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Purifying process of thanatin antimicrobial peptide

A technology of antimicrobial peptide and death factor, applied in the field of protein purification, can solve the problems of long cycle, high cost, unsuitable for large-scale production, etc., and achieve the effect of low cost, short time consumption and simple operation

Inactive Publication Date: 2018-05-18
GUANGDONG HINAPHARM PHARMA CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] In view of the deficiencies of the above-mentioned prior art, the purpose of the present invention is to provide a purification process for deadlyn antimicrobial peptides, which aims to solve the problem of high cost and long cycle of purification methods for deadlyn antimicrobial peptides in the prior art, which are not suitable for large-scale production problem

Method used

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  • Purifying process of thanatin antimicrobial peptide
  • Purifying process of thanatin antimicrobial peptide

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] 1) Expression of deadlyn: use the deadlyn antimicrobial peptide expression strain constructed in our laboratory (Chinese patent application number: 201711083981.0) for fermentation. After the fermentation is completed, filter or centrifuge to obtain the fermentation supernatant. in the supernatant.

[0057] 2) Desalination of fermented liquid: desalting with roll-type membrane, parameter control: inlet pressure 5.0bar, outlet pressure 4.8bar, flow rate 100LPM; add water several times during the process until the ionic strength of the desalination solution is less than 13ms / cm, and use 0.45 um membrane filter to collect and filter the desalted solution.

[0058] 3) Loading the column: equilibrate the CM-Sepharose Fast Flow chromatography column with 10 times the column bed volume of binding buffer, the flow rate is 1 times the column bed volume / min, and then put the filtered desalted solution on the column, the flow rate is 1 times the column bed volume / min, and then w...

Embodiment 2

[0063] 1) Expression of deadlyn: use the deadlyn antimicrobial peptide expression strain constructed in our laboratory (Chinese patent application number: 201711083981.0) for fermentation. After the fermentation is completed, filter or centrifuge to obtain the fermentation supernatant. in the supernatant.

[0064] 2) Desalination of fermented liquid: roll membrane desalination is used, parameter control: inlet pressure 4.8bar, outlet pressure 4.6bar, flow rate 90LPM; add water several times during the process until the ionic strength of the desalted solution is less than 13ms / cm, and use 0.45 um membrane filter to collect and filter the desalted solution.

[0065] 3) Loading the column: equilibrate the CM-Sepharose Fast Flow chromatography column with 9 times the column bed volume of the binding buffer, the flow rate is 1.2 times the column bed volume / min, and then put the filtered desalted solution on the column, the flow rate is 1.2 times the column bed volume / min, and the...

Embodiment 3

[0070] 1) Expression of deadlyn: use the deadlyn antimicrobial peptide expression strain constructed in our laboratory (Chinese patent application number: 201711083981.0) for fermentation. After the fermentation is completed, filter or centrifuge to obtain the fermentation supernatant. in the supernatant.

[0071] 2) Desalination of fermented liquid: desalting with roll-type membrane, parameter control: inlet pressure 5.2bar, outlet pressure 5.0bar, flow rate 110LPM; add water several times during the process until the ionic strength of the desalination solution is less than 13ms / cm, and use 0.45 um membrane filter to collect and filter the desalted solution.

[0072] 3) Loading the column: equilibrate the CM-Sepharose Fast Flow chromatography column with 8 times the column bed volume of binding buffer, the flow rate is 0.8 times the column bed volume / min, and then put the filtered desalted solution on the column, the flow rate is 0.8 times the column bed volume / min, and the...

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Abstract

The invention discloses a purifying process of a thanatin antimicrobial peptide. The purifying process comprises expression of thanatin, desalting of fermented supernatant, column loading, gradient elution, desalting of eluent and freeze drying. According to the purifying process, a CM-Sepharose Fast Flow chromatographic column and the thanatin antimicrobial peptide undergo ionic exchange, and a high-purity thanatin antimicrobial peptide being greater than 95 percent in purity is obtained through gradient elution. The purifying process has the advantages of easiness in operation, low cost, short time, high product purity and suitability for large-scale purification of the thanatin antimicrobial peptide.

Description

technical field [0001] The invention relates to the technical field of protein purification, in particular to a purification process of deadin antibacterial peptide. Background technique [0002] death hormone ( Thanatin ), also known as death peptide, is produced by the spotted-bellied stinkbug ( Podisus maculiventris ) is a cyclic insect antimicrobial peptide induced by 21 amino acid residues. It has a simple structure and a wide antibacterial spectrum. It has inhibitory effects on Gram-positive bacteria, Gram-negative bacteria, some fungi and protozoa. , does not exhibit hemolytic activity to mammalian cells. In view of its superior broad-spectrum antibacterial activity and unique mode of action, deadin is an ideal antibiotic substitute product with broad application prospects. [0003] At present, the main production method of deadin antimicrobial peptides is to biosynthesize deadin through microbial fermentation, and there are mainly two expression systems of Escher...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P21/00C07K1/18
CPCC07K14/43563
Inventor 梁伟凡周玉岩丁小云范惠敏李洪金
Owner GUANGDONG HINAPHARM PHARMA CO LTD