CN1431497A - Method of using nano zeolite molecular sieve assemble material as affinity chromatography filler to separate functional protein molecules - Google Patents
Method of using nano zeolite molecular sieve assemble material as affinity chromatography filler to separate functional protein molecules Download PDFInfo
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- CN1431497A CN1431497A CN 03115232 CN03115232A CN1431497A CN 1431497 A CN1431497 A CN 1431497A CN 03115232 CN03115232 CN 03115232 CN 03115232 A CN03115232 A CN 03115232A CN 1431497 A CN1431497 A CN 1431497A
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Abstract
In the invention, with being fixed in macropore-micropore multistage ordered zeolite structure through the ion exchnage process, the transition metal ions are as the affiliation active spots where the distinctive combination is occurred with the functional biomacromolecules possesisng the sequential histidine structure. Then, using the gradient elution through the buffer solution to reach the goal of enrich and separation. The carrier provides the advantages of even pore passages, good mechanical and chemical stability, easy to modify the surfaces etc. The diatomaceous earth is as the molding board and the molecular sieve of nano Beta zeolite wit surface being modified is as affinity chromatography filler. With the column being filled with the said materials, the satisfying result for separating the functional protein molecules is obtained in different leaching systems.
Description
Technical field
The present invention utilizes the nanosized zeolitic material with the multilevel ordered structure of macropore-micropore, pass through cation exchange, after methods such as chemogenic deposit and surface chemistry grafting are carried out modification to its surface, optionally carry out the separation of functional protein molecule in the complex biological system (as through ion-exchange fixing metal ions Co as a kind of filling carrier of novel affinity chromatography
2+After, be rich in the selenoprotein-P of histidine structure etc. in the affine separation mice plasma).
Background technology
Along with finishing that the human genome total order is measured, the structure of setting forth its coded protein has become the new forward position of life science in function.About nineteen ninety-five, some far-sighted scientists propose the notion of " back genome " in good time.Promptly after the base sequence of genome static state is understood, change the i.e. research of " functional genomics " of research over to the dynamic biological function of genome.
New ideas by the proteomics of propositions such as Australian scientist Wilkins in 1994 are new growing points of genome times afterwards comprehensively (Post-Genome Era) life science.It is the quantitative examination by the whole protein of pair cell on protein level or body gene expression, with process that discloses life and the mechanism of explaining gene expression control.China since last year also be entitled as 973 projects of " proteomics research of human major disease "; one of its research emphasis is the corresponding significant peptide section that has special site of screening function albumen; set up the technical system of protein group component spectrum, the chain spectrum of protein function and the protein function screening of high flux, scale, high sensitivity, high accuracy, and be useful for major disease related protein group's research.One of core topic in this field is to set up biological complex sample protein matter group high efficiency separation, scale authenticate technology and new method.
Zeolite molecular sieve has bigger serface, high hydrothermal stability enriches the nano-pore of homogeneous, good ion-exchange performance and enrich adjustable surface nature, and advantage such as good mechanical stability, there is report to use zeolitic material recently and carries out the separation of amino acid molecular.But to macromolecular system, because traditional its aretation of zeolitic material, diffusional resistance is bigger, and usable range is restricted.Nano molecular sieve not only has most of characteristic of big crystal grain zeolite molecular sieve, and owing to have unique modifiable active outside surface, short diffusion admittance, the ion exchangeable of outside surface, unique absorption of nanometer aperture and reactivity worth, relating to the deficiency that remedies the latter aspect macromolecular chemistry, material and the bioseparation technology.Its external surface area will enlarge markedly after the zeolite size enters nanoscale, by nanometer assembling can obtain the to have various macro morphologies porosint of the assembly hole that contains multilevel ordered macropore or mesoporous yardstick of (as pipe, ball, film and block).This material enriches and controlled outside surface and the assembly hole thereof of character based on it, the biomacromolecule high-affinity is combined with high selectivity is reversible, might be as a class good rigidly, macro morphology is controlled, easy derivatization, the effective parting material that is fit to functional protein under the higher flow velocity, realize that the lip-deep biological function site-specific of target protein also reversibly combines with affinity ligand fixing in the parting material, make the effective enriching and purifying of target molecule in the biological complex system, satisfying the requirement of essential affinity chromatography of proteomics research and micro column high efficiency liquid phase chromatograph technology such as (μ HPLC), and realize the coupling of multiple separate analytical techniques such as microtrabeculae affinity chromatography-μ HPLC-ESI-MS and microtrabeculae affinity chromatography-CE-ESI-MS.Have not yet to see the report of such research method.
Summary of the invention
The objective of the invention is to set up the zeolite molecular sieve material of applying nano assembling carries out the affine separation of functional protein molecule for the affinity chromatography filler carrier new system.
For traditional affinitive material, the selected material of the present invention have the duct even, with low cost, be easy to regeneration, stable in properties, chemical physics good mechanical stability, and be satisfied with the requirement that the screenings of extensive biomacromolecules such as μ HPLC/CE separates.Material makes transition metal ion such as Co through ion exchange process
2+On be fixed in material surface, and utilize the histidine structure that is rich in the structure of some functional protein molecule, mutual complexing with transition metal ion fixing in imidazole ring in the histidine structure side chain and the zeolitic material, have the effect of the protein molecular that enriches the histidine structure with the specific absorption combination of performance transition metal ion, thereby make the functional protein molecule be able to from the living things system of complexity, obtain separate.
The method of technology of the present invention is as follows: (inserts concrete preparation method be that China applies for a patent 02132609.3)
1, selects to have the nanosized zeolitic material of the multilevel ordered structure of macropore-micropore as affine isolated vectors.
2, the nano zeolite of the multi-stage artery structure of some assembling porosint makes transition metal ion be fixed in the material by ion exchange process with certain density transition metal ion such as cobalt nitrate solution reflux.
3, the parting material wet method that processing is obtained is packed in the cylinder.
4, utilize deionized water drip washing cylinder repeatedly.
5, balance cylinder: the plasma sample with animal is an example, available 0.05moll
-1Tris-HCl solution/1.0moll
-1NaCl solution/phosphate buffered solution balance cylinder.
6, after elution process, sample are adsorbed on the post, to remove the material of non-specific binding, when uv absorption detects the baseline that reaches original, finish drip washing with the initial buffer solution drip washing post of 3-5 times of column volume.Selecting best elution requirement that the target protein molecule is reached in the gradient buffer liquor capacity of minimum more completely reclaims.
Actual conditions is as follows:
The nanosized zeolitic material of the multi-stage artery structure that 1, obtains, is being packed in the affinity chromatographic column with wet method fixedly behind transition metal ion such as Co (II) plasma through ion exchange process.This chromatographic column can be made by oneself, also can be commercial pillar.
2, utilize nearly neutral 0.05moll
-1Tris-HCl solution/1.0moll
-1NaCl solution/phosphate buffered solution (3-5 doubly column volume) balance pillar, balance is generally used the solution or the close solution of sample dissolution.
3, sample to be separated is added in the top of pillar.And about about 20-40 minute time of process, make sample to be separated with the abundant combination of the material in the post.Sample concentration is in the 0.1-10mg/ml scope, and high concentration is used for Protein Separation, to raise the efficiency.
4, utilize leacheate such as formic acid-ammonium formate buffer solution in initial pH value 7.0,, remove the material of non-specific binding in cylinder with 2-3 column volume drip washing cylinder doubly.
5, in gradient scope, drip washing cylinder repeatedly in the velocity range of 10-50ml/hr makes the target protein molecule be about at pH at 4.0 o'clock, is leached out specifically.Can suitably pressurize to improve flow velocity.
6, the collection effluent to obtaining is followed the tracks of evaluation target protein molecule with atomic absorption spectrum or electrophoretic techniques.
7, can make packing material obtain repeated regeneration by roasting.
Leacheate of the present invention is with following any equal result that can obtain satisfaction.Formic acid-ammonium formate buffer solution, perhaps acetic acid-sodium acetate buffer solution, phosphoric acid-buffer solution of sodium phosphate or imidazoles solution.Because such leacheate optionally will elute with the target protein molecule of transition metal ion complexing, thereby reach the purpose of separation and concentration.
The cylinder length and the internal diameter of separating column can influence separation efficiency.Column length can 0.5-5m, and internal diameter can 0.5-20cm, is used for separating using longlyer, and the pillar that bore is big is to raise the efficiency.
Above-mentioned separating column can be many short column series connection, be 3m as every 0.5m post series connection, or 3 1m posts is in series.
Collect the wash-out component, can adopt electrophoresis, atomic spectrum, methods such as ultra-violet absorption spectrum detect protein molecular.
The filling material of the inventive method can obtain regeneration by 400 ℃-600 ℃ roasting, uses repeatedly, reduces cost.
The nanosized zeolitic material of multistage pore canal has that the duct is even, external surface area is big, stable in properties, advantage such as with low cost, and its favorable mechanical physical property can satisfy the requirement of microtrabeculae HPLC/CE.
With the inventive method and high performance liquid chromatography or gas chromatography coupling such as multiple isolation technics couplings such as micro column high efficiency liquid phase chromatograph μ HPLC or μ HPLC-ESI-MS, the application of the inventive method will be expanded further.
Traditional affinity chromatography packing material adopts agarose, sephadex etc. as supporting body more, not only costs an arm and a leg, and also not high pressure resistant, after surpassing certain limit, column pressure, stops up cylinder very easily because of crimp, influence separation efficiency.Behind the filling material of the nanosized zeolitic material of multistage pore canal of the present invention as affinity chromatography, its favorable mechanical physical and chemical stability can effectively remedy the deficiency of above-mentioned traditional material; Its relative shirtsleeve operation method has good popularizing application prospect to the separation and concentration of protein molecule.Simultaneously it makes things convenient for the character of repeated regeneration and can introduce the characteristics of other function affinity groups simultaneously by technology such as molecule graftings, will expand the range of application of material in affine separation of this type greatly.
Description of drawings
Fig. 1 is the electron-microscope scanning figure of natural diatomaceous earth material under the low resolution.The pattern of natural diatomaceous earth is the about 1.2 μ m of thickness, the round pie material of internal diameter 20-40 μ m.
Fig. 2 is the electron-microscope scanning figure of natural diatomaceous earth material under the high resolving power.Be evenly distributed with the duct of aperture 300-500nm as can be seen on it.
Fig. 3 represents then that by Electrostatic Absorption one deck nanometer β zeolite is modified the electron-microscope scanning figure in the diatomaceous surface of this kind equably.
Fig. 4 is expression metallic ion Co
2+Enter into the principle schematic of ad-hoc location of the structural framework of nanosized zeolitic material by ion exchange process.
Fig. 5 is fixed on transitional metal ion Co on the carrier of separating for expression by electrostatic interaction
2+Contain the process synoptic diagram of the mutual chelating of target protein molecule of continuous histidine residues with the surface.The effect of this optionally specific bond mainly depends on the same mutual chelating that is fixed on the transition metal ion in the parting material of imidazole ring in the histidine structure continuous in the target protein molecule.
Fig. 6 represents with homemade affine separating column, selects the installation drawing of target protein-selenoprotein-P in the Beta nano zeolite mice plasma that affine separation and concentration experiment is cultivated as filling material.
Fig. 7 forms the mixtures of polypeptides separating resulting figure of structure for the different histidines of having of example 1.The peptide molecule that shows two kinds of synthetic with different continuous histidine structures is an outflow process in the affinity chromatographic column of filling material at the zeolite molecular sieve with multilevel hierarchy.Selecting formic acid-ammonium formate buffer solution in the process is leacheate.The polypeptide B that (the pH distribution range is seen on the figure) as can be seen from the figure has 4 continuous histidine structures than polypeptide A (containing 2 histidines in the structure) with the Co in the column material
2+In conjunction with more firm, thus the delivery time of polypeptide B be later than polypeptide A.
Fig. 8 is the separating resulting figure of example 2.Show mice plasma sample through specific culture, through homemade be that filling material carries out affinity chromatography and separates with multistage zeolite molecular sieve material.And flow out the concentration of the selenium element in the component with alkaline fusion-alkaline hydride generation-ICPAES coupling technique analysis, determine the detachment process of target protein molecule indirectly.As can be seen from the figure component 10 and 11 is about near 4.0 the selenoprotein-P that is corresponding to leacheate pH.And can further be proved by the band that is separated near the obvious intensification the molecular weight 55-60kDa of SDS-PAGE.
Fig. 7, Fig. 8 show to have selenoprotein-P of enriching the histidine structure and the peptide molecule with continuous histidine structure of synthetic can obtain good separation graph by above-mentioned affine pattern.
Embodiment
Below example be nanosized zeolitic material to multistage pore canal provided by the present invention further specifying of carrying out that the affinity chromatography detachment process done.The separation of example 1 histidine or polypeptide
With the filler of the homemade decorated nanometer Beta of 0.1g zeolite appropriate amount of deionized water furnishing slurry, to transfer in the separating column, the pillar bed height is about 1cm.Add 1.0ml equalizing and buffering solution then pillar is carried out balance.With 0.50ml concentration is 124 μ gL
-1Histidine or 1.0mL concentration be 1.5mgL
-1Polypeptide solution sample (sample is the peptide molecule of different histidine structures of containing of synthetic) be moved in the extraction column, treat that the clean back of flow of liquid adds 1.0ml equalizing and buffering solution washing, the histidine or the polypeptide that are adsorbed onto on the pillar are 7.0 by adding pH successively, 6.0,5.0,4.0 formic acid-ammonium formate elution buffer solution gradient elution, drip washing speed is 10ml/hr, detect absorbance value in each collection tube by ultraviolet spectrum then, and draw the effluent distribution plan.Example 2-5
The bed height of adjusting pillar is respectively 1.5,2.0,2.5,3.0cm, and other condition is the same, repeats above-mentioned separating experiment.Example 6-8
Select formic acid-ammonium formate buffer solution system or sodium phosphate-phosphate buffer solution or imidazoles gradient solution to carry out the separation of peptide molecule potpourri as elution system respectively, repeat example 1 step (contain different histidine structures respectively among the peptide molecule A of synthetic and the B, and by 1: 1 mixed in molar ratio).The separation of selenoprotein-P in example 9 mice plasma
The filler of 0.5g being made by oneself decorated nanometer Beta zeolite is transferred in the separating column with appropriate amount of deionized water furnishing slurry, and the pillar bed height is about 1cm.Add 4.0ml equalizing and buffering solution then pillar is carried out balance.Freezing blood plasma is thawed the back at 4000 revolutions per seconds in room temperature, centrifugal 5min, and the membrane filtration by 0.45 μ m.The 2.0ml plasma sample is moved in the extraction column, treat that the clean back of flow of liquid adds 4.0ml equalizing and buffering solution washing, the selenoprotein P that is adsorbed onto on the pillar filler washes out by adding elution buffer solution gradient, drip washing speed is 30ml/hr, (concrete elution mode is identical with separating of histidine and polypeptide).Dripping about 200 μ l concentration in the component of each acceptance is 2.0M HCl, detect total Se content in each pipe by alkaline fusion-alkaline pattern HG-ICP-AES then, and in conjunction with electrophoretic separation technique, with the specific isolation usefulness of definite affine piece-rate system of being set up to selenoprotein-P.Example 10-11
Selecting through the mice plasma of specific culture and the plasma sample of Shaoxing sheldrake respectively is experimental subjects, repeats example 9 steps, separation and concentration selenoprotein-P wherein.Example 12-15
Select formic acid-ammonium formate buffer solution system respectively; Sodium phosphate-phosphate buffer solution, imidazoles gradient solution be as elution system, repeats example 9 steps, carries out the separation and concentration of selenoprotein-P in the plasma sample of mice plasma and Shaoxing sheldrake.Example 16-18
In material support by ion exchange process fixing transition metal ion Zn respectively
2+, Cu
2+, Ni
2+, repeat example 9 steps, carry out the affine detachment process of selenoprotein-P in the mice plasma.Example 19-24
Select the surface to be modified with dissimilar nanometer LTA, FAU, MFI zeolite molecular sieve respectively, repeat example 1 and example 9,, and investigate its different affinity chromatography behavior with the exchange capacity of definite its metallic ion.Example 25
After the material surface modifying of the multistage pore canal zeolite of nanometer assembling is modified gallium ion, has the casein that enriches phosphate groups with separation and concentration.The elute soln of selecting is the imidazoles solution in a series of gradient scope.Example 26
Utilize the exchange capacity of the selected parting material of plasma spectrometry-analytical reagent composition.Promptly after cobalt nitrate solution is handled with material reflow, to obtain the material dress post of modification, and, collect the effluent that obtains with 4M HCl solution drip washing cylinder, determine the absolute concentration of cobalt ions with plasma spectrometry-analytical reagent composition, and calculate the exchange capacity of material.The result shows that the exchange capacity of Beta nano zeolite is about 0.1%.Example 27-28
Compare of the influence of the affine separating column of unlike material to separating effect, as quartz glass, plastics, teflon etc.
Claims (6)
1, the molecular sieve of Nano zeolite assembled material is as the method for affinity chromatograph filling separation function protein molecule, it is characterized in that having the multilevel ordered parting material of molecular sieve of Nano zeolite with finishing, through ion-exchange fixedly behind the transition metal ion, as the affinity chromatography column packing, and the buffer solution drip washing cylinder by certain gradient scope, reach the purpose of separation and concentration protein molecule, actual conditions is:
(1) nano zeolite is evenly modified the surface in porous matrix material, and by ion exchange process, transition metal ion is fixed in the material; Filling carrier dress post as affine separation;
(2) balance packed column cylinder, and will treat that test sample places the top of post bed;
(3) weak acid buffer solution or the imidazoles solution with certain gradient scope is optionally eluted the target protein molecule under the normal temperature;
(4) existence of evaluation target protein molecule.
2, by the described molecular sieve of Nano zeolite assembled material of claim 1 as affinity chromatograph filling separation function method of protein, it is characterized in that leacheate is any in following: formic acid-ammonium formate buffer solution; Acetic acid-sodium acetate buffer solution; Phosphoric acid-buffer solution of sodium phosphate; Or imidazoles solution.
3, press the described molecular sieve of Nano zeolite assembled material of claim 1 as affinity chromatograph filling separation function method of protein, it is characterized in that separating column is series connection.
4, the porosint by the described molecular sieve of Nano zeolite assembling of claim 1 is the filling material method of separating protein of affinity chromatography, it is characterized in that the component of collecting can detect with following any method: electrophoresis; Ultra-violet absorption spectrum; Atomic spectrum.
5, press the described molecular sieve of Nano zeolite assembled material of claim 1 as affinity chromatograph filling separation function method of protein, its feature filling material can be regenerated after roasting or ion-exchange.
6, press the described molecular sieve of Nano zeolite assembled material of claim 1 as affinity chromatograph filling separation function method of protein, it is characterized in that the pattern of the inventive method applicable to high performance liquid chromatography and gas chromatography.
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100374857C (en) * | 2006-01-26 | 2008-03-12 | 复旦大学 | Method for fast enriching trace polypeptide and protein and realizing identification |
| CN100374858C (en) * | 2006-03-30 | 2008-03-12 | 复旦大学 | Method for simultanuously enriching desalting and appraising micro protein or polypeptide solution |
| CN1868576B (en) * | 2005-05-24 | 2010-10-27 | 黄亮 | Biological modification infusorial earth adsorbent, and its prepn. method |
| CN107456780A (en) * | 2017-08-07 | 2017-12-12 | 中国食品发酵工业研究院 | It is a kind of to be used for solid-phase extraction column of saturation mineral hydrocarbon oil measure and its preparation method and application in food |
| CN111530125A (en) * | 2015-07-20 | 2020-08-14 | W.L.戈尔及同仁股份有限公司 | Affinity chromatography device |
| CN112939016A (en) * | 2021-03-10 | 2021-06-11 | 成都理工大学 | Chain-shaped ZSM-5 micro mesoporous molecular sieve formed by egg protein induction and synthesis method thereof |
| CN113457214A (en) * | 2021-06-08 | 2021-10-01 | 上海海路生物技术有限公司 | Solid phase extraction system and method combining syringe type gravity column and solid phase extractor |
| WO2023040006A1 (en) * | 2021-09-14 | 2023-03-23 | 谱天（天津）生物科技有限公司 | Method for increasing mass spectrum identification number of protein and/or peptide fragment group |
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| CN100439918C (en) * | 2006-06-29 | 2008-12-03 | 复旦大学 | Method for enriching, desalting protein or polypeptide in minute quantities, and carrying out analysis directly |
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Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1868576B (en) * | 2005-05-24 | 2010-10-27 | 黄亮 | Biological modification infusorial earth adsorbent, and its prepn. method |
| CN100374857C (en) * | 2006-01-26 | 2008-03-12 | 复旦大学 | Method for fast enriching trace polypeptide and protein and realizing identification |
| CN100374858C (en) * | 2006-03-30 | 2008-03-12 | 复旦大学 | Method for simultanuously enriching desalting and appraising micro protein or polypeptide solution |
| CN111530125A (en) * | 2015-07-20 | 2020-08-14 | W.L.戈尔及同仁股份有限公司 | Affinity chromatography device |
| CN111530125B (en) * | 2015-07-20 | 2022-03-04 | W.L.戈尔及同仁股份有限公司 | Affinity chromatography device |
| CN107456780A (en) * | 2017-08-07 | 2017-12-12 | 中国食品发酵工业研究院 | It is a kind of to be used for solid-phase extraction column of saturation mineral hydrocarbon oil measure and its preparation method and application in food |
| CN112939016A (en) * | 2021-03-10 | 2021-06-11 | 成都理工大学 | Chain-shaped ZSM-5 micro mesoporous molecular sieve formed by egg protein induction and synthesis method thereof |
| CN113457214A (en) * | 2021-06-08 | 2021-10-01 | 上海海路生物技术有限公司 | Solid phase extraction system and method combining syringe type gravity column and solid phase extractor |
| CN113457214B (en) * | 2021-06-08 | 2022-10-04 | 上海海路生物技术有限公司 | Solid phase extraction system and method combining syringe type gravity column and solid phase extractor |
| WO2023040006A1 (en) * | 2021-09-14 | 2023-03-23 | 谱天（天津）生物科技有限公司 | Method for increasing mass spectrum identification number of protein and/or peptide fragment group |
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