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Glass surface grafting method for protein crystallization

A protein crystallization, glass surface technology, applied in animal/human proteins, biochemical equipment and methods, enzymes, etc., can solve the problems of low protein nucleation efficiency and high nucleation potential energy, to improve the success rate of protein crystallization, increase the potential of nucleation. sequence, the effect of increasing protein concentration

Inactive Publication Date: 2015-05-06
NORTHWESTERN POLYTECHNICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] In order to overcome the disadvantages of low protein nucleation efficiency due to the high nucleation potential energy of the prior art, the present invention provides a method for promoting protein nucleation by grafting functional groups to glass for crystallization

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Example 1: Screening of ribonuclease A crystallization conditions.

[0024] The first step: Soak the glass after alcohol ultrasonic treatment in concentrated sulfuric acid (98% concentration) and hydrogen peroxide with a volume ratio of 8:1 for 50 hours, then clean the glass surface with deionized water until the pH is neutral, and then wash it Soak in cetyltrimethoxysilane solution diluted 200 times with toluene for 10 hours, wash the glass with toluene, and prepare a glass sheet with long-chain alkyl groups on the surface.

[0025]Step 2: Dissolve ribonuclease A with a product number of R5500 from Sigma Corporation of the United States in a pH 7, 0.025M HEPES-Na buffer solution to a final concentration of 20 mg / ml, and filter out impurities with a 0.22 μm filter membrane.

[0026] The third step: use 50 kinds of crystallization reagents of the Crystal Screen kit of HR2-110 from Hampton Company to screen the crystallization conditions of ribonuclease A, specifically mi...

Embodiment 2

[0029] Example 2: Screening of insulin crystallization conditions.

[0030] The first step: Soak the glass after alcohol ultrasonic treatment in concentrated sulfuric acid (98% concentration) and hydrogen peroxide at a volume ratio of 5:1 for 28 hours, then clean the surface of the junction glass with deionized water until the pH is neutral, and then wash it Immerse in a 2,3-epoxypropyltrimethoxysilane solution diluted 100 times in xylene for 10 hours, wash the glass with xylene, and dry it to prepare a glass sheet with hydroxyl groups on its surface.

[0031] Step 2: Dissolve the insulin whose product number is I5500 from Sigma Company of the United States in 1M ammonia water to make the final concentration 30 mg / ml, and filter out impurities with a 0.22 μm filter membrane.

[0032] Step 3: Screen the crystallization conditions of insulin with 50 kinds of crystallization reagents from the Crystal Screen kit of Hampton Company with the article number HR2-110, specifically mixi...

Embodiment 3

[0035] Example 3: Screening of pepsin crystallization conditions.

[0036] The first step: Soak the glass after alcohol ultrasonic treatment in 98% concentrated sulfuric acid and hydrogen peroxide with a volume ratio of 8:1 for 50 hours, then clean the surface of the junction glass with deionized water until the pH is neutral, and then immerse it in toluene to dilute it for 50 Soaked in cetyltrimethoxysilane solution twice as much for 10h, cleaned the glass with toluene, and prepared a glass sheet with methyl groups on the surface.

[0037] Step 2: Dissolve pepsin with product number R5500 from Sigma, USA in 0.025M HEPES-Na buffer at pH 7 to make the final concentration 35 mg / ml, and filter out impurities with a 0.22 μm filter membrane.

[0038] Step 3: Screen the crystallization conditions of pepsin with 50 kinds of crystallization reagents from the Crystal Screen kit of Hampton Company, the product number is HR2-110. Specifically, the crystallization reagent and the protein ...

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Abstract

The invention provides a glass surface grafting method for protein crystallization, which comprises the following steps: soaking a glass sheet subjected to alcohol or acetone ultrasonic treatment in concentrated sulfuric acid and oxydol; cleaning the glass surface with deionized water until the pH value is 7, soaking in an silane solution diluted by an organic solvent to obtain a glass substrate, preparing a protein crystallization solution, dropping on the surface of the glass substrate to obtain liquid drops, and standing to crystallize; and observing under a microscope, and making statistics on the condition number of protein crystals. According to the invention, the functional group is grafted on the crystal glass surface, so that the protein is concentrated on the glass surface and simultaneously forms an ordered structure on the bottom surface to lower the surface energy of nucleation, thereby enhancing the success rate of protein crystallization.

Description

technical field [0001] The invention relates to a method for grafting glass surface functional groups for protein crystallization, in particular to a method for screening protein crystallization conditions by grafting functional groups on the glass surface. Background technique [0002] Protein is the carrier of life activities, and elucidating protein-related functions is the key to exploring life phenomena. Protein structure is the premise and foundation of functional research. X-ray diffraction technology is currently the most important and reliable research method for obtaining protein structure. High-quality protein crystals are the prerequisite for obtaining high-resolution structural information. Screening crystallizable conditions is the first step to obtain high-quality protein crystals, and it is one of the important bottlenecks restricting protein structure research. [0003] Protein crystal growth involves two steps: nucleation and growth. Nucleation is the fir...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K1/30C07K14/62C12N9/22C12N9/50C12N9/36C12N9/08
Inventor 尹大川张辰艳王前进沈何放郭云珠何进卢慧甍曹慧玲刘永明
Owner NORTHWESTERN POLYTECHNICAL UNIV
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