CN107754768B - Metal chelating affinity chromatography medium containing barrier layer - Google Patents
Metal chelating affinity chromatography medium containing barrier layer Download PDFInfo
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- CN107754768B CN107754768B CN201711053681.8A CN201711053681A CN107754768B CN 107754768 B CN107754768 B CN 107754768B CN 201711053681 A CN201711053681 A CN 201711053681A CN 107754768 B CN107754768 B CN 107754768B
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- affinity chromatography
- metal chelating
- chelating affinity
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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
- B01J20/02—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising inorganic material
- B01J20/10—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising inorganic material comprising silica or silicate
- B01J20/103—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising inorganic material comprising silica or silicate comprising silica
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- 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 - B01D15/36
- B01D15/3804—Affinity chromatography
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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
- B01J20/24—Naturally occurring macromolecular compounds, e.g. humic acids or their derivatives
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/16—Extraction; Separation; Purification by chromatography
- C07K1/22—Affinity chromatography or related techniques based upon selective absorption processes
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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
- B01J2220/4806—Sorbents characterised by the starting material used for their preparation the starting material being of inorganic character
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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
-
- 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
Abstract
The scheme relates to a metal chelating affinity chromatography medium containing a barrier layer, which takes macroporous silicon dioxide microspheres as an inner core, a mixture composed of three components of sodium alginate, chitosan oligosaccharide and silicon nitride is filled in pore channels of the microspheres, epichlorohydrin is bonded on the surface of the inner core, the epichlorohydrin is connected with a ligand, and the ligand is complexed with metal ions; meanwhile, amylose is adhered to the surface of the inner core to form a resistance layer, and the mass ratio of the epichlorohydrin to the amylose is 1: 4-5; according to the invention, the blocking of nonspecific macromolecules is realized through the blocking layer, nonspecific adsorption is reduced, and the adsorption separation efficiency of target micromolecular polypeptides is improved.
Description
Technical Field
The invention relates to a chromatographic medium, in particular to a metal chelating affinity chromatographic medium containing a barrier layer.
Background
In recent years, the biotechnology has been continuously advanced, and the rapid and effective separation and purification of polypeptides from complex natural mixtures gradually become the key and difficulty in the development of novel drugs. Therefore, whether the polypeptide can be efficiently and stably separated and purified becomes a precondition and basis for research and application of the polypeptide. With the rapid development of chromatographic technology, immobilized metal chelating affinity chromatography is used as a high-efficiency separation technology and can be applied to the separation and purification of polypeptides.
The immobilized metal chelating affinity chromatography is a technology for realizing separation based on coordination of metal ions and amino acid residues, has the advantages of high separation speed, simple operation and the like, is a powerful method for separating and purifying proteins, and is widely applied to separation and purification of proteins, nucleic acids, polypeptides and the like. However, in practical applications, when a target small molecule such as a polypeptide needs to be separated from a mixed system, the adsorption sites of the target small molecule are reduced due to the influence of non-specific adsorption of macromolecules, thereby reducing the adsorption performance of the target small molecule. Therefore, the affinity chromatography medium needs to be modified to some extent so as to resist macromolecules, reduce the nonspecific adsorption of macromolecules and improve the adsorption of target micromolecules. In the prior art, hydrophilic substance polyethylene glycol (PEG) is grafted on the surface of an affinity medium matrix to form a semi-permeable protection barrier to perform size exclusion on macromolecular heteroprotein, so that the separation and purification efficiency of the micromolecular polypeptide is improved, but the method needs to convert a terminal hydroxyl group of the PEG into an aldehyde group, a carboxyl group, a halogen, an amino group and other active groups in advance to graft the terminal hydroxyl group on the matrix, has a complex process, and has a not ideal specific separation effect on target micromolecular polypeptide in a system containing the macromolecular heteroprotein.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide a metal chelating affinity chromatography medium containing a barrier layer.
The technical scheme of the invention is summarized as follows:
the method comprises the steps of taking a macroporous silicon dioxide microsphere as an inner core, filling a mixture consisting of three components of sodium alginate, chitosan oligosaccharide and silicon nitride in a pore channel of the macroporous silicon dioxide microsphere, bonding epoxy chloropropane on the surface of the inner core, connecting epoxy chloropropane with a ligand, complexing metal ions with the ligand, and adhering amylose on the surface of the inner core to form a barrier layer, wherein the mass ratio of the epoxy chloropropane to the amylose is 1: 4-5.
Preferably, the mass fractions of sodium alginate, chitosan oligosaccharide and silicon nitride in the mixture are respectively as follows:
50-55 wt% of sodium alginate;
40-45 wt% of chitosan oligosaccharide;
3-5 wt% of silicon nitride.
Preferably, the mass ratio of the macroporous silicon dioxide to the compound is 1: 4-5.
Preferably, the polymerization degree of the amylose is 900 to 1000.
Preferably, the particle size of the macroporous silicon dioxide microspheres is 50-100 mu m, and the inner diameter of the pore channel is larger than 50 nm.
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: according to the scheme, the macroporous silica microspheres with the pore diameter of more than 50nm are selected as the inner cores, so that the inner surface area and the pore ratio are large, and mixtures such as sodium alginate and the like can be adsorbed and filled more; the sodium alginate has good bonding and adsorption effects on the silicon dioxide, and can increase the bonding strength of the mixture on the silicon dioxide, so that the core structure is compact and firm; the chitosan oligosaccharide is rich in active groups such as amino, hydroxyl and the like, and provides a connecting group for bonding amylose or epichlorohydrin; the silicon nitride has high hardness, and can effectively enhance the mechanical property and the pressure resistance of the affinity chromatography medium.
The amylose adopted by the invention is long-chain polysaccharide formed by spirals, the degree of polymerization is 900-1000, each spiral turn contains 6 glucose groups, and the amylose is rich in hydroxyl and high in viscosity, can be fixed on the surface of the inner core of the microsphere through mutual adhesion with chitosan oligosaccharide or bonding with epoxy chloropropane without group modification to form a flexible barrier, and can effectively block nonspecific macromolecules by controlling proper amylose density, so that nonspecific adsorption is reduced by size exclusion of macromolecular hybrid protein, and the adsorption separation efficiency of target micromolecular polypeptide is improved.
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 containing a barrier layer, which is illustrated by the following examples and comparative examples.
Example 1
The preparation process comprises the following steps:
(1) soaking 20g of macroporous silica microspheres with the particle size of 50-100 mu m and the pore diameter of more than 50nm in 0.5mol/L sodium hydroxide solution, stirring at 50-55 ℃ for 1-1.5 hours to activate the macroporous silica microspheres, and vacuum-drying for 12 hours;
(2) adding 250mL of acetone solution with the volume fraction of 30% into a mixture of 60g of sodium alginate, 54g of chitosan oligosaccharide, 6g of silicon nitride and 10g of sodium hydroxide, and vigorously stirring and ultrasonically treating the mixture to form uniform suspension; taking the macroporous silica microspheres out of the vacuum drying furnace, quickly placing the macroporous silica microspheres into the suspension, stirring for 8-10 hours at 45-50 ℃, washing with deionized water after filtering, and drying to obtain macroporous silica microsphere cores filled with the mixture;
(3) adding 50mL of acetone, 15g of epoxy chloropropane and 5g of sodium hydroxide into the microsphere core obtained in the step (2), stirring for 3-4 hours at 35-40 ℃, washing and drying after the reaction is finished, and obtaining an activated microsphere core with the surface bonded with the epoxy chloropropane;
(4) dissolving 10g of nitrilotriacetic acid, 70g of amylose and 8g of sodium hydroxide in 250mL of 30% ethanol solution at 50-55 ℃, and uniformly stirring to form a mixed solution; placing the activated microsphere core obtained in the step (3) in the mixed solution, stirring and reacting for 6-8 hours at 50-55 ℃, filtering, washing and drying after the reaction is finished;
(5) and (3) soaking the microspheres obtained in the step (4) in 1mol/L copper 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 with deionized water for three times to obtain the metal chelating affinity chromatography medium with the amylose blocking layer.
Example 2
The preparation process comprises the following steps:
the copper acetate solution in step (5) of example 1 was replaced with nickel acetate of the same concentration, and the rest of the preparation process was the same as in example 1.
Comparative example 1
The preparation process comprises the following steps:
example 1 step (4) amylose was not added and the remaining preparation process was the same as in example 1.
Comparative example 2
The preparation process comprises the following steps:
70g of amylose in step (4) of example 1 was replaced with the same mass of formylated polyethylene glycol (PEG), and the rest of the preparation process was the same as in example 1.
Comparative example 3
The preparation process comprises the following steps:
the sodium alginate in step (2) of example 1 was replaced with the same mass of dextran and the rest of the preparation procedure was the same as in example 1.
Comparative example 4
The preparation process comprises the following steps:
the preparation process is the same as that of the example 1 except that the chitosan oligosaccharide is not added in the step (2) of the example 1 and the mass of the sodium alginate is 110 g.
Example 5
Commercially available metal chelating affinity chromatography media that chelate copper ions.
In order to test the affinity adsorption efficiency of the prepared metal chelate affinity chromatography medium on small molecular polypeptides and the rejection effect of the prepared metal chelate affinity chromatography medium on large molecular proteins, bovine serum albumin BSA is used as a large molecular protein model which is composed of 583 amino acid residues, a plurality of amino acid sequences which are easy to chelate with metal ions exist, and the large molecular protein impurities which need to be removed are large molecular protein impurities, one polypeptide fragment VSLPEW (Val-Ser-Leu-Pro-Glu-Try) in α -whey protein hydrolysate is used as target small molecular polypeptide, wherein the small molecular polypeptide has a good blood pressure reducing function, amino nitrogen or carboxyl oxygen in the VSLPEW polypeptide can be chelated with copper ions, so that separation is realized, when the VSLPEW polypeptide is extracted from an actual fermentation or enzymolysis system, the large molecular proteins similar to the BSA can be adsorbed on a metal chelate affinity chromatography column as non-specific impurities to influence the purification effect of the target small molecular polypeptides, and the metal chelate affinity chromatography medium containing the rejection layer can reduce the non-specific adsorption on the large molecular proteins through size exclusion.
Firstly, preparing disodium hydrogen phosphate and sodium dihydrogen phosphate solutions with pH of 6.5 and concentration of 0.2mol/L as buffer solutions, preparing a solution with VSLPEW polypeptide concentration of 0.05mg/mL by using the buffer solutions, adjusting the amount of BSA, and respectively preparing a group of mixed solutions with the concentration ratios of VSLPEW to BSA of 1: 5 (mixed solution A), 1: 10 (mixed solution B) and 1: 100 (mixed solution C); 20mg of the metal chelate affinity chromatography media obtained in examples 1-2 and comparative examples 1-5 were placed in 5mL centrifuge tubes, 3mL of mixed protein solution was added, shaking was performed in a shaker at 30 ℃ for 50min, the affinity chromatography media was magnetically attracted, the supernatant was taken, filtered through a 0.22 μm fiber membrane, and the supernatant was quantitatively measured by HPLC, and the average of the two parallel operations was taken. Table 1 records the respective concentrations of VSLPEW and BSA in the supernatant after each affinity chromatography medium performs affinity adsorption on the mixed solutions a to C, and the smaller the concentration of VSLPEW or BSA in the supernatant is, the more the prepared metal chelate affinity chromatography medium adsorbs; meanwhile, the larger the concentration difference between the two is, the stronger the selective adsorption of the affinity chromatography medium is.
After affinity adsorption is performed on mixed solutions with different concentration ratios, for the test of example 1, the concentration of the target small-molecule polypeptide VSLPEW in the supernatant is reduced by about 0.0400mg/mL, and the concentration of the impurity large protein BSA is reduced by about 0.0030-0.0123 mg/mL, which indicates that the affinity chromatography medium prepared in the example can selectively adsorb the small-molecule polypeptide VSLPEW, and the experimental data of example 2 can lead to a consistent conclusion that the measured concentration of VSLPEW in the supernatant of example 2 is greater than that in example 1 because nickel ions have weaker chelating property to VSLPEW than copper ions; furthermore, amylose is not added into the affinity chromatography medium in the comparative example 1, the concentration of VSLPEW in the supernatant is only reduced by about 0.0070mg/mL, the concentration of BSA is reduced by about 0.0500-0.0700 mg/mL, the affinity chromatography medium in the comparative example 1 mainly adsorbs impurity large protein molecules, and the selective adsorption of the target small molecule polypeptide is very weak, which shows that the addition of amylose forms a barrier layer capable of effectively blocking the large molecule protein on the surface of the affinity chromatography medium, so that the selective adsorption of the small molecule polypeptide is realized; according to example 5, the commercial affinity chromatography media have difficulty in blocking the macromolecular proteins and cannot selectively adsorb the target small-molecular proteins.
Comparative example 2 adopts aldehydized polyethylene glycol to construct a barrier layer, and can also realize selective adsorption on target small-molecule protein, but the separation effect is poorer than that of example 2, the reduction of the VSLPEW concentration in the supernatant of comparative example 2 is about 2-4 times that of the BSA concentration, and the separation effect on the VSLPEW and the BSA is poorer as the BSA concentration in the mixed solution is increased; the VSLPEW concentration in the supernatant of comparative examples 3 and 4 is higher than that of example 1, which shows that the affinity chromatography media of comparative examples 3 and 4 have poor affinity adsorption performance for VSLPEW, which is related to the mixture filled in the macroporous silica microspheres, and sodium alginate, chitosan oligosaccharide and silicon nitride are used as fillers, which can effectively increase the amount of metal ligands in the metal chelate affinity chromatography media, thereby adsorbing more target proteins.
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 containing a barrier layer is characterized in that macroporous silica microspheres are used as an inner core; the pore canal of the macroporous silicon dioxide microsphere is filled with a mixture consisting of three components of sodium alginate, chitosan oligosaccharide and silicon nitride; the surface of the inner core is bonded with epoxy chloropropane, the epoxy chloropropane is connected with a ligand, and the ligand is complexed with metal ions; amylose is adhered to the surface of the inner core to form a barrier layer; wherein the mass ratio of the epichlorohydrin to the amylose is 1: 4-5.
2. The metal-chelating affinity chromatography medium of claim 1, wherein the mass fractions of sodium alginate, chitosan oligosaccharide and silicon nitride in the mixture are respectively:
50-55 wt% of sodium alginate;
40-45 wt% of chitosan oligosaccharide;
3-5 wt% of silicon nitride;
wherein the grain diameter of the silicon nitride is less than 20 nm.
3. The metal chelating affinity chromatography media of claim 1, wherein the mass ratio of the macroporous silica to the mixture is 1: 4-5.
4. The metal chelating affinity chromatography medium of claim 1, wherein the degree of polymerization of the amylose is 900 to 1000.
5. The metal chelating affinity chromatography media of claim 1, wherein the macroporous silica microspheres have a particle size of 50-100 μm and an inner diameter of pore channels of greater than 50 nm.
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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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6277489B1 (en) * | 1998-12-04 | 2001-08-21 | The Regents Of The University Of California | Support for high performance affinity chromatography and other uses |
| CN103406100A (en) * | 2013-07-08 | 2013-11-27 | 武汉金益肽生物有限公司 | Magnetic chelate, and preparation method and application thereof |
| CN103611512A (en) * | 2013-12-05 | 2014-03-05 | 苏州博进生物技术有限公司 | Hydrophilic high mechanical strength chromatography medium and preparation method thereof |
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2017
- 2017-10-31 CN CN201711053681.8A patent/CN107754768B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6277489B1 (en) * | 1998-12-04 | 2001-08-21 | The Regents Of The University Of California | Support for high performance affinity chromatography and other uses |
| CN103406100A (en) * | 2013-07-08 | 2013-11-27 | 武汉金益肽生物有限公司 | Magnetic chelate, and preparation method and application thereof |
| CN103611512A (en) * | 2013-12-05 | 2014-03-05 | 苏州博进生物技术有限公司 | Hydrophilic high mechanical strength chromatography medium and preparation method thereof |
Non-Patent Citations (2)
| Title |
|---|
| A novel matrix derivatized from hydrophilic gigaporous polystyrene-based microspheres for high-speed immobilized-metal affinity chromatography;Jian-Bo Qu et al.;《Journal of Chromatography B》;20110312;第879卷;第1043-1048页 * |
| 固定化金属螯合亲和层析介质及其应用研究进展;景志刚 等;《农产品加工》;20151130(第11期);第54-57页 * |
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