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Method for preparing reductive N-sugar chain through release and purification of Glycosylasparaginase enzymes

A technology for sugar chains and uses, applied in the field of glycobiology, can solve problems such as strong versatility, high enzyme activity, and low cost, and achieve the effects of low cost, high enzyme activity, and mild reaction conditions.

Inactive Publication Date: 2017-11-24
NORTHWEST UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0011] In order to solve the lack of reducing N-sugar chains containing core α-1,3-fucose modification in the prior art, the release reducing N -The problem of enzymatic hydrolysis of sugar chains, on the one hand, the present invention provides a method for releasing reducing N-sugar chains using Glycosylasparaginase enzymes; Purification method

Method used

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  • Method for preparing reductive N-sugar chain through release and purification of Glycosylasparaginase enzymes
  • Method for preparing reductive N-sugar chain through release and purification of Glycosylasparaginase enzymes
  • Method for preparing reductive N-sugar chain through release and purification of Glycosylasparaginase enzymes

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0093] Example 1: Enzymatic release, purification and mass spectrometry analysis of N-sugar chains in horseradish peroxidase

[0094] 1. Release by enzymatic hydrolysis

[0095] Take 0.01 mg of horseradish peroxidase, dissolve it in 100 μL of 5 mM dithiothreitol (DTT) solution, react at 50 ° C for 30 min, and after cooling to room temperature, add 10 μL of 10 mM iodoacetamide, and shake for 30 min in the dark. Blow dry in a nitrogen blow dryer to obtain denatured glycoprotein samples. The denaturation of glycoprotein, that is, the destruction of the secondary structure in the molecule, does not involve the breaking of peptide bonds and disulfide bonds, and the primary structure remains intact.

[0096] Add 1 mL of Tris-HCl buffer solution with pH 8 and 1.0 mM calcium chloride to the denatured glycoprotein sample to fully dissolve the sample, then add 0.05 μg trypsin to it, and place the resulting mixture in a constant temperature water bath at 37 °C During the reaction for...

Embodiment 2

[0125] Example 2: Enzymatic release, purification and mass spectrometry analysis of N-sugar chains in insects

[0126] 1. Enzymatic hydrolysis to release N-sugar chains

[0127] 100 silkworm eggs were washed repeatedly with Tris-HCl (pH 8.0) buffer for 3 to 5 times to remove impurities; then transferred to a tissue homogenizer and added protein extract (RIPA Buffer) 1mL, then add 1 / 10 volume of protease inhibitor (PMSF, 2.0mg / mL, aqueous solution), grind on ice in a grinder for several minutes, centrifuge to get the supernatant, put it in a dialysis bag and place it in a refrigerator at 4°C After dialysis for 3 days (change the water three times a day), the samples in the dialysis bag were recovered, and then placed in a vacuum freeze dryer for freeze-drying for future use.

[0128] Weigh 200 mg silkworm egg protein and silk protein respectively, dissolve in 500 μL 5.0 mM DTT solution, place in 50 ° C water bath for 30 min, wait for the reaction solution to cool to room tem...

Embodiment 3

[0149] Example 3: Enzymatic release, purification and mass spectrometry analysis of N-sugar chains in tobacco

[0150] 1. Enzymatic hydrolysis to release N-sugar chains

[0151] Take 100 mg of tobacco leaves, cut or pulverize the plant leaves, put them in a grinder and grind them in liquid nitrogen, and then dissolve them in 1 mL of 100 mM Tris-HCl (pH 8.0) on ice, at 4 °C, 10000 r / min Centrifuge for 10 minutes, take the supernatant, and discard the precipitate; then add 1 / 3 volume of 50% TCA aqueous solution, stir well, precipitate the protein overnight in the refrigerator at 4°C, and then centrifuge at 6000r / min in the centrifuge for 5min. Take the precipitate, redissolve the precipitate in pH=8 Tris-HCl buffer, add 50% TCA repeatedly for precipitation; repeat the experiment twice to fully remove free polysaccharides; then dissolve the precipitate in 90% (V / V) acetone In the aqueous solution, sonication, centrifugation, discarding the supernatant, and repeating twice to rem...

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Abstract

The invention belongs to the technical field of sugar biology and particularly relates to a method for preparing a reductive N-sugar chain through release and purification of Glycosylasparaginase enzymes. The enzymolysis and release method of the N-sugar chain comprises the following steps that a Tris-HCl buffer solution is added into denatured glycoprotein, a sample is dissolved, and an appropriate amount of trypsin is added to obtain an enzymolysis solution containing N-glycopeptide; and the appropriate number of Glycosylasparaginase enzymes are added to conduct an enzymolysis reaction to obtain the reductive N-sugar chain. Through an experiment, it is verified that the method can be applied to release of N-sugar chains of all types, including release of the N-sugar chain modified by alpha-1, 3-Fuc, operation is easy and convenient, the cost is low, and the method is suitable for large-scale application and has important research and application value in the fields of life science and medicines.

Description

technical field [0001] The invention belongs to the technical field of glycobiology, and in particular relates to a method for releasing and purifying reducing N-sugar chains by using Glycosylasparaginase enzyme and the application of the method. Background technique [0002] More than 50% of the proteins in organisms exist in the form of glycoproteins, which are widely found in animals, plants, bacteria, and fungi. Glycosylation is one of the most important post-translational modifications of proteins, and plays a very important role in protein translation regulation and protein degradation. [0003] In eukaryotes, the classification of glycoproteins is based on the type of connection between sugar chains and proteins, which can be divided into two categories: N-sugar chains and O-sugar chains. Among them, the O-sugar chain of glycoprotein is connected to protein serine or the hydroxyl oxygen of serine through N-acetylglucosamine, and there are also connections through oth...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P19/14C12P21/06C08B37/00C08B37/02
CPCC08B37/0003C08B37/0033C12P19/14C12P21/06
Inventor 王仲孚黄琳娟张英宋晶晶
Owner NORTHWEST UNIV
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