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Oligomeric or polymerized methylene protein and its separation and purification process and use

A technology for separation and purification of proteins, applied in the field of protein preparation, can solve problems that have not yet been seen, and achieve the effects of protecting protein activity and separation and purification effects, reducing losses, and protecting integrity.

Inactive Publication Date: 2003-08-20
INST OF PROCESS ENG CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] But so far, how to stabilize the structure of the protein does not change when separating and purifying the protein from the raw material solution (cell culture fluid, tissue extract), and there are few studies in this area; , polyoligohydric alcohols, monosaccharides, disaccharides, oligosaccharides, and polysaccharides as protective agents to accompany part or the entire separation and purification process; especially for oligomeric or multimeric subunit proteins, how to avoid subunits during separation Dissociation during purification has not been reported

Method used

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  • Oligomeric or polymerized methylene protein and its separation and purification process and use

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Embodiment 1 extracts alcohol dehydrogenase (tetramer protein) from yeast

[0032] Yeast: Commercially available dry baker's yeast (Gist-Brocades Yeast Co., Ltd., Netherlands) was dissolved in distilled water at a yeast concentration of 10% weight / volume (W / V) (dry weight), and 8 liters of yeast suspension was prepared. Stir for one hour at 4 °C to activate the yeast.

[0033] Adding protectant: After one hour, the cell suspension was divided into two parts (4 liters each), and 1 liter of glycerol and 20% (W / V) sucrose was added to one part of the activated yeast suspension under stirring. The solution was mixed to make the suspension volume 5 liters; another cell suspension without any protective agent was supplemented with distilled water to 5 liters as a reference comparison.

[0034] Broken cells: Use a high-pressure homogenizer pump produced by APV Manton Gaulin in the United States to add five liters of raw material solution into the feeding barrel and stir it. P...

Embodiment 2

[0036] Experimental results: the activity of alcohol dehydrogenase obtained without adding any protective agent is 25u / ml, where u represents the unit of enzyme activity, and / ml represents the slurry after per milliliter of cell disruption. Under the same experimental conditions, after adding glycerol and sucrose, the activity of alcohol dehydrogenase obtained is 40u / ml, which is 60% higher than that without protective agent. Example 2 Extracting Human Tumor Necrosis Factor (Trimer) from Genetically Engineered Escherichia coli

[0037] Escherichia coli (E.coli cell) culture: recombinant human tumor necrosis factor (rhTNF-α) Escherichia coli JM103, culture medium is 1.5% weight / volume (W / V)_tryptone, 1.3% weight / volume (W / V ) yeast extract powder, 0.25% weight / volume (W / V) NaCl, 0.2% weight / volume (W / V) glucose, pH 7.5. The final concentration of tetracycline is 12.5 μg / l. The fermentation of the recombinant bacteria was carried out at 37° C. with a 6-liter fermenter (product...

Embodiment 3

[0042] It can be seen from the above table that the addition of protective agents increases the activity of rhTNF-α by more than 30%, and PEG200 has the best effect, increasing the activity by more than 2 times. Example 3 Purification of Genetic Engineering Human Tumor Necrosis Factor by Adsorption

[0043] Adsorption: Anion-exchange medium DEAE-Sepharose Cl-6B is loaded into an adsorption column (16×55mm), fully equilibrated with 0.01M Tris-HCl, pH 8.0 (with or without polyethylene glycol PEG). Add polyethylene glycol PEG200, 600 or 4000 at a concentration of 1 wt% to the cell disruption supernatant, balance solution, and eluate without the presence of polyethylene glycol PEG, respectively. Feed, wash with equilibration buffer to remove unadsorbed protein to baseline, use 75mM NaCl, 10mM Tris-HCl for segmental elution, collect each elution peak and measure protein concentration and rhTNF-α activity.

[0044] Activity analysis: TNF activity analysis refers to the method of Ag...

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Abstract

The present invention relates to oligomeric or polymerized subunit protein, its separation and purification process capable of maintaining its activity and resulting in high yield and use. the oligomeric or polymerized subunit protein has two or more independent subunits polymerized on the intermolecular force. Through adding protecting agent of 0.5-40 wt% of vol% concentration into cell culture liquid, animal, plant tissue extracting liquid, crushing cell supernatant, crushed cell balancing liquid or crushed cell eluting liquid at normal temperature and convenient cell crushing , membrane separation, adsorption or chromatographic separation to purify, oligomeric or polymerized subunit protein is obtained. The protein is used in the process of preparing agent engineering protein or vaccine.

Description

technical field [0001] The invention relates to the technical field of protein preparation, in particular to an oligomeric or multimeric subunit protein, and a method for protecting its activity and improving yield during the process of separating and purifying the oligomeric or multimeric subunit protein and use. Background technique [0002] In the production of genetically engineered pharmaceutical proteins, low yield and low purity are the key problems to be solved urgently. In addition to the complex and difficult separation of biological components, the loss of the activity of many target proteins is also an important reason. Due to the high market price of pharmaceutical proteins, a slight increase in the recovery rate may bring huge economic benefits. Therefore, the inactivation of proteins in separation and purification and its countermeasures have received great attention in recent years. [0003] An oligomeric or multimeric subunit pr...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K47/18A61K47/42C08H1/00
Inventor 苏志国冯小黎修志龙赵东旭
Owner INST OF PROCESS ENG CHINESE ACAD OF SCI
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