CN105111390A - Metal chelating nano medium, preparation method and method applied to strengthen inclusion body protein renaturation and integrated purification - Google Patents
Metal chelating nano medium, preparation method and method applied to strengthen inclusion body protein renaturation and integrated purification Download PDFInfo
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Abstract
The invention relates to a metal chelating nano medium, a preparation method and a method applied to strengthen inclusion body protein renaturation and integrated purification, wherein connection polymers P (GMA-IDA) of iminodiacetic acid and glycidyl methacrylate are grafted on the surfaces of silicon dioxide nanoparticles which are 20-30nm in mean grain sizes, and the electric density of medium is 2520-5680Mu mol/g. Extremely high electric density enhances electrostatic repulsion between the medium and homocharge protein molecules, thereby more effectively restraining gathering of proteins. A large number of metal chelating groups which are arranged on the surface of the nano medium guarantee recombinant target proteins which are marked through poly-L-histidine to be effectively adsorbed and separated after completing renaturation. The metal chelating nano medium, the preparation method and the method applied to strengthen inclusion body protein renaturation and integrated purification do not need expensive large equipment such as chromatographic columns or chromatographic systems and the like, complete integration of inclusion body protein renaturation and purification outside the chromatographic columns, are simple, convenient and easy to operate and low in cost, simplify refinement and purification method, and are more suitable for large scale industrialization production.
Description
Technical field
The invention belongs to the protein renaturation in biological technical field and isolation technique; Relate to a kind of metal-chelating nanometer medium and preparation method and application in the method for strengthening inclusion body protein renaturation and integrated purifying.
Background technology
The develop rapidly of genetic engineering technique, makes the scale operation of genetically engineered drug become possibility.The recombinant gene expression system that the need of production of this type of medicine is suitable, bacterial expression system is expressed target protein and is had quick, a large amount of, cheap advantage, but the overexpression of foreign protein often forms the aggregate of albumen, i.e. inclusion body.Inclusion body does not have biologic activity, needs again refolding could recover its natural structure and biological activity.The gathering that in renaturation process, between folding intermediate, hydrophobic interaction causes is the major reason reducing renaturation yield, and therefore, suppressing to assemble is the key promoting renaturation.Meanwhile, the inclusion body protein after renaturation is difficult to obtain efficient separation and purification, and this is also the important step limiting its scale operation.
Add the micromolecule additive that arrestin matter is assembled in renaturation system, as the denaturing agent of lower concentration, arginine, hailstorm suppression, glycerine etc., effectively can promote protein renaturation.But the use of additive can introduce new impurity in renaturation system, is unfavorable for the separation and purification of later stage target protein; And while many renaturation additives are assembled between Inhibitory molecules, also can the intramolecular fold of arrestin matter, cause reassociation rate to reduce.The method of chromatogram renaturation not only can the gathering of effective arrestin matter, while renaturation, also have certain separation and purification effect to protein.But the adsorption between medium and protein molecule can cause chromatogram renaturation to reach the time lengthening of balance, rate reduction; And the concentration reclaiming product is lower.
The deficiency of renaturation and chromatogram renaturation technology is assisted in order to overcome micromolecule additive, method (method of using ion-exchange chromatography medium for assisting protein renaturation and the application of protein renaturation are assembled, promoted to the charged species arrestin matter having applicant to propose same electric charge, CN101948504A, 2011).Compared with traditional refolding method, promote that the method for protein renaturation both can not pollute target protein with charge mediator, also can not reduce the Folding rate of protein, and simple to operate, medium is easy to reclaim, without the need to the main equipment of costliness.The same charge mediator used in current renaturation research mainly comprises the Ago-Gel medium of porous, the poly (glycidyl methacrylate) microsphere medium of atresia and nanoparticle etc.Wherein non-porous microspheres medium due to specific surface area limited, electric charge modifies density lower (300 μm of ol/g), limit the effect of its auxiliary renaturation, (aglucon is modified and is improved the method that charge density of microspherical medium through ligand modification promotes protein renaturation to need to improve electric density further by the method for secondary modification, CN103601791A, 2014).And although the Ago-Gel medium of porous can reach very high electric density (1200 μm of ol/g), but medium with cause protein molecule cannot enter in duct with the intermolecular electrostatic repulsion of charged proteins, thus outside surface competence exertion effect (Ion-exehangeresinsfacilitatelike-chargedproteinrefolding: the Effectsofporoussolidphaseproperties.JournalofChromatogra phyA of medium is only had, 2012,1225,168-173).And nanoparticle due to specific surface area large, electric density can be modified high, auxiliary with charged proteins renaturation in there is greater advantage (EnhancedProteinAdsorptionandFacilitatedRefoldingofLike-C hargedProteinwithHighlyChargedSilicaNanoparticlesFabrica tedbySequentialDoubleModifications.Langmuir, 2015,31,655 – 658).Recent applicant assists the method for renaturation based on same charge mediator, establish the Ago-Gel medium IDA-SepharoseFF that a kind of iminodiethanoic acid (IDA) modifies and assist the renaturation of the green fluorescent protein inclusion body of same electric charge and method (the Integrativerefoldingandpurificationofhistidine-taggedpro teinbylike-chargefacilitatedrefoldingandmetal-chelateaff inityadsorption.JournalofChromatographyA of integrated purifying, 1344,59 – 65).The method is integrated with same charge mediator and assists renaturation and metal-chelating purification technique, achieves renaturation and the separation and purification of target protein simultaneously.But be subject to the restriction of dielectric surface IDA modifying method, the electric density of dielectric surface is lower (being about 108 μm of ol/g), and the inclusion body therefore for higher concentration assists the effect of renaturation unsatisfactory.
Summary of the invention
The object of the invention is to overcome the deficiencies in the prior art, Nano particles of silicon dioxide (SNPs) the surface grafting polymerization thing that to the present invention proposes in median size be 20-30nm prepares the metal-chelating nanometer medium of high charge density to the method for the renaturation and integrated purifying of strengthening inclusion body protein, the electric density of the metal-chelating nanometer medium that the method obtains reaches 2520-5680 μm of ol/g, far above the electric density of other media used in the research of current same charged proteins renaturation.And compared with metal-chelating microsphere medium IDA-SepharoseFF, the method has that medium consumption is few, renaturation yield is high, be applicable to the feature of greater concn inclusion body protein renaturation, for target protein, there is good purifying and concentration effect simultaneously, and it is easy to operation, with low cost, enormously simplify Dipurification process, be more suitable for being applied to large-scale industrial production.
Technical scheme of the present invention is summarized as follows:
The present invention using the connection product GMA-IDA of iminodiethanoic acid (IDA) and glycidyl methacrylate (GMA) as monomer, utilize atom transfer radical polymerization (ATRP) method, at the Nano particles of silicon dioxide surface Inducing Graft Polymerization that median size is 20-30nm, obtain metal-chelating nanometer medium SNPs-P (GMA-IDA) that electric density is 2520-5680 μm of ol/g.
The structural formula of above-mentioned GMA-IDA is as follows:
The structure representation of above-mentioned metal-chelating nanometer medium SNPs-P (GMA-IDA) is as follows:
Be Nano particles of silicon dioxide (SNPs) surface grafting iminodiethanoic acid (IDA) of 20-30nm and the connection polymer P (GMA-IDA) of glycidyl methacrylate (GMA) in median size; The electric density of metal-chelating nanometer medium is 2520-5680 μm of ol/g.
The electric density that wherein scope of n value need meet metal-chelating nanometer medium reaches 2520-5680 μm of ol/g.
The preparation method of metal-chelating nanometer medium of the present invention, its step is as follows:
1) the connection product GMA-IDA of GMA and IDA is synthesized;
2) initiator 2-bromo isobutyl acylbromide (BIBB) is fixed on the Nano particles of silicon dioxide surface being 20-30nm in median size, is fixed the Nano particles of silicon dioxide SNPs-Br of BIBB;
3) by above-mentioned steps 2) in the Nano particles of silicon dioxide SNPs-Br of fixing BIBB join N, dinethylformamide (DMF) is with the mixing liquid of water, be made into the suspension that concentration is 0.1-0.2g/mL, the volume ratio of DMF and water is 1:2;
4) above-mentioned steps 3) suspension in add GMA-IDA monomer, CuBr catalyzer, 2,2 '-dipyridyl (Bpy) part and CuBr
2inert agents, the concentration of GMA-IDA is 0.2-1.0mol/L, and solution calculates with the mixing liquid of DMF (DMF) with water, and the molar concentration rate of each component is GMA-IDA:CuBr:CuBr
2: Bpy=100:1:0.2:2, polyreaction 1-2h, prepare metal-chelating nanometer medium SNPs-P (GMA-IDA).
Described step 1) in method, the synthetic method of GMA-IDA is shown in document (SynthesisofMagneticChelatorforHigh-CapacityImmobilizedMe talAffinityAdsorptionofProteinbyCeriumInitiatedGraftPoly merization, Langmuir, 2005,21,6987-6994).
Described step 2) in method, the fixing of initiator B IBB carries out according to universal method, concrete steps are shown in document (SynthesisofDualThermosensitiveandpH-SensitiveHollowNanos pheresBasedonPoly (acrylicacid-b-2-hydroxyethylmethacrylate) viaanAtomTransferReversibleAddition-FragmentationRadical Process, 2012,68,159-166).
The electric density of the metal-chelating nanometer medium that the present invention obtains is 2520-5680 μm of ol/g, high electric density enhance medium with the intermolecular electrostatic repulsion of charged proteins, thus the gathering of more effectively arrestin matter; The high specific surface area of nanometer medium makes the protein molecule of same electric charge spatially obtain more effective oriented alignment, is therefore more conducive to the renaturation of same charged proteins; In addition, the metal chelating groups that nanometer dielectric surface is a large amount of, ensure that the target recombinant albumen of polyhistidine tag effectively can be adsorbed and be separated after renaturation completes.
The metal-chelating nanometer medium of high charge density of the present invention is for strengthening the method for inclusion body protein renaturation and integrated purifying, and its step is as follows:
1) joined by metal-chelating nanometer medium in the renaturation buffer of same electric charge inclusion body protein, the inclusion body protein then adding sex change dissolving carries out renaturation;
2) after renaturation completes, in system, add nickel ion, specific adsorption is carried out to target protein;
3) after having adsorbed, centrifugally remove supernatant liquor, rinse nanometer medium with lavation buffer solution;
4) nanometer medium is joined the wash-out carrying out target protein in elution buffer, after wash-out completes, collected by centrifugation elutriant, obtains the target protein of purifying.
Described renaturation buffer is: the mixing solutions of 20-100mmol/L Tutofusin tris (Tris) and 0-2.0mol/L urea; PH to 7.5-9.0 is adjusted with HCl;
Lavation buffer solution is: 50-100mmol/LTris, 10-50mmol/L imidazoles, 0.1-0.5mol/LNaCl, 0.5-1mol/L urea; PH to 7.5-8.5 is adjusted with HCl;
Elution buffer is: 50-100mmol/LTris, 0.5-1.0mol/L imidazoles, 0.5-1mol/L urea; PH to 7.5-8.5 is adjusted with HCl.
Described step 1) method is: metal-chelating nanometer medium joined in renaturation buffer, makes the concentration of nanometer medium in final renaturation system be 16-64mg/mL; It is that the Air oscillator of 20-37 DEG C shakes pre-equilibration until homo(io)thermism that said mixture is placed in temperature; Add the inclusion body protein solution treating renaturation, make protein concn in renaturation system be 0.2-0.8mg/mL; After mixing, be placed in Air oscillator, renaturation under rotating speed is 50-170rpm condition, renaturation temperature is identical with the temperature of renaturation buffer pre-equilibration before this.
Described step 2) method is: add nickel sulfate solution in the system after renaturation completes, nickel ion concentration is 0.25-0.45 μm of ol/mg, after mixing, be placed in Air oscillator, the absorption of target protein is carried out under rotating speed is 100-170rpm condition, adsorption temp is 20-37 DEG C, and adsorption time is 20-30min.
Described step 3) method is: by the mixed system after having adsorbed at 4 DEG C, centrifugal under 8000-10000rpm condition, remove supernatant liquor, by nanometer medium lavation buffer solution at 4 DEG C, centrifuge washing 3-5 time under 8000-10000rpm condition, the volume of the lavation buffer solution that each washing is used is 10-20 times of nanometer medium settling volume.
Described step 4) method is: nanometer medium joined in elution buffer and mixes, be placed in Air oscillator, wash-out is carried out under rotating speed is 100-170rpm condition, the volume of elution buffer is 5-10 times of nanometer medium settling volume, eluting temperature is 20-37 DEG C, and elution time is 15-30min.Mixed system after wash-out completes is at 4 DEG C, centrifugal under 8000-10000rpm condition, collects supernatant liquor.
Method of the present invention, is applied to the renaturation of the inclusion body protein of polyhistidyl tags amalgamation and expression and integrated purifying, and described protein comprises the protein containing disulfide linkage and do not contain the protein of disulfide linkage.For the difference of kinds of protein, be described as follows:
1) method of the present invention can be used for containing disulfide bond protein matter with not containing renaturation and the integrated purifying of the protein of disulfide linkage.For the protein containing disulfide linkage, in renaturation system, suitable redox system should be added, as Sleep-promoting factor B/reduced glutathion (GSSG/GSH); For the protein containing free sulfhydryl group, in renaturation system, suitable reductive agent should be added, as dithiothreitol (DTT) (DTT).
2) when method of the present invention is applied to the protein containing sulfydryl or disulfide linkage, if there is strong reductant in renaturation system, as DTT, before adding adsorption of metal ions, suitable oxygenant need be added, as cystamine, by strong reductant remaining in system removing, the metal ion added is avoided to be reduced.
The metal-chelating nanometer medium strengthening inclusion body protein renaturation of high charge density of the present invention and the method tool of integrated purifying have the following advantages:
The first, compared with microsphere medium, the electric density of metal-chelating nanometer medium of the present invention is significantly improved;
Second, compared with metal-chelating microsphere medium before, metal-chelating nanometer media table of the present invention reveals the better auxiliary effect with electric charge inclusion body protein renaturation, has medium consumption few, renaturation yield is high, is applicable to the feature of greater concn inclusion body protein renaturation.
3rd, metal-chelating nanometer medium of the present invention has good purifying and concentration effect for the target protein after renaturation.
4th, the metal-chelating nanometer medium strengthening inclusion body protein renaturation of high charge density of the present invention and the method for integrated purifying, do not need the main equipment that chromatographic column or chromatographic system etc. are expensive, namely the integrated of inclusion body protein renaturation and purifying is completed outward at post, easy to operation, with low cost, enormously simplify Dipurification process, be more suitable for being applied to large-scale industrial production.
Accompanying drawing explanation
Fig. 1: recombinant enhanced green fluorescent protein (6 × his-EGFP) renaturing inclusion bodies of the auxiliary 0.2mg/mL hexahistine mark of metal-chelating nanometer medium SNPs-P (GMA-IDA) of the different charge densities in embodiment 4;
Fig. 2: the auxiliary 0.4mg/mL6 × his-EGFP renaturing inclusion bodies of metal-chelating nanometer medium SNPs-P (GMA-IDA) of the high charge density in embodiment 5;
Fig. 3: the auxiliary 0.8mg/mL6 × his-EGFP renaturing inclusion bodies of metal-chelating nanometer medium SNPs-P (GMA-IDA) of the high charge density in embodiment 6;
Fig. 4: metal-chelating nanometer medium SNPs-P (GMA-IDA) of the high charge density in embodiment 9 is to the purification effect of 6 × his-EGFP after renaturation.
Embodiment
The present invention using the connection product GMA-IDA of iminodiethanoic acid (IDA) and glycidyl methacrylate (GMA) as monomer, utilize atom transfer radical polymerization (ATRP) method, at the Nano particles of silicon dioxide surface Inducing Graft Polymerization that median size is 20-30nm, obtain the metal-chelating nanometer medium that electric density is 2520-5680 μm of ol/g.
The structural formula of above-mentioned GMA-IDA is as follows:
The structure representation of above-mentioned metal-chelating nanometer medium SNPs-P (GMA-IDA) is as follows:
The electric density that wherein scope of n value need meet metal-chelating nanometer medium reaches 2520-5680 μm of ol/g.
The preparation method of the metal-chelating nanometer medium of high charge density of the present invention, its step is as follows:
1) the connection product GMA-IDA of GMA and IDA is synthesized;
2) initiator 2-bromo isobutyl acylbromide (BIBB) is fixed on the Nano particles of silicon dioxide surface being 20-30nm in median size, is fixed the Nano particles of silicon dioxide SNPs-Br of BIBB;
3) by above-mentioned steps 2) in the Nano particles of silicon dioxide SNPs-Br of fixing BIBB join N, dinethylformamide (DMF) is with the mixing liquid of water, be made into the suspension that concentration is 0.1-0.2g/mL, the volume ratio of DMF and water is 1:2;
4) above-mentioned steps 3) suspension in add GMA-IDA monomer, CuBr catalyzer, 2,2 '-dipyridyl (Bpy) part and CuBr
2inert agents, the concentration of GMA-IDA is 0.2-1.0mol/L, and solution calculates with the mixing liquid of DMF (DMF) with water, and the molar concentration rate of each component is GMA-IDA:CuBr:CuBr
2: Bpy=100:1:0.2:2, polyreaction 1-2h, prepare metal-chelating nanometer medium SNPs-P (GMA-IDA)
Described step 1) in method, the synthetic method of GMA-IDA is shown in document (SynthesisofMagneticChelatorforHigh-CapacityImmobilizedMe talAffinityAdsorptionofProteinbyCeriumInitiatedGraftPoly merization, Langmuir, 2005,21,6987-6994).
Described step 2) in method, the fixing of initiator B IBB carries out according to universal method, concrete steps are shown in document (SynthesisofDualThermosensitiveandpH-SensitiveHollowNanos pheresBasedonPoly (acrylicacid-b-2-hydroxyethylmethacrylate) viaanAtomTransferReversibleAddition-FragmentationRadical Process, 2012,68,159-166).
The metal-chelating nanometer medium strengthening inclusion body protein renaturation of high charge density of the present invention and the method for integrated purifying, step is as follows:
1) joined by metal-chelating nanometer medium in the renaturation buffer of same electric charge inclusion body protein, the inclusion body protein then adding sex change dissolving carries out renaturation;
2) after renaturation completes, in system, add nickel ion, specific adsorption is carried out to target protein;
3) after having adsorbed, centrifugally remove supernatant liquor, rinse nanometer medium with lavation buffer solution;
4) nanometer medium is joined the wash-out carrying out target protein in elution buffer, after wash-out completes, collected by centrifugation elutriant, obtains the target protein of purifying.
Described renaturation buffer is: the mixing solutions of 20-100mmol/L Tutofusin tris (Tris) and 0-2.0mol/L urea; PH to 7.5-9.0 is adjusted with HCl;
Lavation buffer solution is: 50-100mmol/LTris, 10-50mmol/L imidazoles, 0.1-0.5mol/LNaCl, 0.5-1mol/L urea; PH to 7.5-8.5 is adjusted with HCl;
Elution buffer is: 50-100mmol/LTris, 0.5-1.0mol/L imidazoles, 0.5-1mol/L urea; PH to 7.5-8.5 is adjusted with HCl.
Described step 1) method is: metal-chelating nanometer medium joined in renaturation buffer, makes the concentration of nanometer medium in final renaturation system be 16-64mg/mL; It is that the Air oscillator of 20-37 DEG C shakes pre-equilibration until homo(io)thermism that said mixture is placed in temperature; Add the inclusion body protein solution treating renaturation, make protein concn in renaturation system be 0.2-0.8mg/mL; After mixing, be placed in Air oscillator, renaturation under rotating speed is 50-170rpm condition, renaturation temperature is identical with the temperature of renaturation buffer pre-equilibration before this.
Described step 2) method is: add nickel sulfate solution in the system after renaturation completes, nickel ion concentration is 0.25-0.45 μm of ol/mg, after mixing, be placed in Air oscillator, the absorption of target protein is carried out under rotating speed is 100-170rpm condition, adsorption temp is 20-37 DEG C, and adsorption time is 20-30min.
Described step 3) method is: by the mixed system after having adsorbed at 4 DEG C, centrifugal under 8000-10000rpm condition, remove supernatant liquor, by nanometer medium lavation buffer solution at 4 DEG C, centrifuge washing 3-5 time under 8000-10000rpm condition, the volume of the lavation buffer solution that each washing is used is 10-20 times of nanometer medium settling volume.
Described step 4) method is: nanometer medium joined in elution buffer and mixes, be placed in Air oscillator, wash-out is carried out under rotating speed is 100-170rpm condition, the volume of elution buffer is 5-10 times of nanometer medium settling volume, eluting temperature is 20-37 DEG C, and elution time is 15-30min.Mixed system after wash-out completes is at 4 DEG C, centrifugal under 8000-10000rpm condition, collects supernatant liquor.
Method of the present invention, is applied to the renaturation of the inclusion body protein of polyhistidyl tags amalgamation and expression and integrated purifying, and described protein comprises the protein containing disulfide linkage and do not contain the protein of disulfide linkage.
Example below will be further described method provided by the invention.
Embodiment 1:
The preparation method of the metal-chelating nanometer medium of the high charge density in the present embodiment is as follows:
1) connection product GMA-IDA (the SynthesisofMagneticChelatorforHigh-CapacityImmobilizedMe talAffinityAdsorptionofProteinbyCeriumInitiatedGraftPoly merization of GMA and IDA is synthesized, Langmuir, 2005,21,6987-6994);
2) the alpha-brominated isobutyl acylbromide (BIBB) of initiator is fixed on the Nano particles of silicon dioxide surface being 20-30nm in median size, be fixed the Nano particles of silicon dioxide SNPs-Br of BIBB, concrete steps are shown in document (Reversibleimmobilizationofglucoamylaseontometal-ligandfu nctionalizedmagneticFeSBA-15, BiochemicalEngineeringJournal, 2012,68,159-166);
3) by 3.0g above-mentioned steps 2) in the Nano particles of silicon dioxide SNPs-Br of fixing BIBB join in the mixed system of 30mLDMF and water, be made into the suspension that concentration is 0.1g/mL, the volume ratio of DMF and water is 1:2;
4) above-mentioned steps 3) suspension in add GMA-IDA monomer, CuBr catalyzer, 2,2 '-dipyridyl (Bpy) part, CuBr
2inert agents, the concentration of GMA-IDA is 0.2mol/L, and solution calculates with the mixing liquid of DMF (DMF) with water, and the molar concentration rate of each component is GMA-IDA:CuBr:CuBr
2: Bpy=100:1:0.2:2, reaction forward slip value system leads to nitrogen deoxygenation half an hour, post-polymerization 1h, prepare metal-chelating nanometer medium SNPs-P (GMA-IDA).The electric density of metal-chelating nanometer medium SNPs-P (GMA-IDA) prepared in the present embodiment is 2520 μm of ol/g.
Embodiment 2:
The preparation method of the metal-chelating nanometer medium of the high charge density in the present embodiment is as follows:
1) with the step 1 in embodiment 1) identical;
2) with the step 2 in embodiment 1) identical;
3) by 4.5g above-mentioned steps 2) in the Nano particles of silicon dioxide SNPs-Br of fixing BIBB join in the mixed system of 30mLDMF and water, be made into the suspension that concentration is 0.15g/mL, the volume ratio of DMF and water is 1:2;
4) above-mentioned steps 3) suspension in add GMA-IDA monomer, CuBr catalyzer, 2,2 '-dipyridyl (Bpy) part, CuBr
2inert agents, the concentration of GMA-IDA is 0.6mol/L, and solution calculates with the mixing liquid of DMF (DMF) with water, and the molar concentration rate of each component is GMA-IDA:CuBr:CuBr
2: Bpy=100:1:0.2:2, reaction forward slip value system leads to nitrogen deoxygenation half an hour, post-polymerization 1.5h, prepare metal-chelating nanometer medium SNPs-P (GMA-IDA).The electric density of metal-chelating nanometer medium SNPs-P (GMA-IDA) prepared in the present embodiment is 4400 μm of ol/g.
Embodiment 3:
The preparation method of the metal-chelating nanometer medium of the high charge density in the present embodiment is as follows:
1) with the step 1 in embodiment 1) identical;
2) with the step 2 in embodiment 1) identical;
3) by 6.0g above-mentioned steps 2) in the Nano particles of silicon dioxide SNPs-Br of fixing BIBB join in the mixed system of 30mLDMF and water, be made into the suspension that concentration is 0.2g/mL, the volume ratio of DMF and water is 1:2;
4) above-mentioned steps 3) suspension in add GMA-IDA monomer, CuBr catalyzer, 2,2 '-dipyridyl (Bpy) part, CuBr
2inert agents, the concentration of GMA-IDA is 1.0mol/L, and solution calculates with the mixing liquid of DMF (DMF) with water, and the molar concentration rate of each component is GMA-IDA:CuBr:CuBr
2: Bpy=100:1:0.2:2, reaction forward slip value system leads to nitrogen deoxygenation half an hour, post-polymerization 2h, prepare metal-chelating nanometer medium SNPs-P (GMA-IDA).The electric density of metal-chelating nanometer medium SNPs-P (GMA-IDA) prepared in the present embodiment is 5680 μm of ol/g.
Embodiment 4: the auxiliary 0.2mg/mL6 × his-EGFP renaturing inclusion bodies of metal-chelating nanometer medium SNPs-P (GMA-IDA) of different charge densities
Iso-electric point due to 6 × his-EGFP is 6.0, electronegative in neutral and alkaline conditions, and electronegative metal-chelating nanometer medium SNPs-P (GMA-IDA) of therefore the present invention's synthesis can assist 6 × his-EGFP renaturing inclusion bodies.The present embodiment metal-chelating nanometer medium used is the metal-chelating nanometer medium that the three kinds of electric density prepared in embodiment 1-3 are respectively 2520 μm of ol/g, 4400 μm of ol/g and 5680 μm ol/g, and with the renaturation result of not adding nanometer medium under the same terms in contrast.
The inclusion body protein sample of sex change: be precipitated and dissolved in by inclusion body in denaturation buffer (50mmol/LTris-HCl, 10mol/L urea, pH8.5), is placed in 50 DEG C of water-baths and heats 2h, make its complete sex change.Inclusion body after sex change is placed in 4 DEG C, the centrifugal 15min of 12000rpm, discard precipitate the supernatant liquor that obtains be dissolving after denatured protein, adopt Bradford method to measure total protein concentration in sex change liquid, being diluted to total protein concentration with above-mentioned denaturation buffer is afterwards 10mg/mL.Renaturation experiment is carried out in the centrifuge tube of 1.5mL, and renaturation buffer is the 20mmol/LTris-HCl damping fluid containing 20mmol/LDTT, and pH is 7.5.For the 6 × his-EGFP renaturing inclusion bodies not adding nanometer medium, the above-mentioned renaturation buffer of 1mL is directly placed in 20 DEG C of Air oscillator pre-equilibrations; For the 6 × his-EGFP renaturing inclusion bodies adding nanometer medium, adding concentration in the above-mentioned renaturation buffer of 1mL is that the nanometer medium of the different charge densities of 16mg/mL is placed in 20 DEG C of Air oscillators and shakes pre-equilibration until homo(io)thermism.The denaturing soln 50 of 6 × his-EGFP inclusion body is doubly diluted in above-mentioned renaturation buffer, makes the concentration of 6 × his-EGFP inclusion body protein in renaturation system be 0.2mg/mL (containing 0.14mg/mL6 × his-EGFP).Above-mentioned renaturation system adopts fluorescence spectrophotometer to measure renaturation product and excites under 488nm under 20 DEG C and 50rpm condition after renaturation 12h, the fluorescence intensity of launching under 509nm, the fluorescence standard curve of corresponding natural 6 × his-EGFP can calculate the concentration of active 6 × his-EGFP in renaturation system.By the concentration of activated protein compared with the total concn of 6 × his-EGFP in renaturation system, the fluorescence renaturation yield of 6 × his-EGFP in renaturation product can be obtained.
The activity yield of the auxiliary 6 × his-EGFP renaturing inclusion bodies of SNPs-P (GMA-IDA) of different charge densities is added as shown in Figure 1 in renaturation system.As seen from the figure, within the specific limits, metal-chelating nanometer medium promotes to raise with the increase of nanometer medium charge densitv with the activity yield of electric charge inclusion body protein renaturation, for the metal-chelating nanometer medium that electric density is 5680 μm of ol/g, its electric density is the highest, the yield of auxiliary 0.2mg/mL6 × his-EGFP renaturing inclusion bodies reaches 90%, and compared with dilution refolding (39%), yield improves 130%.In addition, the metal-chelating microsphere medium IDA-SepharoseFF used in research before the effect of the auxiliary 6 × his-EGFP renaturing inclusion bodies of SNPs-P (GMA-IDA) will obviously be better than, for 0.2mg/mL6 × his-EGFP renaturing inclusion bodies, IDA-SepharoseFF is when medium consumption is 470mg/mL (460 μ L/mL), maximum renaturation yield is only 63% (Integrativerefoldingandpurificationofhistidine-taggedpro teinbylike-chargefacilitatedrefoldingandmetal-chelateaff inityadsorption.JournalofChromatographyA, 1344, 59 – 65), and the yield of the auxiliary 6 × his-EGFP renaturing inclusion bodies of SNPs-P (GMA-IDA) is 90%, compared with IDA-SepharoseFF, renaturation yield improves 43%, and required medium consumption is only 16mg/mL (6.2 μ L/mL), then 98% is have dropped compared with IDA-SepharoseFF, show that the nanometer medium of high charge density has than the micron medium better auxiliary effect with electric charge inclusion body protein renaturation, be embodied in higher renaturation yield and lower medium consumption.
Embodiment 5: the auxiliary 0.4mg/mL6 × his-EGFP renaturing inclusion bodies of metal-chelating nanometer medium SNPs-P (GMA-IDA) of high charge density
The metal-chelating nanometer medium of metal-chelating nanometer medium used to be the electric density prepared in embodiment 3 be 5680 μm of ol/g is the metal-chelating nanometer medium that the electric density prepared is the highest.
The inclusion body protein sample of sex change: be precipitated and dissolved in by inclusion body in denaturation buffer (50mmol/LTris-HCl, 10mol/L urea, pH8.5), is placed in 50 DEG C of water-baths and heats 2h, make its complete sex change.Inclusion body after sex change is placed in 4 DEG C, the centrifugal 15min of 12000rpm, discard precipitate the supernatant liquor that obtains be dissolving after denatured protein, adopt Bradford method to measure total protein concentration in sex change liquid, being diluted to total protein concentration with above-mentioned denaturation buffer is afterwards 10mg/mL.Renaturation experiment is carried out in the centrifuge tube of 1.5mL, and renaturation buffer is the 50mmol/LTris-HCl damping fluid containing 1.0mol/L urea and 20mmol/LDTT, and pH is 8.5.For the 6 × his-EGFP renaturing inclusion bodies not adding nanometer medium, the above-mentioned renaturation buffer of 1mL is directly placed in 25 DEG C of Air oscillator pre-equilibrations; For the 6 × his-EGFP renaturing inclusion bodies adding nanometer medium, adding concentration in the above-mentioned renaturation buffer of 1mL is that the metal-chelating nanometer medium of 32mg/mL is placed in 25 DEG C of Air oscillators and shakes pre-equilibration until homo(io)thermism.The denaturing soln 25 of 6 × his-EGFP inclusion body is doubly diluted in above-mentioned renaturation buffer, makes the concentration of 6 × his-EGFP inclusion body protein in renaturation system be 0.4mg/mL (containing 0.28mg/mL6 × his-EGFP).Above-mentioned renaturation system adopts fluorescence spectrophotometer to measure renaturation product and excites under 488nm under 25 DEG C and 100rpm condition after renaturation 12h, the fluorescence intensity of launching under 509nm, the fluorescence standard curve of corresponding natural 6 × his-EGFP can calculate the concentration of active 6 × his-EGFP in renaturation system.By the concentration of activated protein compared with the total concn of 6 × his-EGFP in renaturation system, the fluorescence renaturation yield of 6 × his-EGFP in renaturation product can be obtained.
Metal-chelating nanometer medium assists the effect of 0.4mg/mL6 × his-EGFP renaturing inclusion bodies as shown in Figure 2, as seen from the figure, for 0.4mg/mL6 × his-EGFP renaturing inclusion bodies, adding of metal-chelating nanometer medium SNPs-P (GMA-IDA) makes renaturation yield be increased to 70% by 35%, and microsphere medium IDA-SepharoseFF is only better to low concentration inclusion body (0.1mg/mL) renaturation effect, unsatisfactory (the Integrativerefoldingandpurificationofhistidine-taggedpro teinbylike-chargefacilitatedrefoldingandmetal-chelateaff inityadsorption.JournalofChromatographyA of effect for greater concn renaturing inclusion bodies, 1344, 59 – 65).Metal-chelating nanometer medium SNPs-P (GMA-IDA) that this result embodies high charge density is better than a large advantage of microsphere medium IDA-SepharoseFF, is namely applicable to greater concn renaturing inclusion bodies.
Embodiment 6: the auxiliary 0.8mg/mL6 × his-EGFP renaturing inclusion bodies of metal-chelating nanometer medium SNPs-P (GMA-IDA) of high charge density
The metal-chelating nanometer medium of metal-chelating nanometer medium used to be the electric density prepared in embodiment 3 be 5680 μm of ol/g is the metal-chelating nanometer medium that the electric density prepared is the highest.
The inclusion body protein sample of sex change: be precipitated and dissolved in by inclusion body in denaturation buffer (50mmol/LTris-HCl, 10mol/L urea, pH8.5), is placed in 50 DEG C of water-baths and heats 2h, make its complete sex change.Inclusion body after sex change is placed in 4 DEG C, the centrifugal 15min of 12000rpm, discard precipitate the supernatant liquor that obtains be dissolving after denatured protein, adopt Bradford method to measure total protein concentration in sex change liquid, being diluted to total protein concentration with above-mentioned denaturation buffer is afterwards 10mg/mL.Renaturation experiment is carried out in the centrifuge tube of 1.5mL, and renaturation buffer is the 100mmol/LTris-HCl damping fluid containing 2.0mol/L urea and 20mmol/LDTT, and pH is 9.0.For the 6 × his-EGFP renaturing inclusion bodies not adding nanometer medium, the above-mentioned renaturation buffer of 1mL is directly placed in 37 DEG C of Air oscillator pre-equilibrations; For the 6 × his-EGFP renaturing inclusion bodies adding nanometer medium, adding concentration in the above-mentioned renaturation buffer of 1mL is that the metal-chelating nanometer medium of 64mg/mL is placed in 37 DEG C of Air oscillators and shakes pre-equilibration until homo(io)thermism.The denaturing soln 12.5 of 6 × his-EGFP inclusion body is doubly diluted in above-mentioned renaturation buffer, makes the concentration of 6 × his-EGFP inclusion body protein in renaturation system be 0.8mg/mL (containing 0.56mg/mL6 × his-EGFP).Above-mentioned renaturation system adopts fluorescence spectrophotometer to measure renaturation product and excites under 488nm under 37 DEG C and 170rpm condition after renaturation 12h, the fluorescence intensity of launching under 509nm, the fluorescence standard curve of corresponding natural 6 × his-EGFP can calculate the concentration of active 6 × his-EGFP in renaturation system.By the concentration of activated protein compared with the total concn of 6 × his-EGFP in renaturation system, the fluorescence renaturation yield of 6 × his-EGFP in renaturation product can be obtained.
Metal-chelating nanometer medium assists the effect of 0.8mg/mL6 × his-EGFP renaturing inclusion bodies as shown in Figure 3, as seen from the figure, for 0.8mg/mL6 × his-EGFP renaturing inclusion bodies, adding of metal-chelating nanometer medium SNPs-P (GMA-IDA) makes renaturation yield be increased to 48% by 27%, and microsphere medium IDA-SepharoseFF is only better to low concentration inclusion body (0.1mg/mL) renaturation effect, unsatisfactory (the Integrativerefoldingandpurificationofhistidine-taggedpro teinbylike-chargefacilitatedrefoldingandmetal-chelateaff inityadsorption.JournalofChromatographyA of effect for greater concn renaturing inclusion bodies, 1344, 59 – 65).Metal-chelating nanometer medium SNPs-P (GMA-IDA) that this result embodies high charge density is better than a large advantage of microsphere medium IDA-SepharoseFF, is namely applicable to greater concn renaturing inclusion bodies.
Embodiment 7: the absorption rate of recovery after the auxiliary 0.2mg/mL6 × his-EGFP renaturing inclusion bodies of metal-chelating nanometer medium SNPs-P (GMA-IDA) of high charge density
Because 6 × his-EGFP contains hexahistine label, therefore can, after renaturation completes, the metal-chelating character of metal-chelating nanometer medium be utilized to realize specific adsorption.The metal-chelating nanometer medium of metal-chelating nanometer medium used to be the electric density prepared in embodiment 3 be 5680 μm of ol/g is the metal-chelating nanometer medium that the electric density prepared is the highest.
According to the method identical with embodiment 4, carry out the renaturation of 0.2mg/mL6 × his-EGFP inclusion body in the centrifuge tube of 1.5mL, in renaturation system, the concentration of SNPs-P (GMA-IDA) is 16mg/mL.Above-mentioned renaturation system, at 20 DEG C, under 50rpm condition after renaturation 12h, adds 20mmol/L cystamine, makes DTT remaining in system all oxidized, avoids the nickel ion added to be reduced by DTT.After 30min, nickel sulfate solution is added in system, the concentration of nickel ion is 0.25 μm of ol/mg, after mixing, be placed in Air oscillator, at 20 DEG C, the absorption of target protein is carried out under 100rpm condition, after 20min, mixed system is at 4 DEG C, centrifugal 5min under 8000rpm, remove supernatant liquor, by nanometer medium 1mL lavation buffer solution (50mmol/LTris-HCl, 10mmol/L imidazoles, 0.1mol/LNaCl, 0.5mol/L urea, pH7.5) at 4 DEG C, under 8000rpm after centrifuge washing 3 times, join 400 μ L elution buffer (50mmol/LTris-HCl, 0.5mol/L imidazoles, 0.5mol/L urea, pH7.5) mix in, be placed in Air oscillator, at 20 DEG C, the wash-out of target protein is carried out under 100rpm condition, elution time is 15min.Mixed system after wash-out completes is at 4 DEG C, carry out centrifugal under 8000rpm, collect supernatant elutriant, adopt fluorescence spectrophotometer to measure elutriant to excite under 488nm, the fluorescence intensity of launching under 509nm, the fluorescence standard curve of corresponding natural 6 × his-EGFP can calculate the concentration of active 6 × his-EGFP in elutriant.By the concentration of activated protein compared with the total concn of 6 × his-EGFP in renaturation system, the absorption rate of recovery of active 6 × his-EGFP in elutriant can be obtained.
After the auxiliary 0.2mg/mL6 × his-EGFP renaturing inclusion bodies of metal-chelating nanometer medium SNPs-P (GMA-IDA) completes, when adding 0.25 μm of ol/mg nickel ion in renaturation system, fluorescence yield reaches 79%.For 0.2mg/mL6 × his-EGFP inclusion body, the yield of the auxiliary renaturation of 16mg/mLSNPs-P (GMA-IDA) is 90%.Fluorescence yield after nickel ion absorption reaches 79%, and after showing renaturation, the absorption rate of recovery of active 6 × his-EGFP reaches 88%, and the Active Target albumen namely after most of renaturation is recycled by metal-chelating absorption.This illustrates that metal-chelating nanometer medium SNPs-P (GMA-IDA) of high charge density is after the recombinant protein renaturing inclusion bodies of auxiliary polyhistidine tag completes, and can carry out fractionation by adsorption effectively by metal-chelating effect to target protein.
Embodiment 8: the absorption rate of recovery after the auxiliary 0.4mg/mL6 × his-EGFP renaturing inclusion bodies of metal-chelating nanometer medium SNPs-P (GMA-IDA) of high charge density
The metal-chelating nanometer medium of metal-chelating nanometer medium used to be the electric density prepared in embodiment 3 be 5680 μm of ol/g is the metal-chelating nanometer medium that the electric density prepared is the highest.
According to the method identical with embodiment 5, carry out the renaturation of 0.4mg/mL6 × his-EGFP inclusion body in the centrifuge tube of 1.5mL, in renaturation system, the concentration of SNPs-P (GMA-IDA) is 32mg/mL.Above-mentioned renaturation system, at 25 DEG C, under 100rpm condition after renaturation 12h, adds 20mmol/L cystamine, makes DTT remaining in system all oxidized, avoids the nickel ion added to be reduced by DTT.After 30min, nickel sulfate solution is added in system, the concentration of nickel ion is 0.45 μm of ol/mg, after mixing, be placed in Air oscillator, at 25 DEG C, adsorb under 170rpm condition, after 30min, by mixed system at 4 DEG C, centrifugal 5min under 10000rpm, remove supernatant liquor, by nanometer medium 2mL lavation buffer solution (100mmol/LTris-HCl, 50mmol/L imidazoles, 0.5mol/LNaCl, 1mol/L urea, pH8.5) at 4 DEG C, under 10000rpm after centrifuge washing 5 times, join 1000 μ L elution buffer (100mmol/LTris-HCl, 1.0mol/L imidazoles, 1mol/L urea, pH8.5) mix in, be placed in Air oscillator, at 25 DEG C, the wash-out of target protein is carried out under 170rpm condition, elution time is 30min.Mixed system after wash-out completes is at 4 DEG C, carry out centrifugal under 10000rpm, collect supernatant elutriant, adopt fluorescence spectrophotometer to measure elutriant to excite under 488nm, the fluorescence intensity of launching under 509nm, the fluorescence standard curve of corresponding natural 6 × his-EGFP can calculate the concentration of active 6 × his-EGFP in elutriant.By the concentration of activated protein compared with the total concn of 6 × his-EGFP in renaturation system, the absorption rate of recovery of active 6 × his-EGFP in elutriant can be obtained.
After the auxiliary 0.4mg/mL6 × his-EGFP renaturing inclusion bodies of metal-chelating nanometer medium SNPs-P (GMA-IDA) completes, when adding 0.45 μm of ol/mg nickel ion in renaturation system, fluorescence yield reaches 60%.For 0.4mg/mL6 × his-EGFP inclusion body, the yield of the auxiliary renaturation of 32mg/mLSNPs-P (GMA-IDA) is 70%.Fluorescence yield after nickel ion absorption reaches 60%, and after showing renaturation, the absorption rate of recovery of active 6 × his-EGFP reaches 86%, and the Active Target albumen namely after most of renaturation is recycled by metal-chelating absorption.This illustrates that metal-chelating nanometer medium SNPs-P (GMA-IDA) of high charge density is after the recombinant protein renaturing inclusion bodies of auxiliary polyhistidine tag completes, and can carry out fractionation by adsorption effectively by metal-chelating effect to target protein.
Embodiment 9: the SNPs-P (GMA-IDA) of high charge density is to the purification effect after 0.2mg/mL6 × his-EGFP renaturing inclusion bodies
The metal-chelating nanometer medium of metal-chelating nanometer medium used to be the electric density prepared in embodiment 3 be 5680 μm of ol/g is the metal-chelating nanometer medium that the electric density prepared is the highest.
According to the method identical with embodiment 4, carry out the renaturation of 0.2mg/mL6 × his-EGFP inclusion body in the centrifuge tube of 1.5mL, in renaturation system, the concentration of SNPs-P (GMA-IDA) is 16mg/mL.Above-mentioned renaturation system, at 20 DEG C, under 50rpm condition after renaturation 12h, adds 20mmol/L cystamine, makes DTT remaining in system all oxidized, avoids the nickel ion added to be reduced by DTT.After 30min, nickel sulfate solution is added in system, the concentration of nickel ion is 0.3 μm of ol/mg, after mixing, be placed in Air oscillator, at 37 DEG C, adsorb under 120rpm condition, after 25min, mixed system is at 4 DEG C, centrifugal 5min under 9000rpm, remove supernatant liquor, by nanometer medium 1.5mL lavation buffer solution (80mmol/LTris-HCl, 20mmol/L imidazoles, 0.3mol/LNaCl, 0.8mol/L urea, pH8.0) at 4 DEG C, under 9000rpm after centrifuge washing 4 times, join 500 μ L elution buffer (80mmol/LTris-HCl, 0.8mol/L imidazoles, 0.6mol/L urea, pH8.0) mix in, be placed in Air oscillator, at 37 DEG C, the wash-out of target protein is carried out under 120rpm condition, elution time is 20min.Mixed system after wash-out completes is at 4 DEG C, carry out centrifugal under 9000rpm, collect the elutriant in supernatant, polyacrylamide gel electrophoresis (SDS-PAGE) method is adopted to detect the purity of protein in elutriant, result as shown in Figure 4, as seen from the figure, containing a lot of foreign proteins (swimming lane 1) in the renaturation system of 6 × his-EGFP inclusion body, and 6 × his-EGFP has very high purity (96% in final elutriant, swimming lane 2), show that metal-chelating nanometer medium SNPs-P (GMA-IDA) has good purification effect to 6 × his-EGFP after renaturation.In addition, the total protein concentration of elutriant is determined according to Bradford method, and according to the purity of 6 × his-EGFP, obtain the concentration of 6 × his-EGFP in elutriant and be about 0.25mg/mL, compared with the concentration (0.14mg/mL) of 6 × his-EGFP in renaturation buffer, concentration improves 79%, and namely 6 × his-EGFP obtains effective enrichment, and concentration ratio is 1.8.This illustrates that the metal-chelating nanometer medium of high charge density effectively can not only assist the renaturation of the inclusion body protein of polyhistidine tag, also show good purifying and concentration effect to target protein, the metal-chelating nanometer medium embodying high charge density has larger actual application value in renaturing inclusion bodies and integrated purifying.
The present invention proposes and utilize atom transfer radical polymerization method, at Nano particles of silicon dioxide surface grafting GMA-IDA monomer, prepare the metal-chelating nanometer medium of high charge density, and propose the metal-chelating nanometer medium strengthening inclusion body protein renaturation of high charge density and the methods and applications of integrated purifying.Be described by on-the-spot preferred embodiment, person skilled obviously can not depart from content of the present invention, spirit and scope method as herein described is changed or suitably change with combination, realize the technology of the present invention.Special needs to be pointed out is, all similar replacements and change apparent to those skilled in the art, they are deemed to be included in spirit of the present invention, scope and content.
Claims (10)
1. a metal-chelating nanometer medium; It is characterized in that structural formula is:
Be Nano particles of silicon dioxide (SNPs) surface grafting iminodiethanoic acid (IDA) of 20-30nm and the connection polymer P (GMA-IDA) of glycidyl methacrylate (GMA) in median size; The electric density of metal-chelating nanometer medium is 2520-5680 μm of ol/g.
2. the metal-chelating nanometer medium preparation method of claim 1; It is characterized in that step is as follows:
1) the connection product GMA-IDA of GMA and IDA is synthesized;
2) initiator 2-bromo isobutyl acylbromide (BIBB) is fixed on the Nano particles of silicon dioxide surface being 20-30nm in median size, is fixed the Nano particles of silicon dioxide SNPs-Br of BIBB;
3) by step 2) in the Nano particles of silicon dioxide SNPs-Br of fixing BIBB join N, dinethylformamide (DMF) is with the mixing liquid of water, be made into the suspension that concentration is 0.1-0.2g/mL, the volume ratio of DMF and water is 1:2;
4) in step 3) suspension in add GMA-IDA monomer, CuBr catalyzer, 2,2 '-dipyridyl (Bpy) part and CuBr
2inert agents; The concentration of GMA-IDA is 0.2-1.0mol/L, and solution calculates with the mixing liquid of DMF (DMF) with water, and the molar concentration rate of each component is GMA-IDA:CuBr:CuBr
2: Bpy=100:1:0.2:2, polyreaction 1-2h, prepare metal-chelating nanometer medium SNPs-P (GMA-IDA).
3. the metal-chelating nanometer medium of claim 1 is for strengthening the method for inclusion body protein renaturation and integrated purifying, and its step is as follows:
1) joined by metal-chelating nanometer medium in the renaturation buffer of same electric charge inclusion body protein, the inclusion body protein then adding sex change dissolving carries out renaturation;
2) after renaturation completes, in system, add nickel ion, specific adsorption is carried out to target protein;
3) after having adsorbed, centrifugally remove supernatant liquor, rinse nanometer medium with lavation buffer solution;
4) nanometer medium is joined the wash-out carrying out target protein in elution buffer, after wash-out completes, collected by centrifugation elutriant, obtains the target protein of purifying.
4. method as claimed in claim 3, is characterized in that described renaturation buffer is: the mixing solutions of 20-100mmol/L Tutofusin tris (Tris) and 0-2.0mol/L urea; PH to 7.5-9.0 is adjusted with HCl.
5. method as claimed in claim 3, is characterized in that lavation buffer solution is: 50-100mmol/LTris, 10-50mmol/L imidazoles, 0.1-0.5mol/LNaCl, 0.5-1mol/L urea; PH to 7.5-8.5 is adjusted with HCl.
6. method as claimed in claim 3, is characterized in that elution buffer is: 50-100mmol/LTris, 0.5-1.0mol/L imidazoles, 0.5-1mol/L urea; PH to 7.5-8.5 is adjusted with HCl.
7. method as claimed in claim 3, is characterized in that described step 1) method is: metal-chelating nanometer medium joined in renaturation buffer, makes the concentration of nanometer medium in final renaturation system be 16-64mg/mL; Then being placed in temperature is that the Air oscillator of 20-37 DEG C shakes pre-equilibration until homo(io)thermism; Add the inclusion body protein solution treating renaturation, make protein concn in renaturation system be 0.2-0.8mg/mL; After mixing, be placed in Air oscillator, renaturation under rotating speed is 50-170rpm condition, renaturation temperature is identical with the temperature of renaturation buffer pre-equilibration before this.
8. method as claimed in claim 3, it is characterized in that described step 2) method is: add nickel sulfate solution in the system after renaturation completes, nickel ion concentration is 0.25-0.45 μm of ol/mg, after mixing, be placed in Air oscillator, under rotating speed is 100-170rpm condition, carry out the absorption of target protein, adsorption temp is 20-37 DEG C, and adsorption time is 20-30min.
9. method as claimed in claim 3, it is characterized in that described step 3) method is: by the mixed system after having adsorbed at 4 DEG C, centrifugal under 8000-10000rpm condition, remove supernatant liquor, by nanometer medium lavation buffer solution at 4 DEG C, centrifuge washing 3-5 time under 8000-10000rpm condition, the volume of the lavation buffer solution that each washing is used is 10-20 times of nanometer medium settling volume.
10. method as claimed in claim 3, it is characterized in that described step 4) method is: nanometer medium joined in elution buffer and mixes, be placed in Air oscillator, wash-out is carried out under rotating speed is 100-170rpm condition, the volume of elution buffer is 5-10 times of nanometer medium settling volume, eluting temperature is 20-37 DEG C, and elution time is 15-30min; Mixed system after wash-out completes is at 4 DEG C, centrifugal under 8000-10000rpm condition, collects supernatant liquor.
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| CN106268651A (en) * | 2016-08-26 | 2017-01-04 | 云南贵悦金属材料科技有限公司 | Nano SiO 2 particle for adsorbing separation platinum and its preparation method and application |
| CN106268651B (en) * | 2016-08-26 | 2019-01-18 | 云南贵悦金属材料科技有限公司 | Nano SiO 2 particle and its preparation method and application for adsorbing separation platinum |
| CN108003287A (en) * | 2017-12-27 | 2018-05-08 | 南京百赛生物色谱技术有限公司 | A kind of preparation method of albumen based on acrylamide race metal-chelating monomer is affine trace aquogel polymer |
| CN108003287B (en) * | 2017-12-27 | 2020-08-18 | 南京百赛生物色谱技术有限公司 | Preparation method of protein affinity imprinted hydrogel polymer based on acrylamide metal chelating monomer |
| CN111574583A (en) * | 2020-04-10 | 2020-08-25 | 上海海路生物技术有限公司 | Protein renaturation reagent and its application |
| CN111569849A (en) * | 2020-05-18 | 2020-08-25 | 北京石油化工学院 | Immobilized metal chelate chromatography medium and preparation method and application thereof |
| CN112024336A (en) * | 2020-07-29 | 2020-12-04 | 安徽喜宝高分子材料有限公司 | Preparation and spraying process of powder coating for improving metal waterproof effect by constructing nano protective layer |
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