A Method for Improving the Success Rate of Protein Crystallization by Surface Grafting of Cyclodextrins

A technology of protein crystallization and cyclodextrin, which is applied in the field of protein crystallization, can solve the problems of time-consuming and artificial disadvantages, and achieve the effects of avoiding labor and time-consuming, improving the success rate of crystallization, and promoting crystal nucleation

Inactive Publication Date: 2019-04-19
NORTHWESTERN POLYTECHNICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, in the original technology, cyclodextrin needs to be manually added to the crystallization system one by one, which is time-consuming and labor-intensive, and is not conducive to the realization of automation.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025]Example 1: Screening of glucose isomerase crystallization conditions

[0026] Step 1: Obtain a crystalline plate grafted on the surface of methyl cyclodextrin through surface treatment. Specifically, use H 2 O, 98% concentrated sulfuric acid and K 2 Cr 2 o 7 Mix according to the mass percentage of 30:65:5 and configure it as a treatment solution. Soak the PS board with the treatment solution for 40min; then, rinse the surface of the soaked PS board with deionized water; then, put the treated crystallized board in 10wt% dilute sulfuric acid solution at 60°C for 24h; finally wash it with deionized water After cleaning, a PS plate with hydroxyl groups grafted on the surface is obtained. Put the crystalline plate grafted on the surface with acetic acid to adjust the pH to about 4, containing 1vol% 2,3-glycidoxytrimethoxysilane, 1vol% H 2 Soak in an anhydrous ethanol solution of O for 12 hours at room temperature, rinse with ethanol several times after taking it out, an...

Embodiment 2

[0031] Embodiment 2: Screening of lysozyme crystallization conditions

[0032] Step 1: Obtain a crystalline plate grafted on the surface of β-cyclodextrin through surface treatment. Specifically, use H 2 O, 98% concentrated sulfuric acid and K 2 Cr 2 o 7 Mix according to the mass percentage of 50:47:3 to prepare the treatment liquid. Soak the PS plate with the treatment solution for 20min; then, rinse the surface of the treated PS plate with deionized water; then, soak the treated crystallized plate in 15wt% dilute sulfuric acid solution at 60°C for 1h; finally use deionized water After washing with water, a PS plate with hydroxyl groups grafted on the surface is obtained. Put the surface grafted hydroxyl crystal plate into the pH of about 6 with acetic acid, containing 1vol% 2,3-glycidoxytrimethoxysilane, 1.5vol% H 2 Soak in anhydrous ethanol solution of O for 24 hours at room temperature, rinse it with ethanol several times after taking it out, and then clean it with d...

Embodiment 3

[0037] Example 3: Screening of concanavalin crystallization conditions

[0038] Step 1: Obtain a crystalline plate grafted on the surface of p-toluenesulfonyl cyclodextrin through surface treatment. Specifically, use H 2 O, 98% concentrated sulfuric acid and K 2 Cr 2 o 7 Mix according to the mass percentage of 40:58:2 to prepare the treatment liquid. Soak the PS plate with the treatment solution for 140min; then, rinse the surface of the treated PS plate with deionized water; then, soak the treated crystallized plate in 10wt% 55°C dilute sulfuric acid solution for 10h; finally use deionized water After washing with water, a PS plate with hydroxyl groups grafted on the surface is obtained. Put the crystallization plate grafted on the surface with acetic acid to adjust the pH to about 5.5, containing 2vol% 2,3-glycidoxytrimethoxysilane, 0.5vol% H 2 Soak in anhydrous ethanol solution of O for 10h at room temperature, rinse it with ethanol several times after taking it out, ...

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PUM

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Abstract

The invention provides a method for increasing the protein crystallization success rate by cyclodextrin surface grafting. The method comprises the following steps: firstly, treating the surface of a crystallization plate to hydroxylate the surface of the crystallization plate, and modifying the surface of the crystallization plate by adopting cyclodextrin or a derivative thereof; then preparing a protein solution to finally establish a protein crystallization system, and performing crystallization. According to the method, a crystallization plate of which the surface is grafted with cyclodextrin can be obtained by two-step treatment on the crystallization plate; a large quantity of crystallization plates can be treated at a time; meanwhile, the process is simple, and the labor and time consumption of adding heterogeneous nucleating agents one by one is avoided. Due to the method, the crystallization success rate can be twice or above of that in the prior art.

Description

technical field [0001] The invention relates to a method for grafting cyclodextrin or its derivatives on the surface of a crystallization plate for protein crystallization, in particular to a method for improving the success rate of protein crystallization by grafting cyclodextrin or its derivatives on the surface of a crystallization plate. Background technique [0002] As the material basis of life activities, protein has important biological significance in the study of its function. Protein structure is the premise and foundation of functional research, so analyzing protein structure is of great significance for elucidating related biological problems. At present, the technologies for obtaining protein structure mainly include X-ray diffraction technology, nuclear magnetic resonance technology and cryo-electron microscopy, among which X-ray diffraction technology is the most widely used and the highest precision. Obtaining diffractive protein crystals is the premise and...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K1/30C12N9/92C12N9/36C07K14/42C12N9/56C12N9/22C12N9/42
CPCC07K1/306C07K14/42C12N9/22C12N9/2437C12N9/2462C12N9/54C12N9/92C12Y301/27005C12Y302/01004C12Y302/01017C12Y304/21062C12Y503/01018
Inventor 张辰艳杨雪舟尹大川王前进董晨郭云珠闫二开
Owner NORTHWESTERN POLYTECHNICAL UNIV
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