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Method for preparing GLP-1 or GLP-1 analogue polypeptides by using escherichia coli to express tandem sequence

A technology of GLP-1 and Escherichia coli, applied in the biological field, can solve the problems of difficult identification of enzyme cleavage sites, low yield of target protein, incomplete enzyme cleavage, etc. Conducive to the effect of enzyme digestion and separation and purification

Active Publication Date: 2020-04-28
VONSUN PHARMATECH CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, there are no patents or documents on the tandem expression technology of polypeptides or proteins. One reason may be that it is difficult to completely identify the enzyme cleavage sites between the tandem expression target proteins, resulting in incomplete enzyme cleavage, or residual amino acids after enzyme cleavage. , the yield of the target protein is low

Method used

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  • Method for preparing GLP-1 or GLP-1 analogue polypeptides by using escherichia coli to express tandem sequence
  • Method for preparing GLP-1 or GLP-1 analogue polypeptides by using escherichia coli to express tandem sequence
  • Method for preparing GLP-1 or GLP-1 analogue polypeptides by using escherichia coli to express tandem sequence

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0066] 1. Sequence design of GLP-1 analogue triple tandem protein 1

[0067] In order to increase the expression level of the GLP-1 analogue precursor peptide in prokaryotic expression and increase the proportion of the precursor peptide chain in the entire expressed peptide chain, according to the above-mentioned patent idea, the GLP-1 analogue precursor peptide chain was subjected to three steps Tandem design (n=3), and GLP-1 analog precursors are connected by trypsin cleavage site or kex2 enzyme cleavage site, in order to make trypsin or kex2 enzyme better recognize trypsin cleavage site Or kex2 enzyme cleavage site, to improve the efficiency of enzyme cleavage, we designed a key auxiliary sequence (m) at the trypsin cleavage site or kex2 enzyme cleavage site. According to the GLP-1 analogs published by NCBI (semaglutide precursor sequence (9-37) and liraglutide precursor sequence (7-37)), at its N-terminal leader peptide MRLNSA, in GLP-1 natural Between the sequence and t...

Embodiment 2

[0083] Example 2: Construction of GLP-1 analog tandem protein recombinant plasmid and construction of engineering bacteria

[0084] The nucleotide sequences corresponding to the amino acid sequences designed above were optimized according to the codons of Escherichia coli, and the codon-optimized gene synthesis was carried out by chemical synthesis, and then ligated into pET32a through BamHI and XhoI to construct four recombinant plasmids . Pass the above recombinant plasmid through CaCl 2 The transformation method was introduced into BL21 (DE3), and the single clones were screened for resistance to construct multiple strains of semaglutide precursor recombinant 3 and 4 tandem protein engineering bacteria. After sequencing, the sequence in the engineering bacteria was consistent with the design.

Embodiment 3

[0085] Example 3: Induced expression of GLP-1 analog tandem proteins

[0086] 3.1 LB medium formula

[0087] Table 3. LB medium formula

[0088]

[0089] Prepare 11L LB culture medium according to Table 3, after dissolving and mixing, absorb 10ml and 1.7L culture medium and put them into 50ml and 3L Erlenmeyer flasks respectively, prepare 6 bottles each, seal and sterilize (121°C, 25min). use.

[0090] 3.2 Recovery of engineered bacteria

[0091] Take out one semaglutide precursor recombinant 3 and 4 tandem protein glycerol bacteria, respectively insert 6 tubes of 10ml sterilized medium according to 1% inoculation amount, and culture overnight on a shaker at 37°C and 250rpm.

[0092] 3.3 Induction culture of engineered bacteria

[0093] Take out the resuscitated bacteria solution, insert 1.7L sterilized medium into 6 mice according to the inoculum amount of 1%, culture on a shaker at 37°C and 250rpm for about 7-8h, add 0.1mM IPTG, and incubate at 37°C Expression was in...

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Abstract

The invention discloses a method for preparing GLP-1 or GLP-1analogue polypeptides by using escherichia coli to express a tandem sequence. A general formula of a recombinant tandem protein of the GLP-1 or the GLP-1 analogue designed by the invention is X-(Y-Z)n, wherein X is a leader peptide, Y is a trypsin and CPB enzyme double restriction enzyme cutting site or a Kex2 enzyme and CPB enzyme double restriction enzyme cutting site and auxiliary sequence, Z is the GLP-1 or the GLP-1 analogue, and n is a number of tandem repetition. An inclusion body formed by performing fermentation induction expression on the recombinant tandem protein of the GLP-1 or the GLP-1 analogue through an escherichia coli prokaryotic expression system can be completely dissolved without a denaturing agent, and thedissolved solution can be subjected to trypsin and CPB enzyme double digestion or Kex2 enzyme and CPB enzyme double digestion to obtain the GLP-1 or the GLP-1 analogue. The expression level of the GLP-1 or the GLP-1 analogue obtained by the method is high, and the yield of the GLP-1 target protein after enzyme digestion and the purity after enzyme digestion are high.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a method for preparing GLP-1 or its analogue polypeptide using a tandem sequence highly expressed by Escherichia coli. Background technique [0002] Type 2 diabetes mellitus is a highly heterogeneous metabolic disorder syndrome, the basic pathological damage of which is insulin resistance combined with the obvious decline of islet function. According to statistics, there were about 246 million diabetic patients in the world in 2007, and it will reach 380 million in 2025, of which type 2 diabetic patients account for 90% to 95% of the total. At present, the drugs for the clinical treatment of diabetes are mainly concentrated in various types of insulin and four types of oral hypoglycemic drugs. In order to allow diabetic patients to have more choices in treatment, some new anti-diabetic drugs emerged in the early 21st century, such as glucagon-like peptide-1 analogues, wh...

Claims

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Application Information

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IPC IPC(8): C07K19/00C07K14/605C12N15/62C12N15/70C12N1/21C12R1/19
CPCC07K14/605C07K2319/00C12N15/70
Inventor 辛中帅赵珊珊文良柱平康康徐晓峰李夷平
Owner VONSUN PHARMATECH CO LTD
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