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Efficient Separation and Preparation of N-Linked Glycans from Ovalbumin and Glycans

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

Active Publication Date: 2019-08-27
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, the pH of the buffer solution is 7. Add 10 μL of 40 mM dithiothreitol to the denatured ovalbumin, and incubate at 35°C for 1 hour to reduce the disulfide bonds of the denatured ovalbumin, then add 10 μL of 30 mM iodoacetic acid, and incubate at 30°C in the dark. Incubate for 10 minutes to alkylate the reduced disulfide bonds.

[0025] Add 510 μL of Tris-HCl buffer solution to the denatured ovalbumin at a concentration of 0.05M; add 0.5 mg of Pronase E, the pH of the enzymolysis buffer solution is 4, the enzymolysis reaction temperature is 35°C, and the enzymolysis time is After 12 hours of enzymolysis, the enzymolysis solution was boiled in boiling water for 5 minutes to inactivate the enzyme. Centrifuge and concentrate the enzymolysis solution to obtain the enzymolysis concentrate.

[0026] Pack the column with C18 polar copolymer reversed-phase...

Embodiment 2

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

[0029] Add denatured ovalbumin to 1000 μL of Tris-HCl buffer solution with a concentration of 0.5 M, add 100 mg of Pronase E, the pH of the enzymatic hydrolysis buffer solution is 6.8, the enzymatic hydrolysis reaction temperature is 40°C, and the enzymatic hydrolysis time is 24 Hours, after enzymolysis, the enzymolysis solution was boiled in boiling water for 10 minutes to inactivate the enzyme. Centrifuge and concentrate the enzymolysis solution to obtain the enzymolysis concentrate.

[0030] Pack the column with C...

Embodiment 3

[0032] Weigh 50 mg 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 20 μL of 100 mM dithiothreitol to the denatured ovalbumin, and incubate at 37°C for 5 hours to reduce the disulfide bond of the denatured ovalbumin, then add 50 μL of 100 mM iodoacetic acid, and incubate at 30°C under dark conditions Incubate for 20 minutes to alkylate the reduced disulfide bonds.

[0033] Add 1500 μL of Tris-HCl buffer solution to the denatured ovalbumin, and add 250 mg of Pronase E at a concentration of 5M. The pH value of the enzymolysis buffer solution is 6.5, the enzymolysis reaction temperature is 37°C, and the enzymolysis time is 72 hours. After enzymolysis, the enzymolysis solution was boiled in boiling water for 5 minutes to inactivate the enzyme. Centrifuge and concentrate the enzymolysis solution to obtain the enzymolysis concentrate.

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

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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 fractions from proteins, specifically, the preparation of N-linked glycans with an asparagine (Asn) from ovalbumin by enzymatic separation The polysaccharide 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, and are complexes formed by covalently binding sugar chains to proteins. Protein glycosylation is an important post-translational modification of proteins. In eukaryotes, especially mammals, more than half of the proteins are glycosylated, and almost 100% of membrane proteins are glycosylated. But exist (Apweiler R, Hermjakob H, Sharon N, On the frequency of proteinlycosylation, as deduced from analysis of the SWISS-PROT database. Biochim Biophys Acta-Gen Subj, 1999, 1473, 4-8)....

Claims

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Application Information

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Patent Type & Authority Patents(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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