CN105597657B - A kind of functionalization composite metal oxide material and its preparation method and application - Google Patents
A kind of functionalization composite metal oxide material and its preparation method and application Download PDFInfo
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- CN105597657B CN105597657B CN201510612187.5A CN201510612187A CN105597657B CN 105597657 B CN105597657 B CN 105597657B CN 201510612187 A CN201510612187 A CN 201510612187A CN 105597657 B CN105597657 B CN 105597657B
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Abstract
The invention discloses a kind of functionalization composite metal oxide materials and its preparation method and application.The present invention is subject to the reasonable control of settling rate and reaction temperature, prepares the controllable nickel of regular appearance, size, cobalt, manganese metal composite oxide nano-micro level microballoon by organically combining coprecipitation and high temperature solid-state method.Chelation based on metal ion and histidine, polyhistidine fusion protein can be adsorbed on the micro-sphere material surface, and then can be eluted liquid again quickly elutes.The preparation method of the micro-sphere material is simple and easy to do, it is of low cost, convenient for batch production, prepared nickel, cobalt, manganese metal composite oxide nano-micro level micro-sphere material pattern are uniform controllable, it can be used for quickly and efficiently isolating and purifying the polyhistidine fusion protein in complex biological sample, and realize and be recycled for multiple times that there is very high commercial application prospect.
Description
Technical field
The present invention relates to a kind of functionalization composite metal oxide materials, and in particular to a kind of nickel, cobalt, manganese composition metal oxygen
Compound material, the preparation method of the material and its application in hexahistine fusion protein isolates and purifies.
Background technology
Fusion tag technology is risen in twentieth century end, is a kind of recombinant DNA technology based on reporter gene.This technology
Fast development make purifying recombinant proteins more efficiently it is convenient, be widely used in numerous areas such as life science, chemistry.
Currently used protein purification label includes hexahistine, glutathione sulfydryl transferase and epitope tag etc., wherein six is poly-
His fusions are the most commonly used.By a large amount of fusion protein of the expression such as Escherichia coli, then in complicated cell cracking
The hexahistine fusion protein of expression is isolated and purified in liquid system.
Histidine has stronger chelation with metal ion, thus hexahistine fusion protein can be based on immobilization gold
Belong to ion affinity chromatography (IMAC) to be isolated and purified.Utilize complexon I (NTA), iminodiacetic acid at present more
(IDA) etc. chelating aglucon chelates Ni2+IMAC media are synthesized, and then is applied to hexahistine fusion protein and isolates and purifies.
But the shortcomings of this method metal ion is easy to fall off, and is easily led to protein binding capacity and is declined, poor specificity.Traditional IMAC methods
It needs to remove the cell debris in cell pyrolysis liquid before loading, and colloid pollution may be caused.Therefore, a species specificity is developed
By force, the material of rapidly and efficiently separating and purifying hexahistine fusion protein and method are very necessary.Document (L.Zhang,
X.Zhu,D.Jiao,Y.Sun and H.Sun.Efficient purification of His-tagged protein by
superparamagneticFe3O4/Au–ANTA–Co2+nanoparticles.Mat.Sci.Eng.C-Mater.,2013,33,
1989-1992) propose Co2+It is better than Ni with the binding ability of histidine2+, but cost is higher.Therefore, nickel, cobalt, manganese composition metal
Oxide material can integrate the characteristic of various metals ion, improve to the affine specificity of hexahistine fusion protein and selection
Property.
For nano-micro level microballoon due to its regular pattern, the advantages that larger specific surface area, is widely used in separation analysis neck
Domain, therefore, this invention address that developing the synthetic method of a kind of nickel, cobalt, manganese composite metal oxidate microspheres.
Invention content
The purpose of the present invention is to provide a kind of nickel, cobalt, manganese composite metal oxide material and its synthetic methods, and are based on
The chelation of metal ion and histidine is applied to isolating and purifying for polyhistidine fusion protein, realizes to poly
The rapidly and efficiently purifying of His fusions.
In order to improve the combined coefficient and yield of material, the present invention organically combines coprecipitation and high temperature solid-state,
By reasonably controlling precipitation reaction rate and pyroreaction temperature, synthesizing spherical MnCo(2-x)NixO4(x=0-1) material.
Technical scheme is as follows：
A kind of nickel, cobalt, manganese composite metal oxide material, molecular formula MnCo(2-x)NixO4(x=0~1), in the material
The mass percentage of Ni elements is 0%~25% (relative to material gross mass).The nickel, cobalt, the oxidation of manganese composition metal
Object material, microscopic appearance are the controllable microballoon of micron-sized size, and the microballoon is by the nanocrystalline spherical hollow structure formed.
In above-mentioned nickel, cobalt, manganese composite metal oxide material, the mass percentages of Ni elements is preferably 10%~
20%；The preferred diameter of microscopic appearance of the microballoon is about the spherical shape of 500nm~5 μm；The nanocrystalline average-size is usual
In 50~200nm or so, majority is about 100nm.
The synthetic method of nickel of the present invention, cobalt, manganese composite metal oxide material, includes the following steps：
(1) it by the metal salt of Mn, Co, Ni according to the mixed solution of corresponding molar ratio metal ion, and adjusts molten
Liquid pH to 10-12；
(2) certain density precipitant solution is prepared, the metal ion that excessive precipitating reagent is obtained with step (1) is made to mix
Solution reaction keeps its precipitation complete, and reaction is as follows；
M2++CO3 2-——>MCO3(with Na2CO3For precipitating reagent, M represents metallic element)
(3) step (2) is obtained into washing of precipitate for several times with deionized water, then dried；
(4) step (3) precipitation is fully ground, then carries out high-temperature process, reacting final product is obtained after cooling.
Preferably, above-mentioned steps (1) metal salt can select sulfate, nitrate or chloride of the metal etc..
Preferably, the pH that above-mentioned steps (1) adjust solution by the way that appropriate ammonium hydroxide is added is 10~12.
Preferably, the precipitating reagent of above-mentioned steps (2) can be soluble carbonate salt, hydroxide etc., such as sodium carbonate, hydrogen
Sodium oxide molybdena.Coprecipitation reaction can be by controlling the rate of co-precipitation and the pattern of controlling reaction temperature final product.This is heavy
Forming sediment reaction can be in the case where magnetic agitation speed be 600-900r/m, and the mixing that metal ion is added dropwise into precipitant solution is molten
Liquid.Reaction temperature is preferably in room temperature within the scope of 75 DEG C, and when reaction carries out at room temperature, the microscopic appearance of final product is diameter
For the spheric granules of 500nm or so, when coprecipitation reaction temperature is 70 DEG C, final product is the microballoon that diameter is about 2 μm.
Preferably, above-mentioned steps (4) grinding can carry out in the agate mortar, grind 1~2 hour.
Preferably, above-mentioned steps (4) can carry out high temperature solid state reaction in Muffle furnace, and temperature is 700-950 DEG C, heating
Rate is 2 DEG C/min, and the reaction time is 10~20 hours.Up to final product after natural cooling.
Nickel, cobalt, the manganese composite metal oxide material of the present invention can identify the fusion egg with polyhistidine label
In vain, and by the chelation of metal ion and histidine by polyhistidine fusion protein it is enriched in material surface, it therefore, should
The affinity purification material of nickel, cobalt, manganese composite metal oxide material as polyhistidine fusion protein, wherein the poly group
Propylhomoserin is usually hexahistine.
The present invention provides a kind of polyhistidine fusion protein isolation and purification methods as a result, are prepared using the present invention
Nickel, cobalt, manganese composite metal oxide material are identified by metal-chelating action specificity in appropriate buffer solution and are adsorbed more
Then polyhistidine fusion albumen is again eluted polyhistidine fusion protein by eluent appropriate, to be allowed to
It is isolated and purified.
In above-mentioned polyhistidine fusion protein isolation and purification method, preferably with the (optimization of pH=7.40 phosphate buffers
Result afterwards) it is adsorption-buffering liquid.After absorption is completed, the imidazole solution (concentration such as 1mol/L) that can be used is washed
De-, which can be directly used for subsequently in the analysis and research work of albumen after purification, such as polyacrylamide gel electrophoresis
Deng.
It is recyclable to repeat profit after carrying out fully washing ultrasound to used nickel, cobalt, manganese composite metal oxide material
With.The separation and purification of protein material has excellent repeat performance, greatly improves the utilization rate of material.
Coprecipitation is combined by the present invention with high temperature solid-state method, provides a kind of functionalization composite metal oxide material
MnCo(2-x)NixO4(x=0-1) synthetic method, this method is simple and easy to do, of low cost, morphology controllable, and repeatability is good, just
In batch production, there is very high business application potential quality.The nano-micro level spheric granules that final product is regular appearance is synthesized, is passed through
Synthesis condition is controlled, scantling can be controlled in 500nm~5 μm.By means of the chelation of metal ion and histidine, nickel,
Cobalt, manganese composite metal oxide material can be identified selectively in complicated albumen system and adsorb polyhistidine fusion egg
In vain, it is then eluted again, polyhistidine fusion protein can be desorbed from material surface.Used functionalization is compound
Metal oxide materials are high with specificity, repeat performance is good, isolates and purifies the advantages such as efficient, can be complete in 15 minutes
Pairs of hexahistine fusion protein isolates and purifies.
Description of the drawings
Fig. 1 be the embodiment of the present invention 1 prepared by nickel, cobalt, manganese composite metal oxide material scanning electron microscope
Photo；
Fig. 2 be the embodiment of the present invention 1 prepared by nickel, cobalt, manganese composite metal oxide material X ray diffracting spectrum；
Fig. 3 be embodiment 2 measure nickel, cobalt, manganese composite metal oxide material recycling performance figure, on curve
Number is the number recycled；
Fig. 4 is embodiment 3 isolated and purified using nickel, cobalt, manganese composite metal oxide material it is six poly- in cell pyrolysis liquid
The green fluorescent protein of histidine mark, with polyacrylamide gel electrophoresis detection before purification cell pyrolysis liquid (band 1), wash
Wash the result for the green fluorescent protein that hexahistine marks in liquid (band 2,3) and final eluent (band 4).
Specific implementation mode
Below in conjunction with the accompanying drawings, by embodiment, the technical solution that the present invention is further explained, but the protection model of the application
The actual conditions not by these embodiments are enclosed to be limited.
Embodiment 1：
Nickel, cobalt, manganese composite metal oxide material MnCoNiO4Synthesis, including co-precipitation, grinding, high-temperature process three
Step, specific preparation method are as follows：
(1) 10mmol NiSO are separately added into 1000mL beakers4·6H2O、CoSO4·7H2O and MnSO4·H2O, so
500mL deionized waters are added afterwards to being completely dissolved.Appropriate ammonium hydroxide is added and adjusts pH value of solution=10.Configure a concentration of 27.5 μ of 200mL
Then g/mL sodium carbonate liquors are added dropwise under condition of heating and stirring in metal salt solution at 70 DEG C, until precipitation is complete.With
Deionized water washing precipitation 3 times, gained is deposited in 70 DEG C of drying boxes 10 hours dry after filtering；
(2) agate mortar is used to be fully ground the obtained precipitation of step (1) 1 hour；
(3) by grinding obtained by step (2), adequately precipitation is fitted into crucible, is then risen to the heating rate of 2 DEG C/min
800 DEG C, 15 hours are kept the temperature, then natural cooling is to get final product MnCoNiO4。
Prepared nickel, cobalt, manganese composite metal oxide material MnCoNiO4Electron scanning micrograph such as Fig. 1 institutes
Show, which is the uniform spheric granules of pattern, and diameter is about 2 μm.X ray diffracting spectrum is as shown in Fig. 2, the material crystalline degree
Height, two-phase are simultaneously deposited, wherein a phase is Spinel, in addition one is NiO phases.The corresponding crystal face in each peak is marked in Fig. 2 respectively.
Embodiment 2：
Nickel, cobalt, manganese composite metal oxide material MnCoNiO4The yellow that hexahistine marks in separation and concentration solution
Fluorescin is as follows：
200μgMnCoNiO4The yellow fluorescence protein solution room of the hexahistine label of 20 μ g/mL of microballoon and 500 μ L
The lower rotation mixing 5min of temperature, 2min is centrifuged under 10000rpm rotating speeds, discards clear liquid.Then 200 μ L phosphorus are added into centrifuge tube
Hydrochlorate buffer solution (pH=7.40) mixes 1min, liquid is discarded supernatant after centrifugation.The washing process is repeated 2 times.Finally to centrifugation
20 μ L 1mol/L imidazole solutions are added in bottom of the tube, and rotation mixing 5min centrifuges 2min under 10000rpm rotating speeds, retains supernatant
Liquid.The supernatant is to use composite metal oxide material MnCoNiO4The yellow for being enriched with obtained hexahistine label is glimmering
Photoprotein solution.By centrifuge tube base material with after 200 μ L 1mol/L imidazole solution supersound washings 30min, according to above-mentioned step
Suddenly, the separation and concentration that can carry out the yellow fluorescence protein of hexahistine label again, can at least recycle 8 times.
MnCoNiO45min after microballoon is mixed with the yellow fluorescence protein solution that hexahistine marks, surface can be adsorbed
A large amount of yellow fluorescence protein, after the elution of 1mol/L imidazole solutions, the yellow fluorescence protein of hexahistine label is almost
It can be completely from MnCoNiO4Material surface desorbs, and material surface is nearly no detectable fluorescence signal.Fig. 3 shows
MnCoNiO4The recycling performance of material is excellent, recycles 8 times, can still keep 60% adsorption capacity.
Embodiment 3：
Nickel, cobalt, manganese composite metal oxide material MnCoNiO4Hexahistine in cell pyrolysis liquid is isolated and purified to mark
Green fluorescent protein, be as follows：
The Escherichia coli ultrasound cracking that the green fluorescent protein of hexahistine label will be expressed, then with 12000rpm
Centrifugation rate centrifuge 20min, retain supernatant.Then 200 μ gMnCoNiO are added into 200 μ L supernatants4Microballoon, room temperature
Lower rotation mixes 5min, centrifuges 2min under 10000rpm rotating speeds, discards clear liquid.Then 200 μ are added into centrifuge tube
L10mmol/L imidazole solutions mix 1min, liquid are discarded supernatant after centrifugation.The washing process is repeated 3 times.Finally to centrifuge tube bottom
20 μ L 1mol/L imidazole solutions are added in portion, and rotation mixing 5min centrifuges 2min under 10000rpm rotating speeds, retains supernatant.It should
Supernatant is the green fluorescent protein solution of the hexahistine label after isolating and purifying, and can be directly used for follow-up study.
Fig. 4 is to detect lysate, cleaning solution and final stripping liquid before purification using polyacrylamide gel electrophoresis
The result of the green fluorescent protein of middle hexahistine label.Not before purification, a large amount of non-targeted egg is contained in cell pyrolysis liquid
(band 1) in vain, after 3 washings, without apparent protein band in cleaning solution, it was demonstrated that washes clean.In stripping liquid (band
4) in, the green fluorescent protein of the label of the hexahistine after isolating and purifying is detected, and there is no apparent non-specific suctions
Attached albumen, shows MnCoNiO4There are high selectivity and specificity to the albumen of hexahistine label, it can be in the thin of complexity
The albumen that selective absorption hexahistine marks in cellular lysate liquid.
Embodiment 4：
By MnCoNiO4Material is packed into chromatographic column, to the green fluorescent protein of the hexahistine label in cell pyrolysis liquid
Mass purification is carried out, is as follows：
First by MnCoNiO4Material is packed into suitable chromatographic column as filler, with phosphate buffer solution (pH=7.40)
It is rinsed.Then the Escherichia coli for collecting the green fluorescent protein for having expressed hexahistine label, are cracked, with
Supernatant is taken after the centrifugation of 12000rpm rotating speeds.By in the supernatant loading to chromatographic column, flow control was on a 15-20mL/ hours left sides
It is right.It is washed with the phosphate buffer solution (pH=7.40) of 5 times of applied sample amount volumes, flow control was at 30mL/ hours or so.Then
It being eluted with the imidazole solution of certain gradient of 2 times of applied sample amount volumes, the concentration of imidazole solution is respectively 100mmol/L,
200mmol/L, 500mmol/L and 1mol/L.Flow control was at 15-20mL/ hours or so.Eluent is collected, determines six poly groups
Distribution situation of the green fluorescent protein of His tag in eluent.The final imidazole solution for determining suitable concentration is as elution
Liquid is chromatographed according still further to above-mentioned steps, you can the green fluorescent protein of the hexahistine label purified.This method can
Lot splitting purifies hexahistine fusion protein.
Claims (3)
1. a kind of purposes of composite metal oxide material as polyhistidine fusion protein affinity purification material, described compound
The molecular formula of metal oxide materials is MnCoNiO4, the microscopic appearance of the material is the controllable microballoon of micron-sized size, institute
Microballoon is stated by the nanocrystalline spherical hollow structure formed.
2. purposes as described in claim 1, which is characterized in that the microscopic appearance of the composite metal oxide material is diameter
The microballoon of 500nm~5 μm, the nanocrystalline size for forming the microballoon are 50~200nm.
3. purposes as claimed in claim 1 or 2, which is characterized in that in appropriate buffer solution, the metal composite oxide
Material is identified by metal-chelating action specificity and adsorbs polyhistidine fusion protein, then passes through eluent appropriate again
Polyhistidine fusion protein is eluted, realizes isolating and purifying for polyhistidine fusion protein.
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| 孙永亮等.纯化组氨酸标签蛋白金属螯合亲和色谱填料的制备与性能.《陕西师范大学学报（自然科学报）》.2007,第35卷(第2期), * |
| 李琼.固定化金属离子螯合琼脂糖凝胶的制备及其纯化组氨酸标签蛋白的研究.《中国优秀硕士学位论文全文数据库工程科技Ⅰ辑》.2012,(第7期), * |
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