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Refolding method for protein in electrostrictive polymer

An electroshrinkage and protein technology, applied in the field of protein folding research, can solve the problems of inclusion bodies that cannot be further folded, collisions, misfolding, etc., and achieve the effect of simple and effective experimental means

Inactive Publication Date: 2010-02-03
TIANJIN UNIVERSITY OF TECHNOLOGY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The purpose of the present invention is to solve the problems of collision and entanglement in the protein folding process, which affect the normal self-assembly of the protein, even lead to misfolding, and produce inclusion bodies that cannot be further folded. Analytical methods for protein refolding

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] 1. Protein unfolding:

[0023] Dissolve catalase in a phosphate buffer solution with a concentration of 8 mol / L guanidine hydrochloride; place it overnight at 4°C to denature it.

[0024] 2. Protein encapsulation:

[0025] In 4~5mL denatured catalase phosphate buffer solution, add 1.2~1.6g 2-acrylamido-2-methyl 1-propanesulfonic acid, 0.1~0.2g N,N'-methylenebis Acrylamide, 10 mg ammonium persulfate and 4 mg N, N, N', N'-tetramethylethylenediamine; shake well, pour into a quartz cuvette, let stand at room temperature until the gel solidifies; place the gel in a flowing Water dialysis for 3 days to remove guanidine hydrochloride.

[0026] 3. Protein refolding:

[0027] Insert platinum wire electrodes on both sides of the cuvette containing the gel, and apply a constant electric field of 5V / cm; the volume of the gel shrinks slightly, and water is precipitated; Both the excitation slit and emission slit width are 10nm, the scanning range is 300-420nm, and the scanning s...

Embodiment 2

[0029] 1. Protein unfolding:

[0030] Dissolve catalase in phosphate buffer with a concentration of 10mol / L urea; place it overnight at 4°C to denature it.

[0031] 2. Protein encapsulation:

[0032] In 4~5mL of denatured catalase phosphate buffer, add 1.2~1.6g of acrylic acid, 0.1~0.2g of N,N'-methylenebisacrylamide, 10mg of ammonium persulfate and 4mg of N,N,N' , N'-tetramethylethylenediamine; shake well, pour into a quartz cuvette, let it stand at room temperature until the gel solidifies; dialyze the gel in flowing water for 1 day to remove urea.

[0033] 2. Protein refolding:

[0034] Insert platinum wire electrodes on both sides of the cuvette containing the gel, and apply a constant electric field of 5V / cm; the volume of the gel shrinks slightly, and water is precipitated; Both the excitation slit and emission slit width are 10nm, the scanning range is 300-420nm, and the scanning speed is 1,000nm / min, measure the endogenous fluorescence emission spectrum, read 320nm ...

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PUM

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Abstract

The invention relates to an analysis method of protein refolding in electrical contraction polymer. The invention utilizes the crosslink network of electrical contraction polymer to insulate modifiedprotein molecule and removes modifier and contracts polymer at electric field, to loosen the polymer packing modified protein, therefore, modifier protein can fold without interfered and coiled by other proteins, and without the generation of inclusion body. The inventive analysis method builds a model of external molecule mate, for protein folding dynamic research.

Description

【Technical field】: [0001] The invention relates to the field of protein folding research, and provides an experimental method for protein refolding under the condition of mutual non-interference. 【Background technique】: [0002] Protein folding research is a bottleneck problem in biochemistry and molecular biology (Dill KA, et al. Curr. Opin. Struct. Biol. 2007, 17: 342-346). The content includes the problem of protein structure prediction that explores the determination of amino acid sequence to native conformation (Dunbrack RL Jr. W. A. ​​et al. Annu. Rev. Biophys. Biomol. Struct. 2000, 29:327-359). [0003] In experiments, in vitro denaturation and renaturation experiments are commonly used. That is to use the denaturant guanidine hydrochloride or urea to destroy the hydrogen bonds in the protein structure, so that it cannot maintain the natural three-dimensional conformation and become a relatively loose random coil. Then suddenly dilute the denaturant by stop-flow me...

Claims

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Application Information

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IPC IPC(8): C07K1/113
Inventor 黄积涛邢达杰张嘉琪郑嗣华黄卫洪谢秀荣
Owner TIANJIN UNIVERSITY OF TECHNOLOGY
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