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Preparation method of small molecular proteins or polypeptides and fusion protein

A technology of small molecular proteins and fusion proteins, which is applied in the direction of fusion polypeptides, chemical instruments and methods, animal/human proteins, etc., can solve the problems of long production cycle, complicated process, and inability to make medicines, and achieve low cost, simple extraction methods, High safety effect

Inactive Publication Date: 2018-06-29
ZHUHAI JINBAIKANG BIOLOGICAL TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the natural GLP-1 produced by the human body is easily degraded by dipeptidyl peptidase IV (DPP-IV) in the body, and its plasma half-life is less than 2 minutes, so it cannot be used as a drug in clinical practice.
However, the current biosynthesis method has the problems of long production cycle, complicated process and low yield.

Method used

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  • Preparation method of small molecular proteins or polypeptides and fusion protein
  • Preparation method of small molecular proteins or polypeptides and fusion protein
  • Preparation method of small molecular proteins or polypeptides and fusion protein

Examples

Experimental program
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Effect test

Embodiment 1

[0049] Construction of embodiment 1 fusion partner expression plasmid

[0050] The expression plasmid contains a T7lac-type promoter and a T7 terminator, which can shut down expression when there is no inducer, and start expression when there is an inducer. The plasmid also contains a selectable marker for maintaining the stability of the gene of interest in E. coli.

[0051] Using aldosterone isomerase (KSI) as the fusion partner protein, the gene sequence encoding the fusion partner was artificially synthesized, and the start codon ATG was incorporated into its 5' end. In order to facilitate the cloning of different target protein molecules, the start codon was mixed A restriction enzyme NdeI site was incorporated, a restriction enzyme AlwNI site was incorporated at the 3' end, followed by an XhoI site. Synthetic DNA was digested with NdeI and XhoI, and ligated with the digested NdeI-XhoI fragment of the T7lac-type expression plasmid. The above-mentioned plasmids prolifera...

Embodiment 2

[0053] Example 2 Construction of Escherichia coli Expression System and Production of Human Insulin

[0054]Add a suitable connecting peptide to the amino terminal of the human insulin precursor molecule, and obtain the coding gene by artificial synthesis, specifically L-B(1-29)-AAK-A(1-21), L-B(1-30)-KWK - A (1-21), the 3' end of which incorporates a stop codon TAA, a restriction enzyme XhoI site is incorporated after the stop codon, and a restriction enzyme AlwNI site is incorporated at the 5' end. Synthetic DNA was digested with XhoI and AlwNI, and ligated with the digested XhoI-AlwNI fragment of pKSI plasmid. The ligation product was introduced into Escherichia coli, and multiplied in the presence of ampicillin, and the plasmid was isolated by conventional methods. Examination by a suitable restriction nuclease (e.g. XhoI, AlwNI, NdeI) shows that the plasmid DNA contains an inserted sequence, and sequence analysis shows that the plasmid DNA contains the human insulin prec...

Embodiment 3

[0061] Example 3 Construction of Escherichia coli Expression System and Fusion Protein KSI-L-Arg 34 Expression of GLP-1(7-37)

[0062] Add a suitable connecting peptide to the amino terminal of the GLP-1 analog precursor molecule, and obtain the coding gene by artificial synthesis, specifically L-Arg 34 GLP-1 (7-37), the 3' end incorporates the stop codon TAATGA, the restriction enzyme XhoI site is incorporated after the stop codon, and the restriction enzyme AlwNI site is incorporated at the 5' end. Synthetic DNA was digested with XhoI and AlwNI, and ligated with the digested XhoI-AlwNI fragment of plasmid pKSI and plasmid pmKSI. The ligation product was introduced into Escherichia coli, and multiplied in the presence of ampicillin, and the plasmid was isolated by conventional methods. Examination by a suitable restriction nuclease (e.g. XhoI, AlwNI, NdeI) shows that the plasmid DNA contains the inserted sequence, and sequence analysis shows that the plasmid DNA contains th...

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Abstract

The invention provides a preparation method of small molecular proteins or polypeptides. The method comprises the following steps: (a) establishing a gene engineering strain, wherein the gene engineering strain comprises a recombinant expression vector with a fusion protein coding nucleotide sequence, the fusion protein is an F-L-polypeptide, the F is fusion chaperonin and selected from aldosterone isomerase protein, aldosterone isomerase protein mutants, human glucagon-like peptide-1 and human glucagon-like peptide-1 mutants; L is a connecting peptide; the polypeptides are insulin or analogues thereof, GLP-1 or analogues thereof, GLP-2 or analogues thereof, antibacterial peptides or thymosin; (b) culturing the gene engineering strain so as to express the fusion protein; (c) purifying thefusion protein, and performing protease cleavage, thereby obtaining the small molecular proteins or polypeptides with biological activities. The method disclosed by the invention overcomes the defectthat the small molecular proteins or polypeptides are difficultly expressed, and provides a new thought for expressions of the proteins.

Description

technical field [0001] The present invention relates to a preparation method of a small molecule protein or polypeptide, and also relates to a coding sequence, a recombinant expression vector and a fusion protein used in the preparation method. Background technique [0002] According to the Diabetes Map (7th Edition) statistics released by the International Diabetes Federation, the number of diabetics in the world has reached 415 million in 2015, with an average of 1 out of 11 people suffering from the disease. 642 million. In 2015, a total of 5 million people died of diabetes worldwide, an average of 1 person died every 6 seconds. Estimates of the prevalence and incidence trends of various countries and regions show that the number of diabetic patients in China increased by 11.2 million compared with 2013, reaching 109.6 million, ranking first in the world. According to the current development trend, it is estimated that the number of diabetic patients in China will reach...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/70C12N15/81C12N15/80C07K14/605C07K19/00C07K14/47C07K14/66C07K14/62C12N1/21C12N1/19C12N1/15
CPCC07K14/47C07K14/605C07K14/62C07K14/66C07K2319/00C12N9/90C12N15/70C12N15/80C12N15/81
Inventor 赖红星马文柱祝捷周健英姚元锋陈武光夏玉平肖拥军罗湘冀
Owner ZHUHAI JINBAIKANG BIOLOGICAL TECH CO LTD
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