CN104130309A - Pretreatment method used for improving purity of purified protein - Google Patents
Pretreatment method used for improving purity of purified protein Download PDFInfo
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- CN104130309A CN104130309A CN201310756622.2A CN201310756622A CN104130309A CN 104130309 A CN104130309 A CN 104130309A CN 201310756622 A CN201310756622 A CN 201310756622A CN 104130309 A CN104130309 A CN 104130309A
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Abstract
The invention relates to a pretreatment method used for improving the purity of purified protein, which belongs to the technical field of biological medicine. The invention provides optimized steps for protein purification and a protein adsorption material involved in the steps. The main point of the optimized steps for protein purification is that a non-directional protein adsorption carrier provided by the invention is used to remove impurity protein after pretreatment and before crude grading or during crude separation, which is beneficial for subsequent separation and purification and enables purity of a purified product to be improved. The non-directional protein adsorption carrier may be elementary graphene or a compound of graphene and a nano-material; the form of the non-directional protein adsorption carrier may be powder, a two-dimensional plane or a three-dimensional sponge; and the non-directional protein adsorption carrier may be in a solid state or a solution state or in the state of a dispersion liquid. The pretreatment method is applied to the field of protein purification.
Description
Technical field:
The present invention relates to protein separation field, definite says, relates to a kind for the treatment of process of protein extract and the protein adsorbent that the method relates to.
Background technology:
The separation and purification of protein is used extensively in biochemical research application, is an important operative technique.A typical eukaryotic cell can comprise thousands of different proteins, and some content are very abundant, and some only contain several copies.In order to study some protein, must be first by this protein, from other protein and nonprotein molecule, purifying is out.
The general procedure of a certain specified protein of separation and purification can be divided into pre-treatment, rough classification separates and separates three steps with subfractionation.
Pre-treatment, also claims pre-treatment:
Certain protein of separation and purification first will discharge protein keep original native state with the state dissolving from original tissue or cell, does not lose biological activity.Then according to different situations, select appropriate means, will organize and cytoclasis.After tissue and cytoclasis, select suitable damping fluid desired protein extraction out.The insolubless such as cell debris are removed by method centrifugal or that filter.If desired albumen mainly concentrates on a certain cellular component, as nucleus, karyomit(e), rrna or soluble cell matter etc., can utilize the method for differential centrifugation that they are separated, collect the material of this cellular component as lower step purifying.Be combined with cytolemma or membranous organoid if run into wanted albumen, must utilize ultrasonic wave or stain remover to make membrane structure depolymerization, then extract and obtain extracting solution of protein with suitable medium.
Rough classification separates:
After extracting solution of protein (sometimes also mix and have nucleic acid, polysaccharide and so on) obtains, select a set of appropriate means, desired albumen and other foreign proteins are separated.The separation of general this step with saltouing, the method such as isoelectric precipitation and organic solvent fractional separation.The feature of these methods be easy, treatment capacity is large, can remove a large amount of impurity, again can protein concentrate solution.Some protein extract volume is larger, is unsuitable for again concentrating with precipitation or salting-out process, can adopt ultra-filtration, gel-filtration, vacuum freezedrying or additive method to concentrate.
Subfractionation separates:
Sample after rough classification separates, general small volume, foreign protein major part is removed.Be further purified, generally use chromatography to comprise gel-filtration, ion exchange chromatography, adsorption chromatography and affinity chromatography etc.Also can select if desired electrophoretic method, comprise that zone electrophoresis, isoelectric focusing etc. are as last purification step.The general scale of method separating for subfractionation is less, but resolving power is very high.
In real work, even all adopt optimal separation purification method in above-mentioned three steps, be also difficult to obtain highly purified protein.Especially impurity albumen or the contaminating protein of character the unknown, existing technology is to be difficult to one step to remove, and can only take a series of step; And a small amount of existence of these impurity albumen, contaminating protein also can produce impact greatly to the property research of target protein.Therefore, researcher and technologist are also improving energetically existing purification process or are developing new purifying process, constantly promote proteinaceous research with this; The also progress of pusher's separation and purification of protein technology conversely of property research of protein simultaneously.
Summary of the invention:
Juche idea of the present invention:
In above-mentioned albumen sepn step, be mainly after pre-treatment, before rough classification, the non-directional protein adsorption carrier that adopts the present invention to propose, removes impurity albumen, is conducive to next step separation and purification, improves the purity of protein purification product.
The technical scheme the present invention relates to:
Graphene is the novel nano-material of just having found in recent years, has high specific surface area, and theoretical specific surface area is up to 2630m
2/ g.And there is good absorption property and good mechanical property, also obtain good Application and Development as sorbing material.In addition, the synergistic effect between Graphene and nano material makes this matrix material that contains bigger serface have larger superiority than single nano material, and preparing nano adsorption material taking Graphene as carrier also becomes current research focus.
Contriver carries out graphene functionalized modification and Application Areas for a long time.In experimental study, find that the mixture of Graphene or Graphene and other nano material has good non-directional adsorption to protein, all kinds of impurity protein of absorption that can be quick, a large amount of.On the basis of this discovery, contriver, through test of many times, has optimized the step of protein purification, after pre-treatment, before rough classification, or among roughing out, use above-mentioned Graphene sorbing material to process cell pyrolysis liquid or cell culture supernatant, effectively improve the purity of protein purification product.
That is: the invention provides a kind of optimization step of protein purification process, and the protein adsorbent that relates to of this step.
1, the optimization step of protein purification of the present invention, is mainly after pre-treatment, before rough classification, or in roughing out process, the non-directional protein adsorption carrier that adopts the present invention to propose, remove impurity albumen, be conducive to next step separation and purification, improve the purity of protein purification product.
2, non-directional protein adsorption carrier of the present invention, is characterized in that, can be Graphene simple substance, can be also the mixture of Graphene and nano material; Its form can be Powdered, can be two dimensional surface shape, can be also three-dimensional sponge shape; Its existence can be solid-state, can be also solution state or dispersion liquid.
3, the non-directional protein adsorption carrier described in above-mentioned 2, its characteristic is: single carrier is of a size of 10nm to 100 micron.
4, the mixture of the Graphene described in above-mentioned 2 and nano material, is characterized in that: described nano material comprises a kind of or any two kinds and the two or more mixtures in following material: metal nanoparticle, as gold and silver, aluminum oxide, ferric oxide; Semiconductor nano, as CdTe; Carbon nanotube; Nano nonmetal powder or solution; Macromolecular material.
5, the protein purification step of optimization of the present invention is:
(1) prepare Graphene or graphene/nanometer Material cladding material, as non-directional absorption carrier;
(2) carry out the preprocessor of protein purification, obtain cell pyrolysis liquid, or cell culture supernatant;
(3) carrier above-mentioned steps 1 being obtained with in the cell pyrolysis liquid of step 2 or cell culture supernatant mix, wherein Graphene quality is 1% of cell pyrolysis liquid or cell culture supernatant solution quality---20%;
(4) above-mentioned mixed solution is stirred to 15min---1h, make Graphene fully adsorb magazine protein;
(5) mixed solution after absorption impurity is used to filter paper or membrane filtration;
(6) proceed the later separation step of protein.
Advantage of the present invention:
The present invention utilizes the specific surface area that Graphene is huge, and the good feature of absorption property, it is used separately or with the reasonable compound assembling of nano material, can play the effect of albumen non-directional absorption.In albumen sepn process, be mainly after pre-treatment, before rough classification by this material application, a step is removed plurality of impurities albumen, is conducive to next step separation and purification, improves the purity of protein purification product.It is simple that this material has preparation, the advantage of stable in properties; And environmentally friendly, with low cost; Quick adsorption albumen in a large number; And use simple to operately, not needing operator to possess very high scientific and technological level can operate.
Embodiment:
Below by specific embodiment, the present invention is described further.
The method for purifying proteins that adopts Graphene/golden nanometer particle matrix material to carry out non-directional absorption:
(1) prepare Graphene/gold nano-material composite wood, as non-directional absorption carrier: use amino epoxy addition reaction to prepare extra small size graphene oxide (<500nm), Graphene size prepared by the method on probation is little, and toxicity is little, is convenient to modify; Being used as underlying carrier with this Graphene, to carry out golden nanometer particle compound;
(2) carry out the preprocessor of protein purification: will organize and cytoclasis, and select suitable damping fluid desired protein extraction out.The insolubless such as cell debris are removed by method centrifugal or that filter.If desired albumen mainly concentrates on a certain cellular component, as nucleus, karyomit(e), rrna or soluble cell matter etc., can utilize the method for differential centrifugation that they are separated, collect the material of this cell pyrolysis liquid as lower step purifying;
(3) carrier above-mentioned steps 1 being obtained mixes with the cell pyrolysis liquid of step 2, and wherein Graphene quality is 1%20% of cell pyrolysis liquid or cell culture supernatant solution quality;
(4) above-mentioned mixed solution is stirred to 15min1h, make Graphene fully adsorb magazine protein;
(5) by and attached impurity after mixed solution use filter paper or membrane filtration;
(6) proceed the later separation step of protein, finally obtain the target protein of purifying.
Claims (5)
1. the optimization step of a protein purification process, and the protein adsorbent that relates to of this step, its feature is: be mainly after pre-treatment, before rough classification, or in roughing out process, the non-directional protein adsorption carrier that adopts the present invention to propose, remove impurity albumen, be conducive to next step separation and purification, improve the purity of protein purification product.
2. non-directional protein adsorption carrier according to claim 1, is characterized in that, can be Graphene, graphene oxide, reduced graphene, can be also the mixture of Graphene and nano material; Its form can be powdery, can be two dimensional surface shape, can be also three-dimensional sponge shape; Its existence can be solid-state, can be also solution state or dispersion liquid.
3. non-directional protein adsorption carrier according to claim 1, its characteristic is: single carrier is of a size of 10nm to 100 micron.
4. the mixture of Graphene according to claim 2 and nano material, it is characterized in that: described nano material comprises a kind of or any two kinds and the two or more mixtures in following material: metal nanoparticle, as gold and silver, aluminum oxide, ferric oxide; Semiconductor nano, as CdTe; Carbon nanotube; Nano nonmetal powder or solution; Macromolecular material.
5. the protein purification step of optimization according to claim 1 is:
(1) prepare Graphene or graphene/nanometer Material cladding material, as non-directional absorption carrier;
(2) carry out the preprocessor of protein purification, obtain cell pyrolysis liquid, or cell culture supernatant;
(3) carrier above-mentioned steps 1 being obtained with in the cell pyrolysis liquid of step 2 or cell culture supernatant mix, wherein Graphene quality is 1% of cell pyrolysis liquid or cell culture supernatant solution quality---20%;
(4) above-mentioned mixed solution is stirred to 15min---1h, make Graphene fully adsorb magazine protein;
(5) by using Graphene to carry out the mixed solution membrane filtration after impurity absorption, obtain cell pyrolysis liquid after treatment or supernatant liquor;
(6) proceed the later separation step of protein.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104759269A (en) * | 2015-03-31 | 2015-07-08 | 苏州佰锐生物科技有限公司 | Preparation method of graphene microsphere biological separation medium with controllable particle size |
| CN105669827A (en) * | 2014-11-20 | 2016-06-15 | 中国科学院高能物理研究所 | Application of oxidized graphene as protein adsorption medium material and protein separation method |
| CN106622120A (en) * | 2015-10-30 | 2017-05-10 | 江南石墨烯研究院 | Graphene-based protein adsorption sponge |
| CN111548784A (en) * | 2020-05-13 | 2020-08-18 | 合肥福纳科技有限公司 | Post-processing method and preparation method of quantum dots, prepared quantum dots and application |
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| CN102507953A (en) * | 2011-10-20 | 2012-06-20 | 济南大学 | Preparation method of electrochemistry immunosensor for determining alpha fetoprotein |
| CN102895938A (en) * | 2012-11-13 | 2013-01-30 | 武汉大学 | Preparation method of graphene covered silica gel |
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- 2013-12-25 CN CN201310756622.2A patent/CN104130309A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102507953A (en) * | 2011-10-20 | 2012-06-20 | 济南大学 | Preparation method of electrochemistry immunosensor for determining alpha fetoprotein |
| CN102895938A (en) * | 2012-11-13 | 2013-01-30 | 武汉大学 | Preparation method of graphene covered silica gel |
Non-Patent Citations (2)
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| QU W-D等: "Far-Infrared-Assisted Preparation of a Graphene-Nickel Nanoparticle Hybrid for the Enrichment of Proteins and Peptides", 《CHEMISTRY-A EUROPEAN JOURNAL》 * |
| WAN D.等: "A rapid and simple separation and direct detection of glutathione by gold nanoparticles and graphene-based MALDI-TOF-MS", 《JOURNAL OF SEPARATION SCIENCE》 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105669827A (en) * | 2014-11-20 | 2016-06-15 | 中国科学院高能物理研究所 | Application of oxidized graphene as protein adsorption medium material and protein separation method |
| CN104759269A (en) * | 2015-03-31 | 2015-07-08 | 苏州佰锐生物科技有限公司 | Preparation method of graphene microsphere biological separation medium with controllable particle size |
| CN106622120A (en) * | 2015-10-30 | 2017-05-10 | 江南石墨烯研究院 | Graphene-based protein adsorption sponge |
| CN111548784A (en) * | 2020-05-13 | 2020-08-18 | 合肥福纳科技有限公司 | Post-processing method and preparation method of quantum dots, prepared quantum dots and application |
| CN111548784B (en) * | 2020-05-13 | 2023-06-06 | 合肥福纳科技有限公司 | Post-treatment method of quantum dot, preparation method of quantum dot, prepared quantum dot and application of quantum dot |
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Application publication date: 20141105 |