CN107899552B - Metal chelating affinity chromatography medium using magnetic polymer microsphere as matrix - Google Patents
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- 239000004005 microsphere Substances 0.000 claims abstract description 32
- 239000012501 chromatography medium Substances 0.000 claims abstract description 28
- 238000001042 affinity chromatography Methods 0.000 claims abstract description 27
- 229910021645 metal ion Inorganic materials 0.000 claims abstract description 26
- 229920000642 polymer Polymers 0.000 claims abstract description 25
- 239000002184 metal Substances 0.000 claims abstract description 20
- 229910052751 metal Inorganic materials 0.000 claims abstract description 20
- SZVJSHCCFOBDDC-UHFFFAOYSA-N ferrosoferric oxide Chemical compound O=[Fe]O[Fe]O[Fe]=O SZVJSHCCFOBDDC-UHFFFAOYSA-N 0.000 claims abstract description 18
- RQFQJYYMBWVMQG-IXDPLRRUSA-N chitotriose Chemical compound O[C@@H]1[C@@H](N)[C@H](O)O[C@H](CO)[C@H]1O[C@H]1[C@H](N)[C@@H](O)[C@H](O[C@H]2[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O2)N)[C@@H](CO)O1 RQFQJYYMBWVMQG-IXDPLRRUSA-N 0.000 claims abstract description 17
- IXPNQXFRVYWDDI-UHFFFAOYSA-N 1-methyl-2,4-dioxo-1,3-diazinane-5-carboximidamide Chemical compound CN1CC(C(N)=N)C(=O)NC1=O IXPNQXFRVYWDDI-UHFFFAOYSA-N 0.000 claims abstract description 16
- 239000001913 cellulose Substances 0.000 claims abstract description 16
- 229920002678 cellulose Polymers 0.000 claims abstract description 16
- 235000010413 sodium alginate Nutrition 0.000 claims abstract description 16
- 239000000661 sodium alginate Substances 0.000 claims abstract description 16
- 229940005550 sodium alginate Drugs 0.000 claims abstract description 16
- 239000002131 composite material Substances 0.000 claims abstract description 14
- 229920002401 polyacrylamide Polymers 0.000 claims abstract description 14
- RMAQACBXLXPBSY-UHFFFAOYSA-N silicic acid Chemical compound O[Si](O)(O)O RMAQACBXLXPBSY-UHFFFAOYSA-N 0.000 claims abstract description 14
- 235000012239 silicon dioxide Nutrition 0.000 claims abstract description 14
- 239000003446 ligand Substances 0.000 claims abstract description 10
- STMDPCBYJCIZOD-UHFFFAOYSA-N 2-(2,4-dinitroanilino)-4-methylpentanoic acid Chemical compound CC(C)CC(C(O)=O)NC1=CC=C([N+]([O-])=O)C=C1[N+]([O-])=O STMDPCBYJCIZOD-UHFFFAOYSA-N 0.000 claims abstract description 9
- 239000011159 matrix material Substances 0.000 claims abstract description 9
- JPVYNHNXODAKFH-UHFFFAOYSA-N Cu2+ Chemical compound [Cu+2] JPVYNHNXODAKFH-UHFFFAOYSA-N 0.000 claims description 10
- 239000002245 particle Substances 0.000 claims description 7
- MGFYIUFZLHCRTH-UHFFFAOYSA-N nitrilotriacetic acid Chemical compound OC(=O)CN(CC(O)=O)CC(O)=O MGFYIUFZLHCRTH-UHFFFAOYSA-N 0.000 claims description 4
- VEQPNABPJHWNSG-UHFFFAOYSA-N Nickel(2+) Chemical compound [Ni+2] VEQPNABPJHWNSG-UHFFFAOYSA-N 0.000 claims description 3
- RRYKFOOPBCFDAC-UHFFFAOYSA-N [2-[bis(hydroxymethyl)amino]ethylamino]methanol Chemical compound OCNCCN(CO)CO RRYKFOOPBCFDAC-UHFFFAOYSA-N 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
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 claims description 2
- 102000004169 proteins and genes Human genes 0.000 abstract description 19
- 108090000623 proteins and genes Proteins 0.000 abstract description 19
- 230000000536 complexating effect Effects 0.000 abstract 1
- 239000000243 solution Substances 0.000 description 28
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 21
- 230000000052 comparative effect Effects 0.000 description 17
- 238000002360 preparation method Methods 0.000 description 14
- 238000000034 method Methods 0.000 description 13
- 238000003756 stirring Methods 0.000 description 13
- 239000011259 mixed solution Substances 0.000 description 9
- 239000003513 alkali Substances 0.000 description 8
- 229910001431 copper ion Inorganic materials 0.000 description 8
- 238000001914 filtration Methods 0.000 description 8
- 239000007787 solid Substances 0.000 description 7
- 238000005406 washing Methods 0.000 description 7
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 6
- 238000001035 drying Methods 0.000 description 6
- 150000003839 salts Chemical class 0.000 description 6
- MQRWBMAEBQOWAF-UHFFFAOYSA-N acetic acid;nickel Chemical compound [Ni].CC(O)=O.CC(O)=O MQRWBMAEBQOWAF-UHFFFAOYSA-N 0.000 description 5
- 229940078494 nickel acetate Drugs 0.000 description 5
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 4
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 4
- 238000006243 chemical reaction Methods 0.000 description 4
- 238000002474 experimental method Methods 0.000 description 4
- 239000007788 liquid Substances 0.000 description 4
- 238000011068 loading method Methods 0.000 description 4
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 3
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 3
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 3
- 239000011543 agarose gel Substances 0.000 description 3
- 230000009286 beneficial effect Effects 0.000 description 3
- 230000009920 chelation Effects 0.000 description 3
- OPQARKPSCNTWTJ-UHFFFAOYSA-L copper(ii) acetate Chemical group [Cu+2].CC([O-])=O.CC([O-])=O OPQARKPSCNTWTJ-UHFFFAOYSA-L 0.000 description 3
- 239000008367 deionised water Substances 0.000 description 3
- 229910021641 deionized water Inorganic materials 0.000 description 3
- 238000010828 elution Methods 0.000 description 3
- 238000000926 separation method Methods 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 description 2
- 125000003277 amino group Chemical group 0.000 description 2
- 239000006185 dispersion Substances 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 239000012266 salt solution Substances 0.000 description 2
- 238000002791 soaking Methods 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- 229910052938 sodium sulfate Inorganic materials 0.000 description 2
- 235000011152 sodium sulphate Nutrition 0.000 description 2
- 239000002904 solvent Substances 0.000 description 2
- 239000000725 suspension Substances 0.000 description 2
- 239000012670 alkaline solution Substances 0.000 description 1
- 230000008827 biological function Effects 0.000 description 1
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000005611 electricity Effects 0.000 description 1
- 238000011067 equilibration Methods 0.000 description 1
- 230000004907 flux Effects 0.000 description 1
- 230000004927 fusion Effects 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 1
- 230000003100 immobilizing effect Effects 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 238000002386 leaching Methods 0.000 description 1
- 229920002521 macromolecule Polymers 0.000 description 1
- 230000005389 magnetism Effects 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 229910001453 nickel ion Inorganic materials 0.000 description 1
- 238000006116 polymerization reaction Methods 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 230000002035 prolonged effect Effects 0.000 description 1
- 238000000164 protein isolation Methods 0.000 description 1
- 238000001742 protein purification Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000007711 solidification Methods 0.000 description 1
- 230000008023 solidification Effects 0.000 description 1
- 230000003068 static effect Effects 0.000 description 1
- 230000002522 swelling effect Effects 0.000 description 1
- 238000001132 ultrasonic dispersion Methods 0.000 description 1
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
- B01J20/22—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising organic material
- B01J20/26—Synthetic macromolecular compounds
- B01J20/262—Synthetic macromolecular compounds obtained otherwise than by reactions only involving carbon to carbon unsaturated bonds, e.g. obtained by polycondensation
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
- B01D15/08—Selective adsorption, e.g. chromatography
- B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
- B01D15/38—Selective adsorption, e.g. chromatography characterised by the separation mechanism involving specific interaction not covered by one or more of groups B01D15/265 and B01D15/30 - B01D15/36, e.g. affinity, ligand exchange or chiral chromatography
- B01D15/3804—Affinity chromatography
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
- B01J20/02—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising inorganic material
- B01J20/06—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising inorganic material comprising oxides or hydroxides of metals not provided for in group B01J20/04
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
- B01J20/28—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties
- B01J20/28002—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties characterised by their physical properties
- B01J20/28009—Magnetic properties
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J2220/00—Aspects relating to sorbent materials
- B01J2220/40—Aspects relating to the composition of sorbent or filter aid materials
- B01J2220/48—Sorbents characterised by the starting material used for their preparation
- B01J2220/4812—Sorbents characterised by the starting material used for their preparation the starting material being of organic character
- B01J2220/4825—Polysaccharides or cellulose materials, e.g. starch, chitin, sawdust, wood, straw, cotton
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- Chemical & Material Sciences (AREA)
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- Solid-Sorbent Or Filter-Aiding Compositions (AREA)
Abstract
The scheme relates to a metal chelating affinity chromatography medium with magnetic polymer microspheres as a matrix, wherein polymer composite microspheres wrapped with ferroferric oxide magnetic spheres are used as an inner core, and the polymer consists of sodium alginate, polyacrylamide and silicic acid; the surface of the inner core is crosslinked with chitosan oligosaccharide and cellulose, the chitosan oligosaccharide and the cellulose are bonded with allyl glycidyl ether, the allyl glycidyl ether is connected with a ligand, and the ligand is used for complexing metal ions; the metal chelating affinity chromatography medium prepared by the invention can be repeatedly used for many times, has strong mechanical property, can be respectively complexed with different metal ions, has longer service life and high efficiency of separating target protein.
Description
Technical Field
The invention relates to a chromatography medium, in particular to a metal chelating affinity chromatography medium taking magnetic polymer microspheres as a matrix.
Background
Protein is one of the most important biological macromolecules, is the main embodiment and material basis of all life activities, accurate research on protein structure and biological functions must be established on the basis of obtaining high-purity target protein, protein separation and purification is a method for separating and purifying the required target protein from a mixture by using downstream biological engineering technology, and is one of core technologies in the modern biological industry.
The basis for protein isolation and purification generally includes: utilizing solubility differences, molecular size differences, surface charge differences, hydrophilic-hydrophobic property differences, specific biological affinity differences, and the like. The immobilized metal chelating affinity chromatography has high selection specificity to target protein, relatively low economic cost and mild elution condition, and has obvious advantages in the purification of recombinant protein. The immobilized metal chelating affinity chromatography is carried out on the basis of the affinity of target protein and metal ions, the existing metal chelating affinity chromatography medium taking agarose gel as a matrix has lower content of metal ions, is not suitable for separating protein with high flux, and has lower mechanical strength, high price, limited repeated use times and shorter service life.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide a metal chelating affinity chromatography medium taking magnetic polymer microspheres as a matrix.
The technical scheme of the invention is summarized as follows:
taking a polymer composite microsphere wrapped with a ferroferric oxide magnetic sphere as an inner core, wherein chitosan oligosaccharide and cellulose are crosslinked on the surface of the inner core, the chitosan oligosaccharide and the cellulose are bonded with allyl glycidyl ether, the allyl glycidyl ether is connected with a ligand, and the ligand is complexed with metal ions;
preferably, the polymer consists of sodium alginate, polyacrylamide and silicic acid.
Preferably, the polymer comprises the following three components in percentage by mass:
70-75 wt% of sodium alginate;
20-25 wt% of polyacrylamide;
5-10 wt% of silicic acid.
Preferably, the mass ratio of the ferroferric oxide magnetic spheres to the polymer is 1: 10-12.
Preferably, the mass ratio of the chitosan oligosaccharide to the cellulose is 2: 1-1.2.
Preferably, the particle size of the ferroferric oxide magnetic ball is 0.5-1 μm, and the surface of the ferroferric oxide magnetic ball is modified by amino.
Preferably, the particle size of the polymer composite microsphere wrapped with the ferroferric oxide magnetic ball is 30-100 microns.
Preferably, the ligand is one of iminodiacetic acid, trimethylolethylenediamine and nitrilotriacetic acid.
Preferably, the metal ion is Ni2+、Cu2+、Zn2+、Co2+One kind of (1).
The invention has the beneficial effects that: the prepared metal chelating affinity chromatography medium takes the polymer composite microspheres wrapped with the ferroferric oxide magnetic spheres as the matrix inner core, the inner core has magnetism, when the affinity chromatography medium is repeatedly utilized and another metal ion needs to be complexed, the complexed metal ion can be eluted under the washing of weak alkaline solution and a certain magnetic field is applied, the elution of the chromatography medium by strong alkali or high-concentration salt solution is avoided, the loss of ligands and the inner core is reduced, and the service life of the chromatography medium is prolonged.
The polymer wrapping the ferroferric oxide magnetic spheres consists of three components, namely sodium alginate, polyacrylamide and silicic acid, wherein the sodium alginate is rich in carboxyl and is electronegative, and can be well crosslinked with the ferroferric oxide magnetic spheres with amino groups modified on the surfaces of the sodium alginate and the ferroferric oxide magnetic spheres, and the polyacrylamide and the silicic acid in a specific proportion are added to promote the composite microspheres to reach the size of 30-100 microns required by an affinity chromatography medium core through polymerization, fusion and solidification; the chitosan oligosaccharide has good biocompatibility, has active groups such as amino groups and hydroxyl groups, has a large amount of bonding ligand, is beneficial to improving the separation efficiency, has weak cellulose swelling property, is attached to the surface of the microsphere after being mixed with the chitosan oligosaccharide, and is beneficial to maintaining the shape of an inner core of an affinity chromatography medium; the metal chelating affinity chromatography medium prepared by the invention can be repeatedly used and complex different metal ions, the service life is longer, and the efficiency of separating target protein is high.
Detailed Description
The present invention is further described in detail below with reference to examples so that those skilled in the art can practice the invention with reference to the description. The invention provides a metal chelating affinity chromatography medium using magnetic polymer microspheres as a matrix, which is specifically illustrated by the following examples and comparative examples.
Example 1
The preparation process comprises the following steps:
(1) dissolving 18g of sodium alginate, 5g of polyacrylamide and 2g of silicic acid into 200ml of dilute hydrochloric acid solution with the pH value of 3, and fully stirring at room temperature to form a mixed solution A; adding 2.5g of ferroferric oxide magnetic spheres with the particle size of 0.5-1 mu m into 20mL of deionized water, and violently stirring at room temperature to form a suspension B;
(2) slowly adding the suspension B into the mixed solution A under the stirring condition, and then violently stirring for 15-20 minutes to obtain a mixed solution C; slowly stirring the mixed solution C, heating to 80-90 ℃, keeping for 1.5-2 hours, then carrying out ultrasonic dispersion to obtain a dispersion, and centrifuging to remove large-particle precipitates;
(3) adding the dispersion into an injector, fixing the injector on an injection pump, adding 5kV static electricity on a needle of the injector, and then extruding the mixed solution into 0.1mol/L NaOH solution at the speed of 200mL/h to obtain polymer composite microspheres wrapped with ferroferric oxide magnetic spheres;
(4) adding the composite microspheres obtained in the step (3) into dilute hydrochloric acid containing 10g of chitosan oligosaccharide and 5g of cellulose and having a pH value of 50ml of 6, stirring for 2-2.5 hours at the temperature of 40-45 ℃, filtering, washing with deionized water and drying to obtain microsphere cores of surface cross-linked chitosan oligosaccharide and cellulose;
(5) placing the microspheres obtained in the step (4) in 100mL of NaOH solution with the concentration of 0.2mol/L, simultaneously adding 5g of allyl glycidyl ether and 2.5g of sodium sulfate, stirring for 8 hours at 45-50 ℃, filtering and drying in vacuum after the reaction is finished;
(6) adding 5g of nitrilotriacetic acid, 50mL of ethanol and 2g of sodium hydroxide into the microspheres obtained in the step (5), stirring and reacting at 55-60 ℃ for 6-8 h, filtering to remove the solvent after the reaction is finished, washing and drying;
(7) and (3) soaking the microspheres obtained in the step (6) in 1mol/L nickel acetate solution, adjusting the pH of the solution to 4-4.5 by using acetic acid, stirring for 12 hours at 40 ℃, filtering and washing for three times to obtain the target product.
Example 2
The preparation process comprises the following steps:
the nickel acetate solution in step (7) of example 1 was replaced with a copper acetate solution of the same concentration, and the remaining procedure was the same as in example 1.
Comparative example 1
The preparation process comprises the following steps:
(1) adding agarose gel microspheres with the particle size of 30-100 mu m into dilute hydrochloric acid with the pH value of 6 of 50ml containing 10g of chitosan oligosaccharide and 5g of cellulose, stirring for 2-2.5 hours at the temperature of 40-45 ℃, filtering, washing with deionized water and drying to obtain microsphere cores with surface cross-linked chitosan oligosaccharide and cellulose;
(2) placing the microspheres obtained in the step (1) in 100mL of NaOH solution with the concentration of 0.2mol/L, simultaneously adding 5g of allyl glycidyl ether and 2.5g of sodium sulfate, stirring for 8 hours at 45-50 ℃, filtering and drying in vacuum after the reaction is finished;
(3) adding 5g of nitrilotriacetic acid, 50mL of ethanol and 2g of sodium hydroxide into the microspheres obtained in the step (1), stirring and reacting at 55-60 ℃ for 6-8 h, filtering to remove the solvent after the reaction is finished, washing and drying;
(4) and (3) soaking the microspheres obtained in the step (3) in 1mol/L nickel acetate solution, adjusting the pH of the solution to 4-4.5 by using acetic acid, stirring for 2 hours at 40 ℃, filtering and washing for three times to obtain the target product.
Comparative example 2
The preparation process comprises the following steps:
18g of sodium alginate, 5g of polyacrylamide and 2g of silicic acid used in the preparation of the mixed solution A in step (1) of example 1 were replaced with 25g of sodium alginate, and the rest of the preparation was the same as in example 1.
Comparative example 3
The preparation process comprises the following steps:
18g of sodium alginate, 5g of polyacrylamide and 2g of silicic acid used for preparing the mixed solution A in the step (1) of example 1 were replaced by 18g of sodium alginate and 7g of polyacrylamide, and the rest of the preparation process was the same as that of example 1.
Comparative example 4
The preparation process comprises the following steps:
18g of sodium alginate, 5g of polyacrylamide and 2g of silicic acid used for preparing the mixed solution A in the step (1) of example 1 were replaced by 23g of sodium alginate and 2g of silicic acid, and the rest of the preparation process was the same as that of example 1.
Comparative example 5
The preparation process comprises the following steps:
step (4) of example 1 is omitted, namely chitosan oligosaccharide and cellulose are not crosslinked on the inner core of the microsphere, the polymer composite microsphere wrapped with the ferroferric oxide magnetic spheres obtained in step (3) is directly subjected to step (5) of bonding allyl glycidyl ether, and the rest preparation processes are the same as those of example 1.
Comparative example 6
The preparation process comprises the following steps:
10g of chitosan oligosaccharide, 5g of cellulose in step (4) of example 1 were replaced with only 15g of chitosan oligosaccharide, and the rest of the preparation process was the same as in example 1.
In order to test the performance of the metal chelating affinity chromatography media prepared in examples 1-2 and comparative examples 1-6, an affinity chromatography experiment was performed with recombinant protein a having 6 consecutive histidine tags as a target protein, and the immobilization amount of each affinity chromatography medium on the target protein was recorded. After the first chromatographic affinity chromatography experiment is finished, different metal ions are respectively washed and chelated again by the following two methods.
The method comprises the following steps: after the first chromatographic experiment is finished, the mixed solution of 0.05mol/L sodium hydroxide and 0.01mol/L sodium chloride with 3 column volumes is used for showering, a magnetic field of 1000-1500 Gs is applied along the length direction of the chromatographic column, and then the balance liquid with 2 column volumes is used for balancing. For the chromatographic columns prepared by the affinity media in the embodiment 1 and the comparative examples 1-6, 1mol/L copper acetate solution with 5 column volumes is used for slowly showering, then balance liquid with 2 column volumes is used for balancing, the showering liquid and the balance liquid are collected, the copper ion concentration is detected, and the lower the copper ion concentration is, the more the content of the re-chelated copper ions of the affinity chromatographic column is. This step of the test was carried out on example 2 using nickel acetate. And recording the concentration of metal ions in the solution after chelation by the chromatographic column.
The second method comprises the following steps: after the first chromatographic experiment is finished, the mixture solution of 0.5mol/L sodium hydroxide and 0.15mol/L sodium chloride in 3 column volumes is used for showering, and then the balance solution in 5 column volumes is used for balancing. For the chromatographic columns prepared by the affinity media in the example 1 and the comparative examples 1-6, 5 column volumes of 1mol/L copper acetate solution are used for slowly showering, 2 column volumes of equilibrium solution are used for balancing, the showering solution and the equilibrium solution are collected, the copper ion concentration is detected, and the lower the copper ion concentration is, the more the content of the re-chelated copper ions of the affinity chromatographic column is. This step of the test was carried out on example 2 using nickel acetate. And recording the concentration of metal ions in the solution after chelation by the chromatographic column.
Table 1 reports the target protein solid load of each example and comparative example, and the metal ion concentrations in the rinse solution and the equilibrium solution after chelating the metal ions for the second time, respectively. Firstly, analyzing the solid carrying capacity of each chromatography medium on target protein, wherein the chromatography medium in the embodiment 2 chelates the recombinant protein A with 6 continuous histidine tags by copper ions, and the chelating effect of the copper ions on the target protein is poorer than that of nickel ions, so that the solid carrying capacity of the target protein in the embodiment 2 is lower; the difference of the solid loading of the example 1 and the comparative examples 2-4 on the target protein comes from the components in the polymer, the polymer in the comparative example 2 only consists of sodium alginate, and does not contain polyacrylamide and silicic acid, so that the stability of the inner core of the composite microsphere is poor, the mechanical strength is low, and the composite microsphere is easy to deform under certain column pressure, so that the solid loading on the target protein is less, and the solid loading of the example 1 and the comparative examples 3-4 show that when the polyacrylamide and the silicic acid exist simultaneously, the mechanical strength of an affinity chromatography medium can be enhanced, so that the magnetic polymer composite microsphere has larger solid loading and separation efficiency; according to the immobilization amounts of the embodiment 1 and the comparative examples 5-6, the core surface cross-linked shell oligo and the cellulose can enrich the core surface groups, so that more complex metal ions are used for immobilizing the target protein.
The data in the third and fourth columns of Table 1 were then analyzed for the concentration of metal ions in the leachant and equilibration solutions after chelation by the column. For example 1, the metal ion concentrations in the final eluate and equilibrium solution obtained by the first and second methods are almost the same, while the alkali concentration in the eluate in the second method is 10 times the alkali concentration in the first method and the salt concentration is 15 times the salt concentration in the first method, which indicates that if the chromatographic column is eluted to be capable of chelating the same amount of metal ions again, the second method requires high-concentration salt and alkali solution, and it is known that the high-concentration alkali and salt can seriously damage the structure of the affinity chromatographic column matrix and reduce the service life thereof; in the comparative example 1, agarose gel is used as an inner core, magnetic microspheres are not wrapped, and no induction exists on a magnetic field, so that the original complex metal ions cannot be well eluted by the method for one time, the method is characterized in that after the elution is carried out by weak alkali weak salt solution, the chromatographic column is leached by metal ion solution with the same concentration, the concentration of the metal ions in leaching solution and equilibrium solution is high, namely the amount of the metal ions complexed by the chromatographic column is less, and when the method for two times is used, the original complex metal ions can be well eluted by high salt and alkali concentration solution so as to newly adsorb more metal ions, and the embodiment 1 and the comparative example 2 prove that the metal chelating affinity chromatography medium can be cleaned by the magnetic polymer composite microspheres under the condition of the magnetic field and by using lower alkali and salt concentration.
TABLE 1
While embodiments of the invention have been disclosed above, it is not limited to the applications listed in the description and the embodiments, which are fully applicable in all kinds of fields of application of the invention, and further modifications may readily be effected by those skilled in the art, so that the invention is not limited to the specific details without departing from the general concept defined by the claims and the scope of equivalents.
Claims (7)
1. A metal chelating affinity chromatography medium taking magnetic polymer microspheres as a matrix is characterized in that polymer composite microspheres wrapped with ferroferric oxide magnetic spheres are taken as an inner core, and the surfaces of the ferroferric oxide magnetic spheres are modified by amino; the surface of the inner core is crosslinked with chitosan oligosaccharide and cellulose; the chitosan oligosaccharide and the cellulose are bonded with allyl glycidyl ether; the allyl glycidyl ether is connected with a ligand; the ligand complexes the metal ion; the polymer is composed of sodium alginate, polyacrylamide and silicic acid, and the mass fraction is as follows: 70-75 wt% of sodium alginate; 20-25 wt% of polyacrylamide; 5-10 wt% of silicic acid.
2. The metal chelating affinity chromatography medium as claimed in claim 1, wherein the mass ratio of the ferroferric oxide magnetic spheres to the polymer is 1: 10-12.
3. The metal chelating affinity chromatography medium of claim 1, wherein the mass ratio of the chitosan oligosaccharide to the cellulose is 2: 1-1.2.
4. The metal chelating affinity chromatography medium of claim 1, wherein the ferroferric oxide magnetic spheres have a particle size of 0.5-1 μm.
5. The metal chelating affinity chromatography medium of claim 1, wherein the polymer composite microspheres coated with the ferroferric oxide magnetic spheres have a particle size of 30-100 μm.
6. The metal chelating affinity chromatography media of claim 1, wherein said ligand is one of iminodiacetic acid, trimethylolethylenediamine, and nitrilotriacetic acid.
7. The metal chelating affinity chromatography media of claim 1, wherein the metal ion is Ni2+、Cu2+、Zn2+、Co2+One kind of (1).
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