CN110498419A - Nanometer silicon dioxide particle and preparation method thereof for histidine-tagged protein purifying - Google Patents
Nanometer silicon dioxide particle and preparation method thereof for histidine-tagged protein purifying Download PDFInfo
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- CN110498419A CN110498419A CN201810467980.4A CN201810467980A CN110498419A CN 110498419 A CN110498419 A CN 110498419A CN 201810467980 A CN201810467980 A CN 201810467980A CN 110498419 A CN110498419 A CN 110498419A
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- silicon dioxide
- nanometer silicon
- dioxide particle
- histidine
- tagged protein
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- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 title claims abstract description 113
- 239000000377 silicon dioxide Substances 0.000 title claims abstract description 57
- 239000002245 particle Substances 0.000 title claims abstract description 51
- 235000012239 silicon dioxide Nutrition 0.000 title claims abstract description 48
- 102000004169 proteins and genes Human genes 0.000 title claims abstract description 34
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 34
- 238000002360 preparation method Methods 0.000 title claims abstract description 14
- BEOOHQFXGBMRKU-UHFFFAOYSA-N sodium cyanoborohydride Chemical compound [Na+].[B-]C#N BEOOHQFXGBMRKU-UHFFFAOYSA-N 0.000 claims abstract description 5
- 239000008363 phosphate buffer Substances 0.000 claims description 17
- 239000000243 solution Substances 0.000 claims description 15
- 238000003756 stirring Methods 0.000 claims description 15
- 238000005119 centrifugation Methods 0.000 claims description 13
- 239000000872 buffer Substances 0.000 claims description 11
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 claims description 9
- MUBZPKHOEPUJKR-UHFFFAOYSA-N Oxalic acid Natural products OC(=O)C(O)=O MUBZPKHOEPUJKR-UHFFFAOYSA-N 0.000 claims description 6
- 239000006228 supernatant Substances 0.000 claims description 6
- 238000005406 washing Methods 0.000 claims description 6
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 5
- WYTZZXDRDKSJID-UHFFFAOYSA-N (3-aminopropyl)triethoxysilane Chemical compound CCO[Si](OCC)(OCC)CCCN WYTZZXDRDKSJID-UHFFFAOYSA-N 0.000 claims description 4
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 claims description 4
- 150000002460 imidazoles Chemical class 0.000 claims description 4
- 239000000725 suspension Substances 0.000 claims description 4
- -1 2- ethoxy Chemical group 0.000 claims description 3
- 239000007864 aqueous solution Substances 0.000 claims description 3
- LGQLOGILCSXPEA-UHFFFAOYSA-L nickel sulfate Chemical compound [Ni+2].[O-]S([O-])(=O)=O LGQLOGILCSXPEA-UHFFFAOYSA-L 0.000 claims description 3
- 229910000363 nickel(II) sulfate Inorganic materials 0.000 claims description 3
- 235000006408 oxalic acid Nutrition 0.000 claims description 3
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 claims description 2
- 229910000147 aluminium phosphate Inorganic materials 0.000 claims description 2
- 238000006243 chemical reaction Methods 0.000 claims description 2
- NBZBKCUXIYYUSX-UHFFFAOYSA-N iminodiacetic acid Chemical compound OC(=O)CNCC(O)=O NBZBKCUXIYYUSX-UHFFFAOYSA-N 0.000 claims description 2
- 238000002156 mixing Methods 0.000 claims description 2
- 230000010355 oscillation Effects 0.000 claims description 2
- 239000012460 protein solution Substances 0.000 claims description 2
- PXHVJJICTQNCMI-UHFFFAOYSA-N Nickel Chemical compound [Ni] PXHVJJICTQNCMI-UHFFFAOYSA-N 0.000 claims 2
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 claims 2
- JMANVNJQNLATNU-UHFFFAOYSA-N oxalonitrile Chemical compound N#CC#N JMANVNJQNLATNU-UHFFFAOYSA-N 0.000 claims 2
- 150000003851 azoles Chemical class 0.000 claims 1
- 229910052759 nickel Inorganic materials 0.000 claims 1
- 150000003839 salts Chemical class 0.000 claims 1
- 229910000033 sodium borohydride Inorganic materials 0.000 claims 1
- 239000012279 sodium borohydride Substances 0.000 claims 1
- 238000000746 purification Methods 0.000 abstract description 5
- 238000000926 separation method Methods 0.000 abstract description 5
- 229910052751 metal Inorganic materials 0.000 abstract description 4
- 239000002184 metal Substances 0.000 abstract description 4
- 238000000034 method Methods 0.000 abstract description 3
- 238000005576 amination reaction Methods 0.000 abstract description 2
- 229910052757 nitrogen Inorganic materials 0.000 abstract description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N nitrogen Substances N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 abstract description 2
- 229920000642 polymer Polymers 0.000 abstract description 2
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 description 13
- 108010043121 Green Fluorescent Proteins Proteins 0.000 description 7
- 102000004144 Green Fluorescent Proteins Human genes 0.000 description 7
- 230000004048 modification Effects 0.000 description 7
- 238000012986 modification Methods 0.000 description 7
- 238000001042 affinity chromatography Methods 0.000 description 6
- 239000005090 green fluorescent protein Substances 0.000 description 6
- MURGITYSBWUQTI-UHFFFAOYSA-N fluorescin Chemical compound OC(=O)C1=CC=CC=C1C1C2=CC=C(O)C=C2OC2=CC(O)=CC=C21 MURGITYSBWUQTI-UHFFFAOYSA-N 0.000 description 4
- 229910021645 metal ion Inorganic materials 0.000 description 4
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
- RAXXELZNTBOGNW-UHFFFAOYSA-N imidazole Natural products C1=CNC=N1 RAXXELZNTBOGNW-UHFFFAOYSA-N 0.000 description 3
- 238000001179 sorption measurement Methods 0.000 description 3
- 229920000936 Agarose Polymers 0.000 description 2
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 2
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 2
- GWEVSGVZZGPLCZ-UHFFFAOYSA-N Titan oxide Chemical compound O=[Ti]=O GWEVSGVZZGPLCZ-UHFFFAOYSA-N 0.000 description 2
- 230000001580 bacterial effect Effects 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 239000008367 deionised water Substances 0.000 description 2
- 229910021641 deionized water Inorganic materials 0.000 description 2
- 235000013601 eggs Nutrition 0.000 description 2
- 102000037865 fusion proteins Human genes 0.000 description 2
- 108020001507 fusion proteins Proteins 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 239000002609 medium Substances 0.000 description 2
- 239000011856 silicon-based particle Substances 0.000 description 2
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 2
- 230000009182 swimming Effects 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- QCVGEOXPDFCNHA-UHFFFAOYSA-N 5,5-dimethyl-2,4-dioxo-1,3-oxazolidine-3-carboxamide Chemical compound CC1(C)OC(=O)N(C(N)=O)C1=O QCVGEOXPDFCNHA-UHFFFAOYSA-N 0.000 description 1
- 102000002322 Egg Proteins Human genes 0.000 description 1
- 108010000912 Egg Proteins Proteins 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 1
- 102000006830 Luminescent Proteins Human genes 0.000 description 1
- 108010047357 Luminescent Proteins Proteins 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 239000012501 chromatography medium Substances 0.000 description 1
- 239000012539 chromatography resin Substances 0.000 description 1
- 238000002425 crystallisation Methods 0.000 description 1
- 230000008025 crystallization Effects 0.000 description 1
- 125000004093 cyano group Chemical group *C#N 0.000 description 1
- 229960000935 dehydrated alcohol Drugs 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 235000014103 egg white Nutrition 0.000 description 1
- 210000000969 egg white Anatomy 0.000 description 1
- 230000005611 electricity Effects 0.000 description 1
- 229960004756 ethanol Drugs 0.000 description 1
- 125000001301 ethoxy group Chemical group [H]C([H])([H])C([H])([H])O* 0.000 description 1
- 239000000945 filler Substances 0.000 description 1
- 238000001502 gel electrophoresis Methods 0.000 description 1
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 1
- 239000010931 gold Substances 0.000 description 1
- 229910052737 gold Inorganic materials 0.000 description 1
- 238000006197 hydroboration reaction Methods 0.000 description 1
- 230000002209 hydrophobic effect Effects 0.000 description 1
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 1
- 125000002883 imidazolyl group Chemical group 0.000 description 1
- 230000008676 import Effects 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- JEIPFZHSYJVQDO-UHFFFAOYSA-N iron(III) oxide Inorganic materials O=[Fe]O[Fe]=O JEIPFZHSYJVQDO-UHFFFAOYSA-N 0.000 description 1
- 239000003446 ligand Substances 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 238000001000 micrograph Methods 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 239000002086 nanomaterial Substances 0.000 description 1
- SOQBVABWOPYFQZ-UHFFFAOYSA-N oxygen(2-);titanium(4+) Chemical compound [O-2].[O-2].[Ti+4] SOQBVABWOPYFQZ-UHFFFAOYSA-N 0.000 description 1
- BHTJEPVNHUUIPV-UHFFFAOYSA-N pentanedial;hydrate Chemical compound O.O=CCCCC=O BHTJEPVNHUUIPV-UHFFFAOYSA-N 0.000 description 1
- 229920002401 polyacrylamide Polymers 0.000 description 1
- 238000001742 protein purification Methods 0.000 description 1
- 238000005215 recombination Methods 0.000 description 1
- 230000006798 recombination Effects 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 239000011347 resin Substances 0.000 description 1
- 229920005989 resin Polymers 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- HBMJWWWQQXIZIP-UHFFFAOYSA-N silicon carbide Chemical compound [Si+]#[C-] HBMJWWWQQXIZIP-UHFFFAOYSA-N 0.000 description 1
- 229910010271 silicon carbide Inorganic materials 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 239000004408 titanium dioxide Substances 0.000 description 1
- 230000007704 transition Effects 0.000 description 1
- 238000002604 ultrasonography Methods 0.000 description 1
Classifications
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B82—NANOTECHNOLOGY
- B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
- B82Y30/00—Nanotechnology for materials or surface science, e.g. nanocomposites
-
- C—CHEMISTRY; METALLURGY
- C01—INORGANIC CHEMISTRY
- C01B—NON-METALLIC ELEMENTS; COMPOUNDS THEREOF; METALLOIDS OR COMPOUNDS THEREOF NOT COVERED BY SUBCLASS C01C
- C01B33/00—Silicon; Compounds thereof
- C01B33/113—Silicon oxides; Hydrates thereof
- C01B33/12—Silica; Hydrates thereof, e.g. lepidoic silicic acid
- C01B33/18—Preparation of finely divided silica neither in sol nor in gel form; After-treatment thereof
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/16—Extraction; Separation; Purification by chromatography
- C07K1/22—Affinity chromatography or related techniques based upon selective absorption processes
-
- C—CHEMISTRY; METALLURGY
- C01—INORGANIC CHEMISTRY
- C01P—INDEXING SCHEME RELATING TO STRUCTURAL AND PHYSICAL ASPECTS OF SOLID INORGANIC COMPOUNDS
- C01P2004/00—Particle morphology
- C01P2004/01—Particle morphology depicted by an image
-
- C—CHEMISTRY; METALLURGY
- C01—INORGANIC CHEMISTRY
- C01P—INDEXING SCHEME RELATING TO STRUCTURAL AND PHYSICAL ASPECTS OF SOLID INORGANIC COMPOUNDS
- C01P2004/00—Particle morphology
- C01P2004/01—Particle morphology depicted by an image
- C01P2004/03—Particle morphology depicted by an image obtained by SEM
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Nanotechnology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Physics & Mathematics (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Biochemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Inorganic Chemistry (AREA)
- Biophysics (AREA)
- Health & Medical Sciences (AREA)
- Composite Materials (AREA)
- Condensed Matter Physics & Semiconductors (AREA)
- General Physics & Mathematics (AREA)
- Materials Engineering (AREA)
- Crystallography & Structural Chemistry (AREA)
- Analytical Chemistry (AREA)
- Peptides Or Proteins (AREA)
- Solid-Sorbent Or Filter-Aiding Compositions (AREA)
Abstract
The invention discloses a kind of nanometer silicon dioxide particles and preparation method thereof for histidine-tagged protein purifying.The method is first by Nano-meter SiO_22Aldehyde radical again after surface amination restores carbon-to-nitrogen double bon with sodium cyanoborohydride, enhances metal chelating groups and Nano-meter SiO_2 in connection after metal chelating groups2Connective stability.Affinity separation polymer of the nanometer silicon dioxide particle prepared by the present invention as purifying histidine-tagged protein, making separation and purification of protein, operation is simple, cost reduces.
Description
Technical field
The present invention relates to technical field of nano material, are related to a kind of nanometer titanium dioxide for histidine-tagged protein purifying
Silicon particle and preparation method thereof.
Background technique
Histidine tag (His-Tag) is the preferred label of protein purification.Because His-Tag is very small, to destination protein
Self character has little effect, and will not change the solubility of destination protein itself, to protein structure after fusion protein crystallization
It does not influence, is very beneficial for the research of protein structure；In addition His- is purified using immobilized metal ion affinity chromatography
Tagged fusion protein is easy to operate.The imidazole group having on the residue of histidine (His) can be with Ni2+、Co2+Equal transition gold
Belong to ion to form coordinate bond and be selectively bound on metal ion.The chelating ligand connected on affinity chromatography resin can incite somebody to action
Ni2+、Co2+Equal metal ions are fixed on chromatography media, and the layer for combining metal ion is passed through in His-tagged recombinant protein
The combination for the property of can choose is on medium when analysing medium, and other impurity proteins then cannot combine or be only capable of faint combination.
Mostly use commodity Ni- complexon I (Ni-NTA) gel as affinity chromatography tree in protein research and production
Rouge purifies histidine-tagged protein.But the carrier of Ni-NTA gel mostly uses agarose particle, and agarose particle surface is usual
It is more coarse, easily to albumen generate non-specific adsorption, isolated destination protein content it is few or containing hydrophobic amino acid it is more
When non-specific adsorption problem it is especially prominent.In addition, for the resin of affinity chromatography, that there are mechanical strengths is low, easily broken, and big
Need import more, it is expensive.The research of affinity chromatography filler, but chromatography method protein isolate are dedicated to now with some scholars
Than relatively time-consuming, separative efficiency is lower, it is difficult to (the affinity chromatography such as Li Hongli be widely applied in recombinant protein industrialized production
Application and new development ocean lakes and marhshes notification .2005 (03) of the technology in modern protein research: 86-94.).
Summary of the invention
The present invention provides a kind of nanometer silicon dioxide particle and preparation method thereof for histidine-tagged protein purifying.
Technical scheme is as follows:
The preparation method of nanometer silicon dioxide particle for histidine-tagged protein purifying, the specific steps are as follows:
Step 1, in ethanol by nanometer silicon dioxide particle ultrasonic disperse, 3-aminopropyltriethoxysilane is added,
Stirring at normal temperature, centrifugation, obtains amidized nanometer silicon dioxide particle；
Step 2, amidized nanometer silicon dioxide particle is washed with phosphate buffer and removes unreacted 3- amino
Propyl-triethoxysilicane, ultrasonic disperse is in phosphate buffer, addition glutaraldehyde solution, stirring at normal temperature, after reaction,
Phosphate buffer washing, centrifugation obtain the nanometer silicon dioxide particle of aldehyde radical；
Step 3, the nanometer silicon dioxide particle of aldehyde radical is suspended again with phosphate buffer, and imido- diethyl is added
4- (2- ethoxy) -1- piperazine ethanesulfonic acid solution of acid, stirring at normal temperature are washed with Tris-HCl buffer and are suspended again, added
Enter sodium cyanoborohydride solution, stirring at normal temperature is centrifuged, and the washing of Tris-HCl buffer obtains pure for histidine-tagged protein
The nanometer silicon dioxide particle of change.
Preferably, the partial size of the nanometer silicon dioxide particle is 10~500nm.
Preferably, in 4- (2- ethoxy) -1- piperazine ethanesulfonic acid solution of the imido- oxalic acid, imino-diacetic
The mass concentration of acetic acid is 0.05%.
Preferably, the concentration of the sodium cyanoborohydride is 1~10mM.
Preferably, the stirring at normal temperature time is 0.5~12h.
The present invention also provides nanometer silicon dioxide particles made from above-mentioned preparation method.
Further, the answering in histidine-tagged protein purifying the present invention also provides above-mentioned nanometer silicon dioxide particle
With, the specific steps are as follows:
Suspended the nanometer silicon dioxide particle purified for histidine-tagged protein again with Tris-HCl buffer, is added
Nickel sulfate solution, stirring at normal temperature, washing are suspended again with phosphate buffer, nanometer silicon dioxide particle suspension are added
Into the aqueous solution containing histidine-tagged protein, nanometer silicon dioxide particle is collected by centrifugation in room temperature oscillation mixing, with containing 1~
The phosphate buffer of 25mM imidazoles washs, then elutes histidine tag egg with the phosphate buffer of the imidazoles Han 0.4~1.5M
It is white, centrifugation removal nanometer silicon dioxide particle, the histidine-tagged protein solution for the purifying that supernatant is.
Preferably, the time of the stirring at normal temperature is 1~30min.
Preferably, the nickel sulfate solution concentration is 0.1~1M.
Preferably, the phosphate buffer pH is 7.3.
Compared with prior art, the invention has the following advantages that
The present invention using nano silica good biocompatibility, property is stable, adsorption capacity is strong, specific surface area is huge and
There is the features such as great amount of hydroxy group on surface, amination, aldehyde radical is successively carried out, in connection after metal chelating groups, with cyano hydroboration
Sodium reduction carbon-to-nitrogen double bon enhances metal chelating groups and Nano-meter SiO_22Connective stability, preparation purifying histidine-tagged protein
Affinity separation polymer, making separation and purification of protein, operation is simple, cost reduces.
Detailed description of the invention
Fig. 1 is the nanometer silicon dioxide particle structural schematic diagram purified for histidine-tagged protein.
Fig. 2 is the scanning electricity of the nanometer silicon dioxide particle (a) before modification and the nanometer silicon dioxide particle (b) after modification
Sub- microscope figure.
Fig. 3 is the shows fluorescent microscopy images for being combined with the nanometer silicon dioxide particle of histidine tag green fluorescent protein.
Fig. 4 is the SDS-PAGE figure for the histidine tag green fluorescent protein that nanometer silicon dioxide particle obtains after purification.
Specific embodiment
The present invention is described in detail with attached drawing combined with specific embodiments below.
Embodiment 1
By 0.04g nanometer silicon dioxide particle (partial size 20nm) ultrasonic disperse in 4mL dehydrated alcohol.Ultrasound terminates
Afterwards, 200 μ L 3-aminopropyltriethoxysilane are added in silica suspension, are put into 30 DEG C of shaking tables, shake 12h, obtain
To amidized nano silica.Amidized nano silica is collected by centrifugation, with the phosphoric acid buffer of the 10mM of pH=7.3
Liquid is washed three times.The phosphate buffer of the 10mM of the pH=7.3 of 4mL is added in amidized nano silica, and ultrasonic 5min is mixed,
Addition 1mL concentration is 5% glutaraldehyde water solution, and 12h is stirred at room temperature, is washed with deionized water three times, obtains the nanometer two of aldehyde radical
Silica.0.1M 4- (2- the ethoxy) -1- piperazine ethanesulfonic acid solution (pH8.0) for containing 0.05% iminodiacetic acid, shake is added
Suspension is swung, 12h is stirred at room temperature, is washed three times with Tris-HCl buffer.With Tris-HCl buffer again suspended nano titanium dioxide
Silicon particle, is added the sodium cyanoborohydride aqueous solution that 5mL concentration is 1mM, and nano silica is collected by centrifugation in stirring at normal temperature 2h
Grain, is washed three times with Tris-HCl buffer, obtains nano-silica of the surface purified for histidine-tagged protein by modification
Silicon carbide particle.Fig. 2 is seen with the nanometer silicon dioxide particle scanning electron microscope observation result after modification before modification.
Embodiment 2
Expression histidine tag is added in the surface made from 0.04g embodiment 1 in the nano silica by modification green
Color fluorescin recombination bacillus coli is crushed centrifugate supernatant, and room temperature vibrates 5min, and the speed of 4000rpm is centrifuged 5min, is added
The unbonded protein of deionized water washing, the speed of 4000rpm are centrifuged 5min, obtain to surface and are combined with histidine tag green
The nanometer silicon dioxide particle of fluorescin.Surface is combined with the nanometer silicon dioxide particle of histidine tag green fluorescent protein
Fluorescence microscope result is shown in Fig. 3.As can be seen from Figure 3, it is distributed many green fluorescent proteins under fluorescence microscope, demonstrates utilization
The nanometer silicon dioxide particle modified can be very easy rapidly in conjunction with histidine-tagged protein.
Embodiment 3
Surface made from embodiment 1 is combined with the nanometer silicon dioxide particle of histidine tag green fluorescent protein with containing
The phosphate buffer of 5mM imidazoles washs three times, and the imidazole solution of 1mL 500mM is added, and room temperature vibrates 5min, 4000rpm centrifugation
5min.The identification of SDS- polyacrylamide gel electrophoresis is carried out to centrifugation supernatant.
The histidine tag green fluorescent protein SDS- polyacrylamide obtained after purification with nanometer silicon dioxide particle is solidifying
Gel electrophoresis result is shown in that Fig. 4, swimming lane 1 are Marker, and 2 be that the histidine tag green that nanometer silicon dioxide particle obtains after purification is glimmering
Photoprotein, 3 be broken thallus centrifuged supernatant, and 4 be direct sampling liquid after bacterial cell disruption.As can be seen from Figure 4, swimming lane 3,4 is distinguished
It is directly point sample and broken thallus centrifuged supernatant point sample after bacterial cell disruption, the protein band run out of all compares more, illustrates egg
White mixture is more miscellaneous more.But purify histidine tag green fluorescent protein with nanometer silicon dioxide particle after modification, and general
Its band run out of after eluting is just relatively simple clear, embodies nanometer silicon dioxide particle separation histidine tag egg
Bai Kehang.
Claims (10)
1. the preparation method of the nanometer silicon dioxide particle for histidine-tagged protein purifying, which is characterized in that specific steps
It is as follows:
Step 1, in ethanol by nanometer silicon dioxide particle ultrasonic disperse, 3-aminopropyltriethoxysilane, room temperature is added
Stirring, centrifugation, obtains amidized nanometer silicon dioxide particle；
Step 2, amidized nanometer silicon dioxide particle is washed with phosphate buffer and removes unreacted 3- aminopropyl
Triethoxysilane, ultrasonic disperse is in phosphate buffer, addition glutaraldehyde solution, stirring at normal temperature, after reaction, phosphoric acid
W salt buffer washes, centrifugation, obtain the nanometer silicon dioxide particle of aldehyde radical；
Step 3, the nanometer silicon dioxide particle of aldehyde radical is suspended again with phosphate buffer, and imido- oxalic acid is added
4- (2- ethoxy) -1- piperazine ethanesulfonic acid solution, stirring at normal temperature are washed with Tris-HCl buffer and are suspended again, cyanogen is added
Base sodium borohydride solution, stirring at normal temperature, centrifugation, the washing of Tris-HCl buffer are obtained for histidine-tagged protein purifying
Nanometer silicon dioxide particle.
2. preparation method according to claim 1, which is characterized in that the partial size of the nanometer silicon dioxide particle is 10
~500nm.
3. preparation method according to claim 1, which is characterized in that 4- (the 2- hydroxyl second of the imido- oxalic acid
Base) in -1- piperazine ethanesulfonic acid solution, the mass concentration of iminodiacetic acid is 0.05%.
4. preparation method according to claim 1, which is characterized in that the concentration of the sodium cyanoborohydride be 1~
10mM。
5. preparation method according to claim 1, which is characterized in that the stirring at normal temperature time is 0.5~12h.
6. nanometer silicon dioxide particle made from preparation method according to any one of claims 1 to 5.
7. application of the nanometer silicon dioxide particle according to claim 6 in histidine-tagged protein purifying, feature
It is, the specific steps are as follows:
Suspended the nanometer silicon dioxide particle purified for histidine-tagged protein again with Tris-HCl buffer, and sulfuric acid is added
Nickel solution, stirring at normal temperature, washing are suspended again with phosphate buffer, nanometer silicon dioxide particle suspension are added to and is contained
In the aqueous solution of histidine-tagged protein, nanometer silicon dioxide particle is collected by centrifugation, with the miaow containing 1~25mM in room temperature oscillation mixing
The phosphate buffer of azoles washs, then elutes histidine-tagged protein, centrifugation with the phosphate buffer of the imidazoles Han 0.4~1.5M
Remove nanometer silicon dioxide particle, the histidine-tagged protein solution for the purifying that supernatant is.
8. application according to claim 7, which is characterized in that the time of the stirring at normal temperature is 1~30min.
9. application according to claim 7, which is characterized in that the nickel sulfate solution concentration is 0.1~1M.
10. application according to claim 7, which is characterized in that the phosphate buffer pH is 7.3.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810467980.4A CN110498419A (en) | 2018-05-16 | 2018-05-16 | Nanometer silicon dioxide particle and preparation method thereof for histidine-tagged protein purifying |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810467980.4A CN110498419A (en) | 2018-05-16 | 2018-05-16 | Nanometer silicon dioxide particle and preparation method thereof for histidine-tagged protein purifying |
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101745353A (en) * | 2010-02-12 | 2010-06-23 | 上海交通大学 | Magnetic microsphere as well as preparation and preparation method thereof used for purifying histidine tag protein |
| CN104876228A (en) * | 2015-04-14 | 2015-09-02 | 上海大学 | Solid-phase synthesis method for nano silicon dioxide particles based on histidine tag |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101745353A (en) * | 2010-02-12 | 2010-06-23 | 上海交通大学 | Magnetic microsphere as well as preparation and preparation method thereof used for purifying histidine tag protein |
| CN104876228A (en) * | 2015-04-14 | 2015-09-02 | 上海大学 | Solid-phase synthesis method for nano silicon dioxide particles based on histidine tag |
Non-Patent Citations (2)
| Title |
|---|
| LAURA E. VALENTI等: "Ni(II)-modified solid substrates as a platform to adsorb His-tag proteins", 《JOURNAL OF MATERIALS CHEMISTRY B》 * |
| 王立等: "二氧化硅复合微球的制备及其对his-tagged融合蛋白质的分离", 《化学研究》 * |
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Application publication date: 20191126 |