CN101186634A - Refolding method for protein in electrostrictive polymer - Google Patents
Refolding method for protein in electrostrictive polymer Download PDFInfo
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- CN101186634A CN101186634A CNA200710151050XA CN200710151050A CN101186634A CN 101186634 A CN101186634 A CN 101186634A CN A200710151050X A CNA200710151050X A CN A200710151050XA CN 200710151050 A CN200710151050 A CN 200710151050A CN 101186634 A CN101186634 A CN 101186634A
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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 modified protein 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]: the present invention relates to the protein folding research field, under non-interfering condition for folding a kind of experimental technique that provides is provided protein again.
[background technology]: protein folding research be current biological chemistry and a molecular biology bottleneck difficult problem (Dill KA, et al.Curr.Opin.Struct.Biol.2007,17:342-346).Content comprises probes into aminoacid sequence to the conclusive protein structure prediction problem of native conformation (Dunbrack RL Jr.Curr.Opin.Struct.Biol.2006,6:374-384), and the dynamics problem of detecting protein folding procedure and approach (Eaton W.A.et al.Annu.Rev.Biophys.Biomol.Struct.2000,29:327-359).
Experimentally, generally adopt the experiment of external sex change and renaturation.Promptly be with the hydrogen bond in denaturing agent Guanidinium hydrochloride or the urea destruction protein structure, make it can not maintain natural three-dimensional conformation, become comparatively loose random coil.Adopt dilution denaturing agent suddenly such as stopped-flow method then, make protein be folded into again natural conformation (Roder H.Methods.2004,34:15-27).
But in protein folding procedure, the denatured protein chain will collide and tangle inevitably, and this will influence the normal self-assembly of protein, even cause malfolding, and the inclusion body that can not further fold.Organism has produced many molecular chaperoneses (Frieden C.﹠amp for overcoming this problem; Clark A.C.Proc.Natl.Acad.Sci.USA 1997,27:5535-5538).They can cover proteinic some segment or protein is encapsulated in " cage " in a certain stage of protein folding, thereby had avoided other proteinic interference and entanglement.
[summary of the invention]: the present invention seeks to solve the collision and the entanglement that occur in the protein folding procedure, and influence the normal self-assembly of protein, even cause malfolding, and the problem that produces the inclusion body that can not further fold, provide a kind of and cause the analytical procedure of protein refolding in the shrinkable polymer at electricity.
The inventive method utilizes polymer network that protein molecule has been kept apart, and by the contraction of polymkeric substance under electric field, the proteinic duct of lax parcel makes not folding protein carry out renaturation.When this method can overcome protein folding and other protein interactions and mutually tangling, avoid the formation of inclusion body, provide a kind of method for protein refolding unperturbed dynamics research simultaneously.
Electricity provided by the present invention causes proteinic methods for refolding in the shrinkable polymer, comprising:
---protein with denaturing agent (for example Guanidinium hydrochloride, urea) unfolding, is formed loose random coil polypeptide chain structure;
---the polypeptide chain of sex change is dissolved in monomer (for example 2-acrylamido-2-methyl 1-propane sulfonic acid, vinylformic acid), linking agent (N for example, N '-methylene-bisacrylamide) and initiator (ammonium persulphate and N, N, N ', N '-Tetramethyl Ethylene Diamine) in the formed solution, crosslinking polymerization forms transparent gel;
---with flowing water washing gel, the denaturing agent molecule in the gel is removed through dialysis.This moment, not folding protein receptor still can not be by renaturation to the parcel of polymer network.
Insert electrode in the gel both sides, and apply steady electric field.Polymkeric substance shrinks under electric field action, wraps up in the lax immediately expansion of the polymer network that twines the denatured protein molecule in the structure.Protein conformation " thaws ", and folding process begins to carry out.With protein folding dynamic analysis method (fluorescence spectrophotometer, circular dichroism photosensitiveness spectrometer, ultraviolet-visible light spectrophotometer etc.) commonly used it is carried out tracking monitor.
Detailed process of the present invention is as follows:
The first, protein unfolding:
Catalase is dissolved in the phosphate buffered saline buffer that concentration is the 8mol/L Guanidinium hydrochloride, places down for 4 ℃ and spend the night, make its sex change, form loose random coil polypeptide chain structure;
The second, protein encapsulation:
In the catalase phosphate buffered saline buffer of 4～5mL sex change, add 1.2～1.6g 2-acrylamido-2-methyl isophthalic acid-propane sulfonic acid, 0.1～0.2g N, N '-methylene-bisacrylamide, 10mg ammonium persulphate and 4mgN, N, N ', N '-Tetramethyl Ethylene Diamine; Shake up, inject quartz colorimetric utensil, room temperature leaves standstill to gel solidification; Gel is placed the mobile water 3d that dialyses, remove Guanidinium hydrochloride;
Three, protein refolding:
Platinum wire electrode is inserted in the cuvette both sides that filled gel the last step, applies 5V/cm constant electric field; Gel volume is little contraction, and separates out moisture; Wrap up in the lax immediately expansion of the polymer network that twines the denatured protein molecule in the structure, protein conformation " thaws ", and folding process begins to carry out; During this time, use fluorescence spectrophotometer, under 25 ℃, excitation wavelength 280nm excites slit and the wide 10nm of being of emission slit, sweep limit 300～420nm, sweep velocity 1 under the 000nm/min, is surveyed its endogenous fluorescence emission spectrum, behind the endogenous fluorescence emission spectrum of deduction control systems, read the endogenous fluorescence intensity of 320nm and 365nm, make the folding more endogenous fluorescence intensity of catalase curve over time then.
Advantage of the present invention and positively effect: the present invention is aggregated the thing network with protein molecule and isolates, and can not influence each other.The polymkeric substance hole has just hindered the entanglement of polypeptide chain in folding process as natural molecular chaperones, and this provides a kind of simple and effective laboratory facilities for single protein molecule carries out noiseless folding research.
The protein of the present invention after with sex change is encapsulated in the grid of network polymer, to reach isolated purpose.Along with applying of electric field, grid enlarges, and Zhe Die protein will not fold under other protein interferential situations of nothing.
[embodiment]:
Embodiment 1
1, protein unfolding:
Catalase is dissolved in the phosphate buffered saline buffer that concentration is the 8mol/L Guanidinium hydrochloride; Place down for 4 ℃ and spend the night, make its sex change.
2, protein encapsulation:
In the catalase phosphate buffered saline buffer of 4～5mL sex change, add 1.2～1.6g 2-acrylamido-2-methyl isophthalic acid-propane sulfonic acid, 0.1～0.2g N, N '-methylene-bisacrylamide, 10mg ammonium persulphate and 4mgN, N, N ', N '-Tetramethyl Ethylene Diamine; Shake up, inject quartz colorimetric utensil, room temperature leaves standstill to gel solidification; Gel is placed the mobile water 3d that dialyses, remove Guanidinium hydrochloride.
3, protein refolding:
Platinum wire electrode is inserted in the cuvette both sides that fill gel, apply 5V/cm constant electric field; Gel volume is little contraction, and separates out moisture; During this time, use fluorescence spectrophotometer, under 25 ℃, excitation wavelength 280nm excites slit and the wide 10nm of being of emission slit, sweep limit 300～420nm, sweep velocity 1 under the 000nm/min, is surveyed its endogenous fluorescence emission spectrum, behind the endogenous fluorescence emission spectrum of deduction control systems, read the endogenous fluorescence intensity of 320nm and 365nm, make the folding more endogenous fluorescence intensity of catalase curve over time then.
Embodiment 2
1, protein unfolding:
Catalase is dissolved in the phosphate buffered saline buffer that concentration is the 10mol/L urea; Place down for 4 ℃ and spend the night, make its sex change.
2, protein encapsulation:
In the catalase phosphate buffered saline buffer of 4～5mL sex change, add 1.2～1.6g vinylformic acid, 0.1～0.2gN, N ' ,-methylene-bisacrylamide, 10mg ammonium persulphate and 4mgN, N, N ', N '-Tetramethyl Ethylene Diamine; Shake up, inject quartz colorimetric utensil, room temperature leaves standstill to gel solidification; Gel is placed the mobile water 1d that dialyses, remove urea.
2, protein refolding:
Platinum wire electrode is inserted in the cuvette both sides that fill gel, apply 5V/cm constant electric field; Gel volume is little contraction, and separates out moisture; During this time, use fluorescence spectrophotometer, under 25 ℃, excitation wavelength 280nm excites slit and the wide 10nm of being of emission slit, sweep limit 300～420nm, sweep velocity 1 under the 000nm/min, is surveyed its endogenous fluorescence emission spectrum, behind the endogenous fluorescence emission spectrum of deduction control systems, read the endogenous fluorescence intensity of 320nm and 365nm, make the folding more endogenous fluorescence intensity of catalase curve over time then.
Claims (2)
1. one kind causes proteinic methods for refolding in the shrinkable polymer at electricity, comprising:
---protein with Guanidinium hydrochloride or urea denaturing agent unfolding, is formed loose random coil polypeptide chain structure;
---the polypeptide chain of sex change is dissolved in 2-acrylamido-2-methyl isophthalic acid-propane sulfonic acid or Acrylic Acid Monomer, N, N '-methylene-bisacrylamide linking agent and by ammonium persulphate and N, N, N ', N ', in the initiator solution that N '-Tetramethyl Ethylene Diamine constitutes, crosslinking polymerization forms transparent gel;
---with flowing water washing gel, the denaturing agent molecule in the gel is removed through dialysis;
---insert electrode in the gel both sides, and apply steady electric field; With fluorescence spectrophotometer, circular dichroism photosensitiveness spectrometer or ultraviolet-visible light spectrophotometer proteinic folding process is again carried out tracking monitor.
2. according to claim 1ly cause proteinic methods for refolding in the shrinkable polymer, it is characterized in that this method specifically may further comprise the steps at electricity:
The first, protein unfolding:
Catalase is dissolved in the phosphate buffered saline buffer that concentration is the 8mol/L Guanidinium hydrochloride, places down for 4 ℃ and spend the night, make its sex change, form loose random coil polypeptide chain structure;
The second, protein encapsulation:
In the catalase phosphate buffered saline buffer of 4～5mL sex change, add 1.2～1.6g 2-acrylamido-2-methyl isophthalic acid-propane sulfonic acid, 0.1～0.2g N, N '-methylene-bisacrylamide, 10mg ammonium persulphate and 4mg N, N, N, N ', N '-Tetramethyl Ethylene Diamine; Shake up, inject quartz colorimetric utensil, room temperature leaves standstill to gel solidification; Gel is placed the mobile water 3d that dialyses, remove Guanidinium hydrochloride;
Three, protein refolding:
Platinum wire electrode is inserted in the cuvette both sides that filled gel the last step, applies 5V/cm constant electric field; With catalatic folding again being carried out tracking monitor with fluorescence spectrophotometer, circular dichroism photosensitiveness spectrometer or ultraviolet-visible light spectrophotometer.
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| CN200710151050A CN100586956C (en) | 2007-12-14 | 2007-12-14 | Refolding method for protein in electrostrictive polymer |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106243186A (en) * | 2015-06-15 | 2016-12-21 | 张鹏 | Protein renaturation or the circulation How It Works as the leading operation of protein renaturation can be independently used for |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106243186A (en) * | 2015-06-15 | 2016-12-21 | 张鹏 | Protein renaturation or the circulation How It Works as the leading operation of protein renaturation can be independently used for |
| CN106243186B (en) * | 2015-06-15 | 2020-12-25 | 张鹏 | Circulating operation method capable of being independently used for protein renaturation or used as protein renaturation leading operation |
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| CN100586956C (en) | 2010-02-03 |
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