Method for selecting buffer solution for protein crystallization

A protein crystallization and buffer technology, which is applied in the preparation methods of peptides, chemical instruments and methods, biochemical equipment and methods, etc., can solve the problems of fixed protein solubility, small nucleation interval, and low success rate of protein crystallization. The effect of increasing the probability of protein crystal nucleation and improving the crystallization efficiency

Inactive Publication Date: 2013-10-02
NORTHWESTERN POLYTECHNICAL UNIV
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  • Summary
  • Abstract
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Problems solved by technology

The reason is that this method does not take into account the properties of the protein itself, the solubility of the pro

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0013] Example 1: The crystallization conditions of lysozyme protein were screened by selecting two kinds of protein buffers whose pH was evenly distributed in the range of pH 1-14.

[0014] The first step: with the lysozyme whose product number is 100940 of Japanese Seikagaku Company through six times of recrystallization, according to the method proposed by the present invention, select two kinds of pH conditions evenly distributed in the range of 1~14pH interval, be that pH is 1 and 14 respectively. 14. Combine 0.05mol / L sodium acetate buffer to dissolve the protein so that the final concentration of the protein is 20mg / ml.

[0015] The second step: In this embodiment, 24 kinds of crystallization reagents using polyethylene glycol as a precipitating agent in the Crystal Screen kit of Hampton Company, whose article number is HR2-110, are used to screen the crystallization conditions of lysozyme. :1 After mixing, the crystallization conditions of lysozyme protein were screene...

Embodiment 2

[0018] Example 2: Within the range of protein stability, select a buffer pH far away from the isoelectric point of the protein to screen the crystallization conditions for catalase protein.

[0019] The first step: the catalase whose article number is C0615 from Sigma Company of the United States, the isoelectric point of catalase is 5, and the protein stable pH range is 4~8.5, according to the method proposed by the present invention, within the range of protein stable range Choose a pH condition far away from the isoelectric point of the protein, which is 8.5. The catalase was dissolved in 0.1mol / L PB buffer at pH 8.5 to make the final protein concentration 30mg / ml.

[0020] The second step: In this embodiment, 24 kinds of crystallization reagents using polyethylene glycol as a precipitating agent are used in the Crystal Screen kit of Hampton Company with a product number of HR2-110. After mixing the crystallization reagent and the protein solution at a ratio of 0.5:1, the ...

Embodiment 3

[0023] Example 3: Three kinds of protein buffer solutions evenly distributed in the pH range of 1-14 were selected to screen the crystallization conditions of catalase protein.

[0024] The first step: use the catalase with the product number C0615 of Sigma Company of the United States to select three pH conditions evenly distributed in the range of 1-14pH interval according to the strategy proposed by the present invention, which are 1.5, 6.5 and 13.5 respectively. The protein was dissolved in the buffer combination of 0.1mol / L sodium acetate, HEPES-Na and glycine, and the pH values ​​were 1.5, 6.5 and 13.5.

[0025] The second step: In this example, 24 kinds of crystallization reagents using polyethylene glycol as a precipitating agent are used in the Crystal Screen kit of Hampton Company with a product number of HR2-110. After mixing the crystallization reagent and protein solution according to 20:1, the Catalase protein crystallization conditions were screened.

[0026] S...

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PUM

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Abstract

The present invention provides a method for selecting a buffer solution for protein crystallization. The method comprises: preparing a uniformly distributed buffer solution to dissolve protein; mixing a crystallization reagent and the protein solution or mixing a crystallization reagent, the buffer solution and a protein sample to prepare a protein crystallization solution; and standing in a sealed crystallization plate until crystallization. According to the present invention, the appropriate protein crystallization buffer solution pH value condition is selected, such that protein solubility is changed in a wide range, and protein crystal nucleation probability is increased so as to increase crystallization efficiency.

Description

technical field [0001] The invention relates to a method for selecting a buffer for protein crystallization, in particular to a method for screening protein crystallization conditions by selecting a buffer. Background technique [0002] Protein is the basis of life activities, and protein structure is an important prerequisite for determining its physiological function. X-ray diffraction technique is currently the most important and reliable research method to obtain protein structure. High-quality protein crystals are the basis of this technology, and how to screen and obtain protein crystallization conditions is the first and most important step in obtaining high-quality protein crystals, which is the main bottleneck restricting protein structure analysis. [0003] The success rate of protein crystallization is affected by many factors. According to literature 1 "Crystallization and evaluation of hen egg-white lysozyme crystals for protein pH titration in the crystalline ...

Claims

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Application Information

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IPC IPC(8): C07K1/14C12N9/36C12N9/08C07K14/415
Inventor 尹大川张辰艳吴子庆沈何放卢慧甍郭云珠周仁斌
Owner NORTHWESTERN POLYTECHNICAL UNIV
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