CN116440879A - Novel streptavidin filler and preparation method thereof - Google Patents
Novel streptavidin filler and preparation method thereof Download PDFInfo
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- CN116440879A CN116440879A CN202310666611.9A CN202310666611A CN116440879A CN 116440879 A CN116440879 A CN 116440879A CN 202310666611 A CN202310666611 A CN 202310666611A CN 116440879 A CN116440879 A CN 116440879A
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- streptavidin
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- alkaline solution
- filler
- epoxy
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- 108010090804 Streptavidin Proteins 0.000 title claims abstract description 36
- 239000000945 filler Substances 0.000 title claims abstract description 23
- 238000002360 preparation method Methods 0.000 title claims abstract description 11
- 229920000642 polymer Polymers 0.000 claims abstract description 12
- 230000004048 modification Effects 0.000 claims abstract description 8
- 238000012986 modification Methods 0.000 claims abstract description 8
- MYRTYDVEIRVNKP-UHFFFAOYSA-N 1,2-Divinylbenzene Chemical compound C=CC1=CC=CC=C1C=C MYRTYDVEIRVNKP-UHFFFAOYSA-N 0.000 claims abstract description 6
- 229920000193 polymethacrylate Polymers 0.000 claims abstract description 4
- 239000004793 Polystyrene Substances 0.000 claims abstract description 3
- 229920002223 polystyrene Polymers 0.000 claims abstract description 3
- 239000011159 matrix material Substances 0.000 claims description 24
- 239000004593 Epoxy Substances 0.000 claims description 21
- 239000012670 alkaline solution Substances 0.000 claims description 19
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 18
- 238000000034 method Methods 0.000 claims description 12
- 239000003795 chemical substances by application Substances 0.000 claims description 11
- 238000006243 chemical reaction Methods 0.000 claims description 10
- KWYUFKZDYYNOTN-UHFFFAOYSA-M Potassium hydroxide Chemical compound [OH-].[K+] KWYUFKZDYYNOTN-UHFFFAOYSA-M 0.000 claims description 9
- 229920002307 Dextran Polymers 0.000 claims description 8
- 230000008878 coupling Effects 0.000 claims description 8
- 238000010168 coupling process Methods 0.000 claims description 8
- 238000005859 coupling reaction Methods 0.000 claims description 8
- 238000001914 filtration Methods 0.000 claims description 7
- 238000003756 stirring Methods 0.000 claims description 7
- 239000003153 chemical reaction reagent Substances 0.000 claims description 6
- 238000004140 cleaning Methods 0.000 claims description 6
- 229920001503 Glucan Polymers 0.000 claims description 5
- 230000004913 activation Effects 0.000 claims description 5
- 239000011148 porous material Substances 0.000 claims description 5
- 239000007864 aqueous solution Substances 0.000 claims description 4
- SHKUUQIDMUMQQK-UHFFFAOYSA-N 2-[4-(oxiran-2-ylmethoxy)butoxymethyl]oxirane Chemical compound C1OC1COCCCCOCC1CO1 SHKUUQIDMUMQQK-UHFFFAOYSA-N 0.000 claims description 3
- BRLQWZUYTZBJKN-UHFFFAOYSA-N Epichlorohydrin Chemical compound ClCC1CO1 BRLQWZUYTZBJKN-UHFFFAOYSA-N 0.000 claims description 3
- 239000007853 buffer solution Substances 0.000 claims description 3
- 230000001105 regulatory effect Effects 0.000 claims description 3
- NOYXQFBTCCSKQG-UHFFFAOYSA-N 2-[[2-(oxiran-2-ylmethoxy)cyclohexyl]oxymethyl]oxirane Chemical compound C1OC1COC1CCCCC1OCC1CO1 NOYXQFBTCCSKQG-UHFFFAOYSA-N 0.000 claims description 2
- 150000007529 inorganic bases Chemical class 0.000 claims description 2
- 239000004005 microsphere Substances 0.000 claims description 2
- 238000002156 mixing Methods 0.000 claims description 2
- 238000012856 packing Methods 0.000 claims description 2
- UWFRVQVNYNPBEF-UHFFFAOYSA-N 1-(2,4-dimethylphenyl)propan-1-one Chemical compound CCC(=O)C1=CC=C(C)C=C1C UWFRVQVNYNPBEF-UHFFFAOYSA-N 0.000 claims 1
- FGPFIXISGWXSCE-UHFFFAOYSA-N 2,2-bis(oxiran-2-ylmethoxymethyl)propane-1,3-diol Chemical compound C1OC1COCC(CO)(CO)COCC1CO1 FGPFIXISGWXSCE-UHFFFAOYSA-N 0.000 claims 1
- 150000001412 amines Chemical class 0.000 claims 1
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 abstract description 24
- 229960002685 biotin Drugs 0.000 abstract description 12
- 235000020958 biotin Nutrition 0.000 abstract description 12
- 239000011616 biotin Substances 0.000 abstract description 12
- 239000003513 alkali Substances 0.000 abstract description 7
- 239000002585 base Substances 0.000 abstract description 3
- 229920002401 polyacrylamide Polymers 0.000 abstract description 2
- 125000003700 epoxy group Chemical group 0.000 abstract 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 6
- 230000027455 binding Effects 0.000 description 6
- 230000003993 interaction Effects 0.000 description 4
- 239000000243 solution Substances 0.000 description 4
- 238000001179 sorption measurement Methods 0.000 description 4
- 239000000126 substance Substances 0.000 description 4
- 238000012360 testing method Methods 0.000 description 4
- CUGZWHZWSVUSBE-UHFFFAOYSA-N 2-(oxiran-2-ylmethoxy)ethanol Chemical compound OCCOCC1CO1 CUGZWHZWSVUSBE-UHFFFAOYSA-N 0.000 description 3
- 108090001008 Avidin Proteins 0.000 description 3
- 239000003446 ligand Substances 0.000 description 3
- 229910021642 ultra pure water Inorganic materials 0.000 description 3
- 239000012498 ultrapure water Substances 0.000 description 3
- 102000004190 Enzymes Human genes 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 238000001042 affinity chromatography Methods 0.000 description 2
- 238000004587 chromatography analysis Methods 0.000 description 2
- 230000000052 comparative effect Effects 0.000 description 2
- 150000001875 compounds Chemical class 0.000 description 2
- 238000010828 elution Methods 0.000 description 2
- 239000002245 particle Substances 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- PLDLPVSQYMQDBL-UHFFFAOYSA-N 2-[[3-(oxiran-2-ylmethoxy)-2,2-bis(oxiran-2-ylmethoxymethyl)propoxy]methyl]oxirane Chemical compound C1OC1COCC(COCC1OC1)(COCC1OC1)COCC1CO1 PLDLPVSQYMQDBL-UHFFFAOYSA-N 0.000 description 1
- 241001655322 Streptomycetales Species 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 230000006978 adaptation Effects 0.000 description 1
- 239000000427 antigen Substances 0.000 description 1
- 102000036639 antigens Human genes 0.000 description 1
- 108091007433 antigens Proteins 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000006287 biotinylation Effects 0.000 description 1
- 238000007413 biotinylation Methods 0.000 description 1
- 238000003759 clinical diagnosis Methods 0.000 description 1
- 230000001808 coupling effect Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000010494 dissociation reaction Methods 0.000 description 1
- 230000005593 dissociations Effects 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000011049 filling Methods 0.000 description 1
- 229960000789 guanidine hydrochloride Drugs 0.000 description 1
- PJJJBBJSCAKJQF-UHFFFAOYSA-N guanidinium chloride Chemical compound [Cl-].NC(N)=[NH2+] PJJJBBJSCAKJQF-UHFFFAOYSA-N 0.000 description 1
- 238000009396 hybridization Methods 0.000 description 1
- 238000003384 imaging method Methods 0.000 description 1
- 238000003364 immunohistochemistry Methods 0.000 description 1
- 238000011031 large-scale manufacturing process Methods 0.000 description 1
- 238000011068 loading method Methods 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 230000009871 nonspecific binding Effects 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 230000006916 protein interaction Effects 0.000 description 1
- 238000001742 protein purification Methods 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 229910000029 sodium carbonate Inorganic materials 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Classifications
-
- 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
- B01J20/265—Synthetic macromolecular compounds modified or post-treated polymers
-
- 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
- B01D15/3823—Affinity chromatography of other types, e.g. avidin, streptavidin, biotin
-
- 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
- B01J20/28011—Other properties, e.g. density, crush strength
-
- 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
- B01J20/30—Processes for preparing, regenerating, or reactivating
- B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
- B01J20/3231—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the coating or impregnating layer
- B01J20/3242—Layers with a functional group, e.g. an affinity material, a ligand, a reactant or a complexing group
- B01J20/3268—Macromolecular compounds
- B01J20/328—Polymers on the carrier being further modified
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02W—CLIMATE CHANGE MITIGATION TECHNOLOGIES RELATED TO WASTEWATER TREATMENT OR WASTE MANAGEMENT
- Y02W10/00—Technologies for wastewater treatment
- Y02W10/10—Biological treatment of water, waste water, or sewage
Landscapes
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Organic Chemistry (AREA)
- Solid-Sorbent Or Filter-Aiding Compositions (AREA)
Abstract
The invention discloses a novel streptavidin filler and a preparation method thereof, wherein polymer base balls such as polystyrene/divinylbenzene, polymethacrylate, polyacrylamide and the like are used as matrixes, hydrophilic modification is carried out on the surfaces of the polymer base balls, epoxy groups are introduced, and then streptavidin is bonded on the activated matrixes to form the Streptavidin (SA) affinity filler. The SA affinity filler has better alkali resistance and high affinity to biotin or biotinylated samples.
Description
Technical Field
The invention relates to the technical field of biochemical separation media, in particular to a novel streptavidin filler and a preparation method thereof.
Background
Affinity chromatography is a chromatographic method for separating and purifying substances by specific recognition and interaction between molecules, such as specific interaction between enzyme and substrate, antigen and antibody, etc., and the corresponding biological molecules can be adsorbed from the initial sample by fixing the enzyme as ligand on the filler, and then the biological molecules are dissociated by proper elution to achieve the aim of purification. Affinity chromatography has the advantages of high selectivity, high resolution and high binding capacity, and can often achieve purity of more than 90% by one-step purification, and can complete separation of substances which are difficult to complete by common methods.
Streptavidin (SA) is one type of proteinTetrameric protein from streptomycete, 66kDa in size, has high Biotin binding capacity and dissociation constant up to 10 -14 Is one of the strongest interactions between biomolecules known to date. The interaction between streptavidin and biotin is widely used in many fields of nucleic acid detection, single molecule imaging, protein interaction, clinical diagnosis and the like, and is a key research means of biotechnology.
Streptavidin, like avidin, can bind specifically to biotin, but compared to avidin, streptavidin has a low isoelectric point (isoelectric point 6.0), contains no sugar groups, and its non-specific binding is far lower than that of avidin, especially in immunohistochemistry and DNA molecular hybridization assays, with little background color development.
At present, some streptavidin fillers are available on the market, but the biotin binding amount is low, about 300nmol/mL, and the alkali resistance is low. Therefore, the streptavidin filler which has high pressure resistance, alkali resistance, low non-specific adsorption, higher biotin binding capacity, simple preparation process and capability of being produced in a rapid and amplified way is also an urgent requirement of the current market.
Disclosure of Invention
In order to solve the technical problems, the invention aims to provide a novel streptavidin filler and a preparation method thereof, wherein the novel streptavidin filler has better alkali resistance and high affinity to biotin or a biotinylation sample.
The invention firstly provides a preparation method of a novel streptavidin filler, which comprises the following steps:
1) Hydrophilic modification of a matrix: dispersing a polymer matrix in an alkaline solution 1, adding an epoxy reagent 1 and glucan, stirring for reaction, and filtering and cleaning to obtain an intermediate 1;
2) Epoxy activation: dispersing the intermediate 1 in an alkaline solution 2, adding an epoxy reagent 2, stirring for reaction, filtering and cleaning to obtain an intermediate 2;
3) Coupling streptavidin: and mixing the intermediate 2, PB buffer solution and streptavidin aqueous solution, regulating pH, and reacting to obtain the novel streptavidin filler.
The hydrophilic modification of the matrix is carried out by dispersing the polymer matrix in alkaline solution 1 for 0.5-1 hr, adding epoxy agent 1 and dextran, stirring at 25-70deg.C for 8.0-40 hr, preferably at 25-45deg.C for 10-24 hr, filtering, and cleaning to obtain intermediate 1. The dextran molecules are introduced on the surface of the matrix through chemical bonding, so that a large amount of-OH is enriched on the surface of the matrix, the matrix shows good hydrophilicity, and the specific adsorption is reduced.
The condition of epoxy activation is that the intermediate 1 is dispersed in alkaline solution 2 for 0.5-2 hours, then epoxy reagent 2 is added, stirred for 4-24 hours at 40-105 ℃, preferably for 4-6 hours at 60-75 ℃, and filtered and cleaned to obtain intermediate 2.
Further, the invention is not limited to polymer matrices, most polymers can be realized, and preferred polymer matrices are polystyrene/divinylbenzene, polymethacrylate, polyacrylamide; the matrix is porous microsphere with pore diameter in the following rangeThe grain diameter is 5.0-100 μm.
Preferably, the pore size of the matrix isThe grain diameter is 80 mu m; optimized to pore size->Particle size 60 μm is optimized again to pore size +.>The particle size was 50. Mu.m.
Further, the alkaline solution 1 and the alkaline solution 2 are inorganic bases including sodium hydroxide, potassium hydroxide and the like, the concentration of the alkaline solution 1 is 0.5-2.0M, the concentration of the alkaline solution 2 is 0.5-6.0M, and preferably, the concentration of the alkaline solution 2 is 0.5-4.0M.
The alkaline solution 1 and the alkaline solution 2 may be the same inorganic alkaline solution or different alkaline solutions.
Further, the epoxy agent 1 and the epoxy agent 2 are selected from epichlorohydrin, ethylene glycol glycidyl ether, 1, 4-butanediol diglycidyl ether, 1, 2-cyclohexanediol diglycidyl ether or pentaerythritol glycidyl ether, and the epoxy agent 1 and the epoxy agent 2 may be the same compound or different compounds.
Further, the molecular weight of the glucan is 2000-200000, for example, 2000, 6000, 20000, 40000, 200000, etc.
Further, the mass ratio of the matrix to the glucan is 1.0:0.02-0.4, and the mass ratio of the matrix to the epoxy agent 1 is 1.0:1.0-1.5.
Further, the mass ratio of the matrix to the epoxy agent 2 is 1.0:2.0-2.5.
Further, the pH is in the range of 5.0 to 12.0, and the preferred pH is in the range of 6.5 to 9.0. When coupling streptavidin, the reaction environment can be under acidic or alkaline conditions, but under alkaline conditions, the coupling effect is better; the reagent used for regulating the pH is inorganic alkali such as sodium hydroxide, potassium hydroxide, sodium carbonate and the like.
Further, when coupling streptavidin, it is reacted at 25-45℃for 16-24 hours, preferably at 25-40℃for 16-18 hours.
Further, the intermediate 2 is dispersed in water for 0.5-2 hours before the coupling streptavidin is added. In general, the dispersing is not needed first, but the dispersing is needed first and then the coupling is needed, so that the effect is better.
In addition, the invention also provides a novel streptavidin filler prepared by the scheme.
The beneficial effects are that:
the novel streptavidin filler has high affinity to biotin or biotinylated substances, and the binding capacity of the biotin can reach 377nmol/mL. Because the polymer matrix is adopted for the surface of Kong Jiqiu, the surface is subjected to hydrophilic treatment, so that the polymer has good alkali resistance, and the biotin binding capacity is reduced by less than 0.8 percent after the polymer matrix is soaked in 0.1M NaOH solution for 48 hours. In addition, the filler also has better pressure resistance, higher mass transfer efficiency, excellent hydrophilicity and extremely low nonspecific adsorption; in addition, the invention has simple process and is easy to realize large-scale production.
Drawings
FIG. 1 is a graph of purified biotin with respect to the filler prepared in example 1.
Detailed Description
The present invention will be described in further detail with reference to specific examples. The present invention may be embodied in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.
Example 1
S1, hydrophilic modification of matrix
10g of polymethacrylate-based ball is accurately weighed by a balance, added into a 500mL three-neck flask, 200mL of 0.5mol/LNaOH solution is added into a reaction flask, stirred for 1.0 hour, 10g of epichlorohydrin and 4.0g of dextran 2000 are added, stirred for 10.0 hours at 25 ℃, filtered by a Buchner funnel after the reaction is finished, washed by 200mL of ethanol, washed by ultrapure water until pH test paper shows neutrality, and pumped for later use, thus obtaining an intermediate 1.
S2, epoxy activation
Transferring the intermediate 1 into a 500mL three-neck flask, adding 200mL of 1.5mol/L sodium hydroxide aqueous solution, stirring and reacting for 1.0 hour, then adding 20g of ethylene glycol glycidyl ether, stirring for 4 hours at 25 ℃, filtering by using a Buchner funnel after the reaction is finished, and washing to be neutral by using ultra-pure water to obtain the intermediate 2.
S3, coupling ligand
The intermediate 2 is added into a 250mL three-neck flask, 50mL 20mM PB buffer solution is added, 4.0mL 2.0mg/mL Streptavidin (SA) aqueous solution is added, the pH is adjusted to 8.0, the reaction is carried out for 20 hours at 40 ℃, after the reaction is finished, a Buchner funnel is used for filtering, 500mL ethanol and 500mL ultrapure water are used for cleaning the filler in sequence, finally the filler is stored in 20% ethanol, and the temperature is kept at 2-8 ℃.
Example 2
The difference from example 1 is that in the hydrophilic modification of the S1 matrix, dextran 2000 is replaced by dextran 4000, and the rest of the procedure is the same as in example 1.
Example 3
The difference from example 1 is that in the hydrophilic modification of the S1 matrix, dextran 2000 is replaced by dextran 6000, and the rest of the procedure is the same as in example 1.
Example 4
The difference from example 1 is that in the S2 epoxy activation, ethylene glycol glycidyl ether is replaced by 1, 4-butanediol diglycidyl ether, and the rest of the procedure is the same as in example 1.
Comparative example
In comparison with example 1, the difference is that no SA is added in the S3 ligand coupling step, and the rest of the procedure is the same as in example 1. Adsorption and elution test of SA affinity packing
Chromatography column: taking SA affinity filler prepared in the example 1 and filling a chromatographic column with the size of 4.6 mm by 50 mm;
instrument: a protein purification system SCG;
flow rate: 0.34mL/min;
a detector: UV 280nm;
mobile phase: a:20mM NaH 2 PO 4 +150mM NaCl，pH 7.4；
B:8M guanidine hydrochloride, pH 1.5;
sample loading amount: 5. Mu.L (3.4 mg/mL biotin);
column temperature: room temperature;
the method comprises the following steps:
balance: 100% A,12min;
eluting: 100% B,16min;
table 1 below shows the test results of examples 1-4 and comparative examples
TABLE 1
Table 2 below the results of the bubble base test of example 1 and example 4
(bubble alkali condition: immersing the filler in a 0.1M NaOH solution for 48 hours)
TABLE 2
The above is only a preferred embodiment of the present invention, and the protection scope of the present invention is not limited to the above examples, and all technical solutions belonging to the concept of the present invention belong to the protection scope of the present invention. It should be noted that modifications and adaptations to the invention without departing from the principles thereof are intended to be within the scope of the invention as set forth in the following claims.
Claims (9)
1. A method for preparing a novel streptavidin filler, which is characterized by comprising the following steps:
1) Hydrophilic modification of a matrix: dispersing a polymer matrix in an alkaline solution 1, adding an epoxy reagent 1 and glucan, stirring for reaction, and filtering and cleaning to obtain an intermediate 1;
2) Epoxy activation: dispersing the intermediate 1 in an alkaline solution 2, adding an epoxy reagent 2, stirring for reaction, filtering and cleaning to obtain an intermediate 2;
3) Coupling streptavidin: and mixing the intermediate 2, PB buffer solution and streptavidin aqueous solution, regulating pH, and reacting to obtain the novel streptavidin filler.
2. The method of claim 1, wherein the polymer matrix is selected from the group consisting of polystyrene/divinylbenzene, polymethacrylate, and polyacrylAn amine; the matrix is porous microsphere with pore diameter in the following rangeThe grain diameter is 5.0-100 μm.
3. The preparation method according to claim 1, wherein the alkaline solution 1 and the alkaline solution 2 are inorganic bases including sodium hydroxide and potassium hydroxide, the concentration of the alkaline solution 1 is 0.5-2.0M, and the concentration of the alkaline solution 2 is 0.5-6.0M.
4. The preparation method according to claim 1, wherein the epoxy agent 1 and the epoxy agent 2 are selected from epichlorohydrin, ethylene glycol diglycidyl ether, 1, 4-butanediol diglycidyl ether, 1, 2-cyclohexanediol diglycidyl ether, or pentaerythritol diglycidyl ether.
5. The method according to claim 1, wherein the dextran has a molecular weight of 2000-200000.
6. The preparation method according to claim 1, wherein the mass ratio of the matrix to the glucan is 1.0:0.02-0.4, and the mass ratio of the matrix to the epoxy agent 1 is 1.0:1.0-1.5.
7. The preparation method according to claim 1, wherein the mass ratio of the matrix to the epoxy agent 2 is 1.0:2.0-2.5.
8. The method of claim 1, wherein the pH is in the range of 5.0 to 12.0.
9. A novel streptavidin packing prepared by the method of any one of claims 1-8.
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| CN202310666611.9A CN116440879A (en) | 2023-06-07 | 2023-06-07 | Novel streptavidin filler and preparation method thereof |
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| CN108084252A (en) * | 2017-12-26 | 2018-05-29 | 常州天地人和生物科技有限公司 | A kind of new Streptavidin purification filler and preparation method thereof |
| CN112341663A (en) * | 2020-10-28 | 2021-02-09 | 苏州纳微科技股份有限公司 | ProteinA affinity chromatography medium of PMMA matrix and preparation method and application thereof |
| CN115990464A (en) * | 2021-10-18 | 2023-04-21 | 马杰 | Hydrophilic modified hydrophobic polymer matrix Protein A affinity chromatography media |
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