Preparation and application of immobilized protease reagent

A technology of immobilized enzymes and proteases, which is applied in the field of preparation of immobilized protease reagents, can solve the problems affecting the identification coverage of proteins and peptides, the enzymatic hydrolysis of proteins is not comprehensive enough, and the unavoidable partiality of enzymatic hydrolysis, etc., to speed up sample processing The effect of speed, increased collision probability, and large degrees of freedom

Active Publication Date: 2014-06-25
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 t

Method used

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

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0113] Example 1. Synthesis of carrier material for immobilized protease

[0114] The SI-ATRP method prepares the technical scheme of hydrophilic and hydrophobic dual carrier immobilized trypsin and the flow chart of the dual carrier immobilized enzyme enzymatic protein hydrolysis figure 1 Shown.

[0115] 1. Synthesis of SI-ATRP initiator

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

Embodiment 2

[0138] Example 2. Immobilization of trypsin

[0139] 1. Aldehydrylization of magnetic nanoparticles

[0140] The aldehyde groups of the polymer side chains of the GMA-G polymer chain-modified magnetic nanoparticles are functionalized as follows: mix a 10mM sodium periodate aqueous solution with the GMA-G polymer chain-modified magnetic nanoparticles evenly at 20 React for 2 hours in the dark at -30°C. After the completion of the reaction, use a methanol aqueous solution with a volume percentage of 50% to repeatedly wash and remove the remaining reactants.

[0141] The aldehyde groups of the polymer side chains of the GMA polymer chain-modified magnetic nanoparticles are functionalized as follows: Use 0.2M dilute sulfuric acid to mix the GMA polymer chain-modified magnetic nanoparticles uniformly, and react at room temperature away from light 2 hour. After the completion of the reaction, use a methanol aqueous solution with a volume percentage of 50% to repeatedly wash and remove t...

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 whole yeast protein

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

[0149] (2) Add 10μl of "cocktail" protease inhibitor (purchased from Roche Germany) to 500μl of the whole protein extract, mix well and add it to a test tube containing yeast, and ultrasonically disrupt the cells. After centrifugation at 20000g at 4°C for 20 minutes, unbroken cells and debris are removed, and the solution is the yeast whole protein.

[0150] (3) Add 4-5 times the volume of cold acetone solution (pre-cooled at -20°C) to the yeast whole protein solution, place at -20°C for more than 2 hours and centrifuge at 12000g at 4°C for 10 minutes, carefully aspirate the supernatant, and save the precipitate .

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

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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-genomic 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 of pathogenic pathogens and the development of new drugs. Since being named as the six hot research areas of the 21st century by the "Science" magazine in 2001, proteomics research has received extensive attention from scientists. At present, 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 peptides of the corresponding enzymatic hydrolysis products. Therefore, rapid, efficient and complete protein enzymatic hydrolysis becomes accurate, high-throughput identifica...

Claims

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Application Information

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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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