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Preparation method of micro-and nanoscale protein crystals capable of stably existing in air

A micro-nano-scale, protein technology, applied in the field of protein crystallization methodology, can solve the problems of crystal solidification method in the preparation method, the influence of diffraction data quality, and the existence of background scattering, so as to facilitate long-term storage, save experimental costs, and eliminate background scattering. Effect

Active Publication Date: 2016-04-06
NORTHWESTERN POLYTECHNICAL UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

When the crystals obtained by these methods are diffracted, not only will a large amount of protein crystals be wasted, but also there will be background scattering, which will affect the quality of diffraction data
In addition, due to the small size of tiny crystals, there are major technical difficulties in their preparation methods and crystal solidification methods

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Example 1: Preparation of micro-nanoscale lysozyme crystals that can exist stably in the air.

[0022] The first step: preparing lysozyme crystallization solution. First, prepare two pH 3 acetic acid-sodium acetate buffer solutions. Then, the No.100940 lysozyme of Japan Seikagaku Company was dissolved in a buffer solution, and the initial concentration was 80mgml -1 . Secondly, dissolve the superior grade pure sodium chloride in another buffer solution to prepare a precipitant solution, the initial concentration is 80mgml -1 . Finally, equal volumes of lysozyme solution and sodium chloride solution were mixed to obtain a protein crystallization solution.

[0023] The second step: prepare glutaraldehyde crosslinking solution. Glutaraldehyde with a volume fraction of 50% was added dropwise to the NaCl precipitant so that the final volume fraction was 5%.

[0024] The third step: preparing tiny crystals of lysozyme. The lysozyme crystal solution is placed in a tempe...

Embodiment 2

[0029] Example 2: Preparation of micro-nanoscale lactate dehydrogenase crystals that can exist stably in the air.

[0030] The first step: preparing lactate dehydrogenase crystallization solution. First, prepare two pH 8.000.1M phosphate buffer solutions. Then, the No.61309 lactate dehydrogenase of Sigma Company of the United States was dissolved in a buffer solution, and the initial concentration was 10mgml -1 . Secondly, ammonium sulfate was dissolved in another buffer solution to prepare a precipitant solution with a volume fraction of 70%. Finally, equal volumes of the lactate dehydrogenase solution and the precipitant solution are mixed to obtain a protein crystallization solution.

[0031]The second step: prepare the cross-linking solution. First prepare a 0.5M tris-hydroxyethylamine buffer solution with a pH of 7.50, and then dissolve glutaraldehyde in the buffer solution with a concentration of 15mM.

[0032] The third step: preparing tiny crystals of lactate dehy...

Embodiment 3

[0037] Example 3: Preparation of micro-nano-scale proteinase K crystals that can exist stably in the air

[0038] The first step: preparing proteinase K crystallization solution. First, prepare two buffer solutions of 0.08M magnesium acetate, 0.05M sodium cacodylate, pH 4.60. Then, the No.P6556 proteinase K of Sigma Company of the United States was dissolved in a buffer solution, and the initial concentration was 20mgml -1 . Secondly, PEG8000 was dissolved in another buffer solution to prepare a precipitant solution with a volume fraction of 20%. Finally, equal volumes of proteinase K solution and PEG8000 precipitant were mixed to prepare protein crystallization solution.

[0039] The second step: prepare the cross-linking solution. Glutaraldehyde with a volume fraction of 50% was added dropwise to 20% PEG8000 precipitant, so that the final volume fraction was 1%.

[0040] The third step: preparing tiny crystals of lysozyme. The proteinase K crystallization solution is p...

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Abstract

The invention provides a preparation method of micro-and nanoscale protein crystals capable of stably existing in air. Protein mini-crystals grow through the simple and feasible method, chemical cross-linking is conducted on the mini-crystals through a cross-linking agent, and the protein mini-crystals capable of stably existing in air are obtained. The method is convenient to operate, economical and practical; the prepared protein mini-crystals do not need crystal rings or capillary tubes when used for free electron laser diffraction, and background scattering can be eliminated; when the crystals are used as a catalyst, a sample can not be dissolved and can be repeatedly used.

Description

technical field [0001] The invention relates to a method for preparing protein microcrystals, in particular to a method for preparing micro-nano-scale protein microcrystals that can exist stably in air, and belongs to the field of protein crystallization methodology. Background technique [0002] At present, in the protein structure database, more than 88% of the protein structures are obtained by X-ray single crystal diffraction analysis. Obtaining high-quality protein single crystals is the key to this technology, and it is also the bottleneck of this technology. Because many proteins are difficult to crystallize or obtain single crystals of appropriate size, breaking through the traditional X-ray single crystal diffraction technology to analyze the structure has become a new direction of effort. Recently, the method of using free electron lasers (free electron lasers, XFELs) to diffract a large number of micro-nano scale protein crystals to obtain diffraction data has be...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/36C12N9/04C12N9/50C12N9/08
CPCC12N9/0006C12N9/0065C12N9/2462C12N9/50C12Y101/01027C12Y101/01028C12Y101/03004C12Y111/01006C12Y302/01017
Inventor 尹大川闫二开张辰艳周仁斌陈达刘悦李大为刘雅丽
Owner NORTHWESTERN POLYTECHNICAL UNIV
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