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Protein separating and purifying method

A technology for separation and purification of proteins, applied in the separation and purification of hepatocyte growth-promoting hormone, and the separation and purification of small molecular proteins, it can solve the problems of long production cycle, no discovery, low pHGF yield, etc., to maintain biological activity, The effect of reducing input and operating costs, and simple process operation

Inactive Publication Date: 2006-03-22
王玉亭
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the extraction process of this method is simple, it also has obvious defects: low pHGF yield, low protein concentration, high production environment requirements, and long production cycle
And to carry out the separation of target objects with different molecular weights, it is difficult to rely on the above-mentioned two-phase extraction method.
[0010] The research has also shown (document "Journal of Northeast Normal University Natural Science Edition" "Research Progress of Two-Phase Extraction Technology", 2000, 32(3), 34), although the two-phase extraction method was first found to be able to extract proteins Separation and extraction. In recent years, it has also been found that this method can also be used to extract antibiotics. However, it has not been found that this method can also extract other impurities including endotoxin

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] A mixture of PEG400 and PEG6000 and PEG2000, ammonium sulfate and phosphate were used to form a two-phase aqueous phase, and combined with DEAE-dextran chromatography, the pHGF was prepared.

[0027] Take fresh suckling pig liver, cut out the outer membrane, mince, add purified water, and perform high-pressure homogenization before use. Take PEG400 and PEG6000, according to the mass composition ratio of 1.0:2.8 (w / w), dissolve it with a little purified water and mix it with the cell homogenate, then add ammonium sulfate, so that after mixing, the PEG and ammonium sulfate including the cell homogenate The ratio is 9% / 11% (w / v), and the concentration is adjusted with purified water, shaken to make it fully mixed, and then left to stand for 1.2h. At this point the upper phase was separated and the lower phase was discarded to obtain the first crude extract.

[0028] Take PEG2000, dissolve it with a little purified water, add it to the crude extract for the first time, mix...

Embodiment 2

[0031] The mixture of PEG20000 and PEG1000 and PEG4000, ammonium sulfate and phosphate were used to form a two-phase aqueous phase, combined with DEAE-dextran chromatography, to prepare pHGF.

[0032] Take fresh suckling pig liver, cut off the outer membrane, mince, and homogenize under high pressure for later use. Take PEG20000 and PEG1000, according to the mass composition ratio of 2.7:1.3 (w / w), after dissolving, mix with the homogenate, add ammonium sulfate, so that the ratio of PEG and ammonium sulfate in the homogenate is 8.2% / 12.3% (w / v), adjusting the concentration with water, after being fully mixed and standing for 1.3 hours, the upper phase was separated to obtain the first crude extract.

[0033] Take PEG4000 again, after dissolving, add in the crude extract for the first time, then add 0.76% (w / v) NaCl solution, after this, add sodium dihydrogen phosphate, form two-phase system, make PEG4000 and phosphate in total The composition ratio in the system reaches 10.3...

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PUM

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Abstract

The protein separating and purifying method includes twice extraction in polymer / salt double water phase system, and chromatographic separation with anionic exchange resin to obtain protein of molecular weight 10,000-30,000. The method can eliminate great amount of impurity and cell segment simultaneously, has the features of maintaining protein activity, short phase separating time, no residue of organic solvent, easy engineering amplification and control operation, and can eliminate endotoxin to affect product quality. The method of the present invention is especially suitable for the separation and purification of hepatocytokine.

Description

Technical field: [0001] The invention relates to the field of biotechnology, in particular to a method for separating and purifying small molecular proteins, in particular to separating and purifying hepatocyte growth factor (pHGF). Background technique: [0002] Aqueous two-phase extraction technology is an extraction method of biologically active substances that has been developed rapidly in recent years. The basic principle is to use polymers, salts, etc. under a certain composition and concentration to form two mutually incompatible water phases in the same water phase, which are called two-water phases. Using such two-water phases, To extract the target product from the cell homogenate or fermentation broth, to achieve the extraction and separation of the target product, its biggest advantage is that the separation is simple, the operation cost is low, and the biological activity of the target product can be effectively maintained. At present, a variety of aqueous two-...

Claims

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Application Information

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IPC IPC(8): C07K1/18C07K1/36
Inventor 王玉亭
Owner 王玉亭
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