitation for 8 to 12 hours (1:1 volume ratio; -20°C). 12000 revs / separation centrifuge collects precipitate, and secondary centrifugation precipitate is ...

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Abstract

The invention discloses a method for preparation of GLP-1 polypeptide or an analogue thereof through MFH fusion protein and application of the GLP-1 polypeptide or the analogue thereof, and belongs to the field of biotechnology. The structural formula of the MFH fusion protein of the GLP-1 polypeptide or the analogue thereof is MFH-DP-H6-E7-PEP, wherein the MFH is a protein fusion vector, DP is a formic acid hydrolysis site, H6 is a histidine label, E7 is a special protease cutting site and PEP is the GLP-1 polypeptide or the analogue thereof. The DNA of the code DP-H6-E7-PEP is connected to pMFH plasmid to obtain pMFH-PEP plasmid and then the pMFH-PEP plasmid is transferred into escherichia coli, MFH-DP-H6-E7-PEP fusion protein is obtained through prokaryotic expression, and formic acid hydrolysis, special protease hydrolysis and affinity chromatography are performed, so that GLP-1 polypeptide or the analogue thereof is obtained. According to the invention, the purposes of large-scale efficient expression of polypeptide, less steps of a peptide release process, mild condition and low production cost are achieved, and the GLP-1 polypeptide or the analogue thereof is suitable for industrial production.

Description

technical field [0001] The invention belongs to the field of biotechnology and relates to the preparation of recombinant polypeptides, in particular to a method and application of using MFH fusion protein to prepare GLP-1 polypeptides or analogs thereof. Background technique [0002] Peptides are an important class of biomolecules, a class of compounds that are connected by less than 80 amino acids and have a certain spatial structure. Polypeptide participates in the biochemical reaction process of various cells in the organism, and is one of the most important function regulators of the human body. As biologically active substances of various cell functions in organisms, polypeptides are involved in various fields such as hormones, nerves, cell growth and reproduction. Compared with small molecule chemical drugs, peptide drugs are often safer, have fewer side effects, and rarely cause serious immune reactions. Peptide substances have small molecular structure, easy modifi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/70C07K1/30C07K1/22C07K1/20C07K14/605C12P21/06A61K38/26A61P3/10
Inventor 苏正定吴刚赵以军李祝
Owner HUBEI UNIV OF TECH
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