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Preparation and application of a kind of immobilized protease reagent

An immobilized enzyme and protease technology, applied in the preparation of immobilized protease reagents, can solve the problems of limiting enzymatic hydrolysis efficiency, inevitable enzymatic hydrolysis bias, affecting the identification coverage of proteins and peptides, etc.

Active Publication Date: 2016-10-19
INST OF RADIATION MEDICINE ACAD OF MILITARY MEDICAL SCI OF THE PLA
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the currently widely used method of monolayer enzyme immobilization on the surface of the matrix material limits the enzyme immobilization capacity per unit mass of the immobilized enzyme material by the surface area of ​​the matrix material, thus limiting the further improvement of the enzymatic hydrolysis efficiency.
At the same time, the existing immobilized enzyme reagents all use a single carrier material, which inevitably causes the enzymatic hydrolysis bias caused by the difference in the affinity of the carrier itself for the protein, resulting in insufficient enzymatic hydrolysis of the protein and affecting the coverage of protein and peptide identification.

Method used

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  • Preparation and application of a kind of immobilized protease reagent
  • Preparation and application of a kind of immobilized protease reagent
  • Preparation and application of a kind of immobilized protease reagent

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0113] Embodiment 1, the synthesis of the carrier material of immobilized protease

[0114] The technical scheme of preparing trypsin with hydrophilic and hydrophobic dual carriers by SI-ATRP method and the flow chart of enzymatic protein hydrolysis with dual carrier immobilized enzymes are as follows figure 1 shown.

[0115] 1. Synthesis of SI-ATRP initiator

[0116] SI-ATRP initiator was synthesized by reacting 3-aminopropyltriethoxysilane with 2-bromoisobutyryl bromide. One end of the initiator was a silane coupling agent bound to the surface of silica-wrapped magnetic nanoparticles, and the other was One end is an ATRP initiator. The specific steps are as follows: add 8mmol 3-aminopropyltriethoxysilane and 10mmol triethylamine to 12.5ml tetrahydrofuran, mix and pass through nitrogen to deoxygenate while ice bathing for 30min to obtain a silane coupling agent, then add 10mmol2 -Bromoisobutyryl bromide (ATRP initiator) was slowly added dropwise to the mixture, stirred vig...

Embodiment 2

[0138] Embodiment 2, the immobilization of trypsin

[0139] 1. Aldehydization of Magnetic Nanoparticles

[0140] The aldehyde group functionalization of the polymer side chain of the magnetic nanoparticle modified by GMA-G polymer chain, the specific method is as follows: 10mM sodium periodate aqueous solution is mixed with the magnetic nanoparticle modified by GMA-G polymer chain, at 20 React at -30°C for 2 hours in the dark. After the reaction is completed, use 50% methanol aqueous solution to wash repeatedly to remove the remaining reactants.

[0141] The aldehyde group functionalization of the polymer side chain of the GMA polymer chain-modified magnetic nanoparticles, the specific method is as follows: mix the GMA polymer chain-modified magnetic nanoparticles with 0.2M dilute sulfuric acid, and react at room temperature in the dark for 2 Hour. After the reaction is completed, use 50% methanol aqueous solution to wash repeatedly to remove the remaining reactants.

[01...

Embodiment 3

[0146] Example 3. Functional Analysis of GMA Polymer Chain Modified Magnetic Particle Immobilized Enzyme (GMA-Trypsin) and GMA-G Polymer Chain Modified Magnetic Particle Immobilized Enzyme (GMA-G-Trypsin)

[0147] 1. Extraction of Yeast Whole Protein

[0148] (1) Whole protein extract: 50mM Tris-HCl (pH=8.0), 8M urea, 2mM EDTA.

[0149] (2) Add 10 μl of “cocktail” protease inhibitor (purchased from Roche, Germany) to 500 μl of whole protein extract, mix well, add to a test tube containing yeast, and ultrasonically disrupt the cells. Then centrifuge at 20,000 g for 20 minutes at 4°C to remove unbroken cells and debris, and the solution is yeast whole protein.

[0150] (3) Add 4-5 times the volume of cold acetone solution (pre-cooled at -20°C) to the whole yeast protein solution, place it at -20°C for more than 2 hours, then centrifuge at 12,000g at 4°C for 10 minutes, carefully absorb the supernatant, and keep the precipitate .

[0151] (4) Place the precipitate in a fume ho...

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Abstract

The invention discloses preparation and application of an immobilized protease reagent. An immobilized enzyme disclosed by the invention is composed of a hydrophobic carrier material and a protease immobilized on the hydrophobic carrier material. Two different-property, namely one hydrophilic and one hydrophobic, polymer chain modified magnetic nanoparticle immobilized trypsins prepared independently by using a surface initiate atom transfer radical polymerization (SI-ATRP) realize quick, efficient and complete enzymolysis of a protein, and the two, one hydrophilic and one hydrophobic, immobilized proteases are combined to use so that complementary enzymolysis is realized, and therefore, enzymatic bias caused by the selectivity of the carrier can be effectively reduced, the comprehensiveness of protein enzymolysis can be improved, and then the identification number of proteins and peptide fragments can be remarkably increased.

Description

technical field [0001] The invention relates to the preparation and application of an immobilized protease reagent. Background technique [0002] As a key research field in the post-genome era, proteomics research can not only provide a theoretical basis for the elucidation of the laws of life activities, but also play a role in the diagnosis, treatment, prevention, pathogenesis and pathogenesis of diseases, and the development of new drugs. Since 2001, it was rated as the six hot research fields in the 21st century by "Science" magazine, and proteomics research has received extensive attention from scientists. Currently the most widely used proteomics research strategy is the "shot-gun method" strategy. In this strategy, the qualitative and quantitative information of the protein is obtained by analyzing the corresponding peptides of the enzymatic hydrolysis product. Therefore, fast, efficient and complete protein enzymatic hydrolysis has become an accurate, high-throughpu...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N11/14C12N11/08C12P21/06
Inventor 钱小红秦伟捷范超
Owner INST OF RADIATION MEDICINE ACAD OF MILITARY MEDICAL SCI OF THE PLA
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