CN107754768A - A kind of metal chelate affinity chromatography medium that layer is refused containing resistance - Google Patents
A kind of metal chelate affinity chromatography medium that layer is refused containing resistance Download PDFInfo
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- CN107754768A CN107754768A CN201711053681.8A CN201711053681A CN107754768A CN 107754768 A CN107754768 A CN 107754768A CN 201711053681 A CN201711053681 A CN 201711053681A CN 107754768 A CN107754768 A CN 107754768A
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- Prior art keywords
- affinity chromatography
- chromatography medium
- metal chelate
- chelate affinity
- epoxychloropropane
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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
-
- 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
-
- 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
-
- 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
-
- 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
-
- 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
This case is related to a kind of metal chelate affinity chromatography medium that layer is refused containing resistance, using macroporous silica microsphere as kernel, filled with the mixture being made up of three kinds of sodium alginate, chitosan oligosaccharide, silicon nitride components in the duct of microballoon, in kernel surface bond epoxychloropropane, epoxychloropropane connects aglucon, aglucon complexation of metal ions；Resistance being formed in kernel surface adhesion amylose simultaneously and refusing layer, the mass ratio of epoxychloropropane and amylose is 1: 4~5；The present invention refuses layer by resistance realizes barrier to non-specific macromolecular, reduces non-specific adsorption, improves the adsorption separation efficiency to target micromolecule polypeptide.
Description
Technical field
The present invention relates to a kind of chromatography media, and in particular to a kind of metal chelate affinity chromatography medium that layer is refused containing resistance.
Background technology
In recent years, biotechnology is constantly progressive, and fast and effectively separation, purification polypeptide are gradual from complicated natural mixture
As the key and difficult point of exploitation newtype drug.Therefore, if efficient stable can be carried out to polypeptide and isolated and purified, become
Polypeptide is studied and the premise of application and basis.With developing rapidly for chromatographic technique, immobilization metal chelating affinity chromatography conduct
Efficient isolation technics, it can apply to isolating and purifying for polypeptide.
Immobilization metal chelating affinity chromatography is that a kind of realized based on metal ion with amino acid residue coordination is separated
Technology, due to fast with separating rate, it is simple to operate many advantages, such as, become since proposition to isolate and purify the strong of albumen
Powerful approach, it is widely used in the isolating and purifying of protein, nucleic acid, polypeptide etc..However, in practical application, when need from
In mixed system during the target small molecule such as isolated polypeptide, due to the influence of macromolecular non-specific adsorption be present, cause target small
The adsorption site of molecule is reduced, so as to reduce the absorption property to target small molecule.Therefore need to carry out affinity chromatography medium
Certain modification, macromolecular is refused to reach resistance, reduce the non-specific adsorption of macromolecular, improve the suction of target small molecule
It is attached.In the prior art by affinity media stromal surface scion grafting hydroaropic substance polyethylene glycol (PEG), to form semi-permeable guarantor
Protect barrier and volume exclusion is carried out to macromolecular foreign protein, so as to improve the efficiency that isolates and purifies to micromolecule polypeptide, but the party
Method need in advance by PEG terminal hydroxy group be converted into aldehyde radical, carboxyl, halogen, amino isoreactivity group could scion grafting its in matrix, work
Skill is cumbersome, and the specific isolation effect of target micromolecule polypeptide is not very managed in the system containing macromolecular foreign protein
Think.
The content of the invention
In view of the deficiencies of the prior art, it is an object of the invention to provide a kind of immobilized metal ion affinity that layer is refused containing resistance
And chromatography media.
Technical scheme is summarized as follows：
Using macroporous silica microsphere as kernel, in the duct of macroporous silica microsphere be filled with by sodium alginate,
The mixture that three kinds of chitosan oligosaccharide, silicon nitride components are formed, in kernel surface bond epoxychloropropane, epoxychloropropane connection is matched somebody with somebody
Base, aglucon complexation of metal ions, while core surface are stained with amylose formation resistance and refuse layer, wherein, the epoxychloropropane
Mass ratio with amylose is 1: 4~5.
Preferably, sodium alginate, chitosan oligosaccharide, the mass fraction of silicon nitride are respectively in the mixture：
50~55wt% of sodium alginate；
40~45wt% of chitosan oligosaccharide；
3~5wt% of silicon nitride.
Preferably, the mass ratio of the macroporous silica and compound is 1: 4~5.
Preferably, the degree of polymerization of the amylose is 900~1000.
Preferably, for the particle diameter of the macroporous silica microsphere at 50~100 μm, duct internal diameter is more than 50nm.
Preferably, the aglucon is one kind in iminodiacetic acid, trihydroxy methyl ethylenediamine, complexon I.
Preferably, the metal ion is Ni2+、Cu2+、Zn2+、Co2+In one kind.
The beneficial effects of the invention are as follows：This case is from macroporous silica microsphere of the aperture more than 50nm as kernel, tool
There are larger internal surface area and void ratio, the mixtures such as sodium alginate can be filled with more absorption；Sodium alginate is to silica
Suction-operated is bonded with good, adhesion strength of the mixture to silica can be increased, make inner core closely solid；
Chitosan oligosaccharide provides linking group rich in amino, hydroxyl isoreactivity group for bonding amylose or epoxychloropropane；Silicon nitride
Hardness is big, can effectively strengthen the mechanical performance of affinity chromatography medium and pressure-resistant performance.
Amylose of the present invention is the long chain polysaccharides formed with spiral shape, and the degree of polymerization is 900~1000, each
Spiral revolution include 6 glucosyl groups, because amylose is rich in hydroxyl and sticky larger, it is not necessary to by base group modification with regard to energy
It is enough to be fixed on microballoon core surface, formation flexible screen by mutually bonding with chitosan oligosaccharide or being bonded with epoxychloropropane
Barrier, controls appropriate amylose density effectively to realize the barrier to non-specific macromolecular, by the miscellaneous egg of macromolecular
The white volume exclusion that carries out reduces non-specific adsorption, improves the adsorption separation efficiency to target micromolecule polypeptide.
Embodiment
With reference to embodiment, the present invention is described in further detail, to make those skilled in the art with reference to specification
Word can be implemented according to this.The invention provides a kind of metal chelate affinity chromatography medium that layer is refused containing resistance, pass through following realities
Apply example and comparative example is specifically described.
Embodiment 1
Preparation process is as follows：
(1) 20g particle diameters are immersed in 0.5mol/L in 50~100 μm, macroporous silica microsphere of the aperture more than 50nm
Sodium hydroxide solution in, being stirred 1~1.5 hour at 50~55 DEG C activates macroporous silica microsphere, vacuum drying 12
Hour；
(2) to 60g sodium alginates, 54g chitosan oligosaccharides, 6g silicon nitrides, 10g sodium hydroxides mixture in add 250mL bodies
Fraction is 30% acetone soln, is stirred vigorously and is ultrasonically formed uniform suspension；By macroporous silica microsphere from true
It is immediately placed in after being taken out in empty drying oven in the suspension, is stirred 8~10 hours at 45~50 DEG C, deionized water is used after filtering
Rinse and be dried to obtain the macroporous silica microsphere kernel filled with mixture；
(3) 50mL acetone, 15g epoxychloropropane and 5g sodium hydroxides are added into step (2) thus obtained microsphere kernel, 35
Stirred 3~4 hours at~40 DEG C, reaction washs drying after terminating, and obtains the activation microballoon kernel of surface bond epoxychloropropane；
(4) 10g complexon Is, 70g amyloses, 8g sodium hydroxides are dissolved in 250mL matter at 50~55 DEG C
Measure in the ethanol solution that fraction is 30%, stir to form mixed solution；Activation microballoon kernel obtained by step (3) is placed in
Among the mixed solution, stirring reaction 6~8 hours at 50~55 DEG C, reaction is filtered after terminating and washs drying；
(5) step (4) thus obtained microsphere is soaked in 1mol/L acetic acid copper solution, with acetic acid regulation pH value of solution be 4~
4.5, stirred 12 hours at 40 DEG C, filter and be washed with deionized the metal chelating for obtaining that there is amylose resistance to refuse layer three times
Close affinity chromatography medium.
Embodiment 2
Preparation process is as follows：
Acetic acid copper solution in (5) the step of embodiment 1 is replaced with the nickel acetate of same concentrations, remaining preparation process and reality
It is identical to apply example 1.
Comparative example 1
Preparation process is as follows：
Amylose is added without in the step of embodiment 1 (4), remaining preparation process is same as Example 1.
Comparative example 2
Preparation process is as follows：
70g amyloses in the step of embodiment 1 (4) are replaced with the aldehyde radical polyethylene glycol (PEG) of phase homogenous quantities, its
Remaining preparation process is same as Example 1.
Comparative example 3
Preparation process is as follows：
Sodium alginate in the step of embodiment 1 (2) is replaced by the glucan of phase homogenous quantities, remaining preparation process and embodiment
1 is identical.
Comparative example 4
Preparation process is as follows：
Chitosan oligosaccharide will be added without in the step of embodiment 1 (2), while it is 110g to add sodium alginate quality, remaining was prepared
Journey is same as Example 1.
Embodiment 5
The metal chelate affinity chromatography medium of commercially available chelating copper ion.
In order to test affine adsorption efficiency of the metal chelate affinity chromatography medium prepared by the present invention to micromolecule polypeptide,
And effect is refused in the resistance to macro-molecular protein, here using ox albumin BSA as macro-molecular protein model, it is by 583
Individual amino acid residue is formed, and a variety of easily amino acid sequences with metal ion-chelant be present, is the macromolecular egg for needing to remove
White matter impurity；Made with a polypeptide fragment VSLPEW (Val-Ser-Leu-Pro-Glu-Try) in alpha lactalbumin hydrolysate
For target micromolecule polypeptide, the wherein micromolecule polypeptide has a good antihypertensive function, in VSLPEW polypeptides ammonia nitrogen or
Carboxyl oxygen can separate with chelating copper ions so as to realize；Being extracted from the fermentation of reality or enzymatic hydrolysis system, VSLPEW is more
During peptide, can usually non-specific impurity absorption be used as to chelate affinity column, shadow in relatives similar to BSA macro-molecular protein
Ring the purification effect to target micromolecule polypeptide；It is situated between using the metal chelate affinity chromatography that layer is refused containing resistance prepared by the present invention
Matter can reduce the non-specific adsorption to macro-molecular protein by volume exclusion.
First prepare pH be 6.5 concentration be 0.2mol/L disodium hydrogen phosphate, sodium dihydrogen phosphate as buffer it is molten
Liquid, the solution for being 0.05mg/mL with buffer preparation VSLPEW peptide concentrations, adjusts BSA amount, prepares VSLPEW respectively
It is 1: 5 (mixed liquor A) with BSA concentration ratios, 1: 10 (mixed liquid B), 1: 100 (mixed liquor C) one group of mixed solution；Respectively will
The metal chelate affinity chromatography medium of 20mg embodiments 1~2 and the gained of comparative example 1~5 is placed in 5mL centrifuge tubes, is added 3mL and is mixed
Hop protein solution, 30 DEG C of isothermal vibration 50min in shaking table, magnetic affinity chromatography medium, takes supernatant, with 0.22 μm of fiber
Membrane filtration, and with high performance liquid chromatography to supernatant quantitative test, take the average of operation repetitive twice.Table 1 have recorded each parent
With chromatography media to the affine absorption of mixed solution A~C after, VSLPEW and the respective concentration of BSA in supernatant, in supernatant
VSLPEW or BSA concentration is smaller, and it is more to illustrate that prepared metal chelate affinity chromatography medium adsorbs to it；Meanwhile two
The concentration difference of person is bigger, illustrates that the Selective adsorption of affinity chromatography medium is stronger.
For various concentrations than the affine absorption of mixed solution after, for the test to embodiment 1, target is small in supernatant
Molecular polypeptide VSLPEW concentration reduces about 0.0400mg/mL, and impurity large protein BSA concentration only reduces about 0.0030
~0.0123mg/mL, illustrate that the affinity chromatography medium prepared by embodiment is capable of the absorption micromolecule polypeptide VSLPEW of selectivity,
Consistent conclusion is can be derived that by the experimental data of embodiment 2, due to nickel ion to VSLPEW chelating ability compared with copper ion
It is weak, so measured VSLPEW concentration is big compared with embodiment 1 in the supernatant of embodiment 2；Further, affinity chromatography medium pair
Without amylose is added in ratio 1, VSLPEW concentration only reduces about 0.0070mg/mL in its supernatant, and BSA's is dense
Degree reduces about 0.0500~0.0700mg/mL on the contrary, the affinity chromatography medium primary attachment of comparative example 1 impurity large protein point
Son, the selective absorption to target micromolecule polypeptide is extremely weak, and the addition of this explanation amylose is in affinity chromatography medium surface shape
Into the barrier layer that can effectively obstruct macro-molecular protein, the selective absorption to micromolecule polypeptide is realized；According to embodiment
5, commercially available affinity chromatography medium is difficult to the barrier to macro-molecular protein, it is impossible to selective absorption target small molecular protein
Matter.
Comparative example 2 can also realize the selection to target small protein using aldehyde radical polyethylene glycol structure barrier layer
Property absorption, but separating effect is poor compared with embodiment 2, and the VSLPEW concentration reduction levels in the supernatant of comparative example 2 are about that BSA concentration subtracts
A small amount of 2~4 times, and with the increase of BSA concentration in mixed solution, both separating effects are deteriorated；Comparative example 3 and right
VSLPEW concentration is higher than the VSLPEW concentration in embodiment 1 in supernatant in ratio 4, illustrates the parent of comparative example 3 and comparative example 4
Poor to the affine absorption properties of VSLPEW with chromatography media, this is relevant with the mixture filled in macroporous silica microsphere,
Sodium alginate, chitosan oligosaccharide and silicon nitride can be effectively increased metal in metal chelate affinity chromatography medium and match somebody with somebody as filler
The amount of base, so as to adsorb more target protein.
Table 1
Although embodiment of the present invention is disclosed as above, it is not restricted in specification and embodiment listed
With it can be applied to various suitable the field of the invention completely, can be easily for those skilled in the art
Other modification is realized, therefore under the universal limited without departing substantially from claim and equivalency range, it is of the invention and unlimited
In specific details.
Claims (7)
1. a kind of refuse the metal chelate affinity chromatography medium of layer containing resistance, it is characterised in that using macroporous silica microsphere is interior
Core；It is filled with the duct of the macroporous silica microsphere and is mixed by what three kinds of sodium alginate, chitosan oligosaccharide, silicon nitride components were formed
Compound；The core surface is bonded epoxychloropropane, and the epoxychloropropane connects aglucon, the aglucon complexation of metal ions；
The core surface adhesion amylose forms resistance and refuses layer；Wherein, the mass ratio of the epoxychloropropane and amylose is 1: 4
~5.
2. metal chelate affinity chromatography medium according to claim 1, it is characterised in that alginic acid in the mixture
Sodium, chitosan oligosaccharide, the mass fraction of silicon nitride are respectively：
50~55wt% of sodium alginate；
40~45wt% of chitosan oligosaccharide；
3~5wt% of silicon nitride；
Wherein, the particle diameter of silicon nitride is less than 20nm.
3. metal chelate affinity chromatography medium according to claim 1, it is characterised in that the macroporous silica is with answering
The mass ratio of compound is 1: 4~5.
4. metal chelate affinity chromatography medium according to claim 1, it is characterised in that the degree of polymerization of the amylose
For 900~1000.
5. metal chelate affinity chromatography medium according to claim 1, it is characterised in that the macroporous silica microsphere
Particle diameter at 50~100 μm, duct internal diameter is more than 50nm.
6. metal chelate affinity chromatography medium according to claim 1, it is characterised in that the aglucon is iminodiacetic acid (salt)
One kind in acid, trihydroxy methyl ethylenediamine, complexon I.
7. metal chelate affinity chromatography medium according to claim 1, it is characterised in that the metal ion is Ni2+、
Cu2+、Zn2+、Co2+In one kind.
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| CN201711053681.8A CN107754768B (en) | 2017-10-31 | 2017-10-31 | Metal chelating affinity chromatography medium containing barrier layer |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711053681.8A CN107754768B (en) | 2017-10-31 | 2017-10-31 | Metal chelating affinity chromatography medium containing barrier layer |
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| CN107754768B CN107754768B (en) | 2020-03-31 |
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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 |
-
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 |
|---|
| JIAN-BO QU ET AL.: "A novel matrix derivatized from hydrophilic gigaporous polystyrene-based microspheres for high-speed immobilized-metal affinity chromatography", 《JOURNAL OF CHROMATOGRAPHY B》 * |
| 景志刚 等: "固定化金属螯合亲和层析介质及其应用研究进展", 《农产品加工》 * |
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