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High-efficiency separation preparation method for preparing N-linked polysaccharide from ovalbumin, and polysaccharide

A technology of ovalbumin and polysaccharides, applied in the direction of fermentation, etc., to achieve the effect of high degree of automation, convenient operation and high purity

Active Publication Date: 2016-07-13
TAIZHOU GUOKEHUAWU BIOMEDICAL TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] So far, although there have been many studies on the release and structural characterization of glycoprotein sugar chains, the enrichment and separation of Asn-linked sugar chains from enzymatic hydrolysis solutions, and the separation and preparation by two-dimensional liquid chromatography have relatively high purity. Asn sugar chain components, has not been reported

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Weigh 1 mg of ovalbumin, add 150 μL of 4M urea in 30 mM ammonium bicarbonate buffer solution, incubate at 20° C. for 1 hour, and the pH of the buffer solution is 7. Add 10μL of 40mM dithiothreitol to the denatured ovalbumin, and incubate at 35°C for 1 hour to reduce the disulfide bond of the denatured ovalbumin, and then add 10μL of 30mM iodoacetic acid, 30°C under dark conditions Incubate for 10 minutes to alkylate the reduced disulfide bonds.

[0025] Add 510μL of Tris-HCl buffer solution to the denatured ovalbumin at a concentration of 0.05M; add 0.5mg of PronaseE, the pH of the enzymatic hydrolysis buffer solution is 4, the enzymatic hydrolysis reaction temperature is 35℃, and the enzymatic hydrolysis time is 12 Hours, after enzymolysis, the enzymolysis solution is boiled in boiling water for 5 minutes to inactivate the enzyme. The enzymatic hydrolysis solution is concentrated by centrifugation to obtain an enzymatic hydrolysis concentrate.

[0026] Pack the column wit...

Embodiment 2

[0028] Weigh 10 mg of ovalbumin, add 105 μL of 8M urea in 50 mM ammonium bicarbonate buffer solution, incubate at 30° C. for 5 hours, and the pH of the buffer solution is 9. Add 40μL of 30mM dithiothreitol to the denatured ovalbumin, and incubate at 40°C for 2 hours to reduce the disulfide bond of the denatured ovalbumin, and then add 20μL of 50mM iodoacetic acid, under dark conditions at 25°C Incubate for 30 minutes to alkylate the reduced disulfide bonds.

[0029] Add the denatured ovalbumin to 1000μL of Tris-HCl buffer solution with a concentration of 0.5M, add 100mg of PronaseE, the pH of the enzymatic buffer solution is 6.8, the enzymatic reaction temperature is 40℃, and the enzymatic hydrolysis time is 24 hours After enzymolysis, the enzymolysis solution is boiled in boiling water for 10 minutes to inactivate the enzyme. The enzymatic hydrolysis solution is concentrated by centrifugation to obtain an enzymatic hydrolysis concentrate.

[0030] Pack the column with C18 polar ...

Embodiment 3

[0032] Weigh 50 mg of ovalbumin, add 200 μL of 10 M urea in 50 mM ammonium bicarbonate buffer solution, incubate at 25° C. for 2 hours, and the pH of the buffer solution is 7. Add 20μL of 100mM dithiothreitol to the denatured ovalbumin, and incubate at 37°C for 5 hours to reduce the disulfide bond of the denatured ovalbumin, and then add 50μL of 100mM iodoacetic acid at 30°C under dark conditions Incubate for 20 minutes to alkylate the reduced disulfide bonds.

[0033] Add the denatured ovalbumin to 1500μL of Tris-HCl buffer solution, add 250mg of PronaseE at a concentration of 5M, the pH of the enzymatic buffer solution is 6.5, the enzymatic reaction temperature is 37℃, the enzymatic hydrolysis time is 72 hours, After hydrolysis, the enzymatic hydrolysate is boiled in boiling water for 5 minutes to inactivate the enzyme. The enzymatic hydrolysis solution is concentrated by centrifugation to obtain an enzymatic hydrolysis concentrate.

[0034] Pack the column with the C18 polar c...

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Abstract

The invention provides a high-efficiency separation preparation method for preparing an N-linked polysaccharide from ovalbumin. The main component of the N-linked polysaccharide is an N-linked polysaccharide component linked with only one amino acid asparagine (Asn). The method comprises the following steps: carrying out enzymatic hydrolysis on the ovalbumin by using a non-specific protease, centrifuging the obtained enzymatic hydrolysis solution, concentrating the centrifuged solution, removing polypeptides and residual proteins obtained after the ovalbumin enzymatic hydrolysis on the concentrated solution through using a one-dimensional chromatographic column, and collecting the target fraction; and centrifuging the collected fraction, concentrating the centrifuged fraction, and allowing the concentrated fraction to go through a two-dimensional chromatographic column to remove salt, 2-4 amino acids-linked N-connected polysaccharide components and micro-molecular amino acids in the fraction in order to obtain the N-linked polysaccharide component only linked with asparagines. A result of high resolution mass spectrum identification of the Asn linked N-linked polysaccharide component prepared in the invention shows that the N-linked polysaccharide component has clear composition, and the extraction and separation method has the advantages of high repeatability and good maneuverability, and is suitable for large-scale separation and preparation of the N-linked polysaccharide component from the ovalbumin.

Description

Technical field [0001] The present invention relates to the extraction, separation and preparation of N-linked glycan components separated and prepared from protein, in particular, the N-linked glycan group with an asparagine (Asn) is obtained by enzymatic separation and separation from ovalbumin The glycan in this component is composed of 5-15 mannose, galactose or N-acetylglucosamine, and the molecular weight is 1000-3000. Background technique [0002] Glycoproteins are widely found in animals, plants, microorganisms and viruses. They are complexes formed by covalently bonding sugar chains with proteins. Protein glycosylation is an important post-translational modification of proteins. In eukaryotes, especially mammals, more than half of the proteins are glycosylated, and membrane proteins are almost 100% in the form of glycosylation. And there is (ApweilerR, HermjakobH, SharonN, Onthe frequency of protein glycosylation, as deduced from analysis of the SWISS-PROT database. Bio...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P19/28C08B37/00
Inventor 梁鑫淼付冬梅于龙肖远胜曹翠岩
Owner TAIZHOU GUOKEHUAWU BIOMEDICAL TECH CO LTD
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