CN105498701A - High-load agarose chromatography media and preparation method thereof - Google Patents
High-load agarose chromatography media and preparation method thereof Download PDFInfo
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Abstract
The present invention relates to a high-load agarose chromatography media comprising a shell and an inner core, the shell portion is covalently bonded with linear macromolecules such as glucan and the like, the shell portion and the inner core portion are coupled with ligands, and ligand species include metal ions, hydrophobic ligands, ion exchange groups and proteins, and the like. The present invention also includes a preparation method of the high-load agarose chromatography media. For example, the load of histidine tagged (His-tagged) recombinant protein of a metal chelated chromatography media prepared by the method is about 2.5 times of that of conventional media. The high-load agarose chromatography media has good prospects in the fields of biological separation and purification.
Description
Technical field
The invention belongs to biological technical field, specifically relate to a kind of high carrying capacity agarose chromatography media and preparation method thereof, and the application in purifying protein and other biological macromolecule purifying field.
Background technology
Separation and purification of protein is one of the key factor in protein science and product engineering field, the protein complicated in source, structure and properties is different and relevant biological activity material sphere, how realizing efficient separation and purification is the problem that researchers endeavour to solve always.Chromatography media is the most element of chromatographic technique, and its structure and properties is one of factor affecting albumen final separation and purification effect most critical.Along with the development of the subjects such as engineered protein, protein engineering, vaccine engineering science and application thereof, extensive efficient protein separation and purification is had higher requirement to high flux chromatographic technique, medium needs to have higher carrying capacity, i.e. the more target protein of separation and purification under identical even less consumption.
For metal chelate chromatography.Metal chelate chromatography is also called immobilized metal ion affinity chromatography, is a kind of chromatography method that 20 century 70s grow up mid-term.Its separation principle is mainly according to the histidine, tryptophan, cysteine etc. of protein surface, can interact specifically with metal ions such as Ni2+, Cu2+ and Co2+, thus good separation is realized (as document 1:KaraA to albumen, OsmanB, YavuzH, etal.Immobilizationof-AmylaseonCu2+ChelatedPoly (ethyleneglycoldimethacrylaten-vinylimidazole) MatrixviaAdsorption [J] .React.Funct.Polym., 2005,62 (1): 6168).Metal chelate chromatography has the features such as selective height, dielectric capacity is large, versatility good, separation condition is gentle, has become protein purification particularly one of the most effective technology in recombinant protein purification field.Such as, utilize technique for gene engineering by histidine-tagged with target protein amalgamation and expression, by a single metal chelate chromatography, the very high target recombinant albumen of purity can be obtained (as document 2:Guti é rrezR, DelValleEMM, Gal á nMA.CharacterizationofMassTransportProcessesinIMACChroma tographybyDynamicsMethods [J] .Biochem.Eng.J., 2007,35 (3): 264272).
Metal chelate chromatography medium generally by matrix, chelation group and metal ion three part form.Agarose has good hydrophily, porous, can supply the features such as derivative hydroxyl, stability, non-specific adsorption are low in a large number, is the matrix for the preparation of chromatography media be most widely used at present.Agarose matrix, after overactivation, reacts with chelating agents such as iminodiacetic acid (IDA), nitrilotriacetic acid (NTA) or carboxymethyl asparagic acids (CM-ASP), then chelated metal ions, prepares metal-chelating medium.Except protein classes, the main and medium self structure of the carrying capacity of metal-chelating medium, particularly amasss these two parameters closely related with metal ion density and albumen accessible surface.Metal chelate chromatography medium is the critical material affecting metal chelate chromatography effect, and its performance particularly carrying capacity, intensity etc. all directly affects final purification effect.Wherein, adopt the medium of high carrying capacity significantly can promote the disposal ability of purge process, reduce medium use cost, shorten the processing time, thus greatly improve purification efficiency.
It is that a kind of important method of its carrying capacity of raising is (as document 3:LenhoffAM.ProteinAdsorptionandTransportinPolymer-funct ionalizedIon-exchangers [J] .J.Chromatogr.A that medium is introduced glucan, 2011,1218 (49): 87488759).Document finds, introduce glucan in strong cation media manufacturing process after, the static adsorbance of medium and dynamic adsorbance improve 54-78% and 150-188% respectively (as document 4:ShiQH, JiaGD, SunY.Dextran-graftedCationExchangerBasedonSuperporousAga roseGel:AdsorptionIsotherms, UptakeKineticsandDynamicProteinAdsorptionPerformance [J] .J.Chromatogr.A, 2010,1217 (31): 50845091).This method is introduced the preparation process of metal chelate chromatography medium by us, found that, prepared medium to the carrying capacity of histidine mark lactic dehydrogenase comparatively commodity medium improve 26.6%.But, we find simultaneously, the glucan introduced through the method is that homogeneous phase is distributed in whole medium, whole grafting process at increase medium and protein binding site with while improving its carrying capacity, also ponding is had to media interior duct to a certain extent, this blocking hinders albumen and enters media interior, have a negative impact (as document 5: Zhang Jingfei to improving media charge further, Zhao Lan, Huang Yongdong, Zhang Zhigang, horse radiance, Su Zhiguo. the fabrication & properties of glucan graft type high carrying capacity metal-chelating medium. process engineering journal [J], 2015, 15 (1): 111-118).Forefathers by prepare shell/nuclear structure and only at shell grafting glucan and the method for coupling aglucon improve media charge (as gloomy in document 6:A Acker, the special human relations of J Burgers, G lattice rad, B-L Johnson, N norman. isolation medium, CN101765458A).How in glucan grafting process, both played and introduced glucan to the castering action of media charge, avoided again the phenomenon that it hinders albumen to enter because blocking duct.Based on this, be badly in need of proposing new glucan grafting method, to prepare high carrying capacity chromatography media.
Because above-mentioned defect, the design people, actively in addition research and innovation, to founding a kind of high carrying capacity agarose chromatography media and preparation method thereof, make it have more value in industry.
Because above-mentioned defect, the design people, actively in addition research and innovation, to founding to founding a kind of high carrying capacity agarose chromatography media and preparation method thereof, make it have more value in industry.
Summary of the invention
Based on above-mentioned consideration, the object of this invention is to provide a kind of high carrying capacity agarose chromatography media and preparation method thereof, by introducing and the regulation and control of linear glucan, make it while maintenance glucan produces facilitation to protein binding site, avoid it to the ponding in duct, thus farthest improve media charge.For metal chelate chromatography medium, the carrying capacity of medium to histidine mark lactic dehydrogenase prepared by the method is adopted to be about 2.5 times of commercial goods medium.The present invention takes following thinking: first, adopts Heterobifunctional group activator to activate agarose matrix; Secondly, introduce glucan to activated substrate, by controlling activation degree, Effective Regulation glucan access degree in the substrate, the composite construction of the shell forming bonding glucan and the kernel not containing glucan; Again, with the compound agarose microbeads of this shell/core for matrix, continue derivative, aglucon comprises metal ion, hydrophobic aglucon, ion-exchange group and albumen etc., finally prepares high carrying capacity agarose chromatography media.For metal-chelating medium, continue activation this shell/core compound agarose matrix, and react with chelating agent successively, chelated metal ions, finally prepare high carrying capacity metal-chelating medium.
The present invention is achieved by the following technical solutions.
The invention provides a kind of high carrying capacity agarose chromatography media, its structural formula as shown in Figure 1.This medium is that a kind of shell/core of matrix is composite structured with agarose, wherein shell through chemical bonding glucan, kernel is not containing glucan.The shell of medium and kernel equal chelating aglucon, shell and this two-part ligand density differentiation of kernel.
The invention provides a kind of preparation method of above-mentioned high carrying capacity agarose chromatography media, comprise the following steps:
S1: the Heterobifunctional group activator activated agarose matrix adopting active one end, one end inertia, makes agarose matrix is connected with inertia group.
S2: activate this inertia group, by regulating and controlling the activation degree of inertia group, the access degree of regulation and control glucan, forms shell thickness and all controlled composite construction of kernel size.
S3: with this shell/shell-core composite structure being connected with glucan for matrix, continue derivatization reaction, prepare high carrying capacity chromatography media.For metal-chelating medium, continue activation this shell/shell-core composite structure being connected with glucan, then react with chelating agent successively, chelated metal ions, prepare metal-chelating medium.
Further, in described step S1, described agarose matrix, its agarose concentration scope 2%-15%.
Further, in described step S1, described agarose matrix can be crosslinked matrix, also can be uncrosslinked matrix.
Further, in described step S1, activator system of described Heterobifunctional group is containing allyl compound, and described is one in allyl glycidyl ether and allyl bromide, bromoallylene or mixture containing allyl compound.
Further, in described step S2, the access content range 0.1-600mg/ml of described regulation and control glucan
Further, in described step S2, described composite construction molecular weight ranges 2kDa-150kDa.
Further, in described step S3, described chelating agent is selected from the one in iminodiacetic acid, carboxymethyl asparagic acid, nitrilotriacetic acid.
Further, in described step S3, described metal ion is selected from the one of Ni2+, Cu2+, Co2+, Fe2+.
Further, in described step S3, except metal ion, can also be ion-exchange group, hydrophobic grouping, albumen etc.
By such scheme, the present invention at least has the following advantages:
(1) by introducing glucan in media manufacturing process, considerably increasing medium can protein-bonded number of loci, the protein-bonded ability of medium thus significantly improve, and the carrying capacity of final medium to albumen effectively improves.
(2) by adopting Heterobifunctional group activator, inertia group is introduced to agarose matrix, this group of reactivation.The activation degree of this inertia group can accurately regulate and control, thus Effective Regulation glucan access degree, form shell bonding glucan, the shell/shell-core composite structure of kernel not containing glucan, finally form shell and the kernel chelating chromatography media of different densities aglucon.Shell thickness and the kernel size of this shell/shell-core composite structure all can realize good regulation and control, the density contrast alienation of shell/core two parts chelating aglucon controllable, contribute to improving media charge.
(3) by regulating shell/core thickness, dextran molecule amount, agarose matrix concentration etc., effective adjustment glucan introduces the pore passage structure after matrix, duct is avoided to block, simultaneously for the pore passage structure that albumen size Selection is suitable, amass to realize optimum protein accessible surface, increase media charge further.
Above-mentioned explanation is only the general introduction of technical solution of the present invention, in order to better understand technological means of the present invention, and can be implemented according to the content of description, coordinates accompanying drawing to be described in detail as follows below with preferred embodiment of the present invention.
Accompanying drawing explanation
Fig. 1 is the structural representation of agarose chromatography media of the present invention;
The dynamic carrying capacity of Fig. 2 metal-chelating medium to His-tagged lactic dehydrogenase measures curve.
Detailed description of the invention
The present invention includes a kind of high carrying capacity agarose chromatography media and preparation method thereof.
Below in conjunction with embodiment, technical solution of the present invention is further described.But the present invention is not limited to this.Comparative example 1 and 2 is chelated metal ions and only shell grafting glucan chelated metal ions more again after whole pearl body grafting glucan respectively, and comparative example 3 is commodity media.
Embodiment 1
Once activate: accurately take 10gSepharose6FF (6% agarose solution, crosslinked matrix), add in three mouthfuls of stills, add 0.01g sodium borohydride successively, 1g anhydrous sodium sulfate, 2mL30%NaOH solution, 1mL allyl glycidyl ether and 3mL water, system is in 50 DEG C of reaction 18h.Reaction adopts a large amount of deionized water cyclic washing medium after stopping.Obtained pi-allyl activated media, its pi-allyl density is that 150 μm of ol/g drain wet glue.
A bromination: add water 10g pi-allyl activated media 20mL, and sodium acetate 1.2g, bromine water 2mL, react 1h under room temperature.Reaction terminates rear massive laundering and washs medium.Obtain part bromination agarose medium, its pi-allyl density is that 105 μm of ol/g drain wet glue.
Grafting glucan: get 10g part bromination agarose medium, add 15mL0.6g/ml dextran solution (Dextran T 40, molecular weight 40kDa), 7mL water, 15mL1MNaOH solution (3.6mg sodium borohydride), at 30 DEG C, react 18h, reaction terminates a large amount of deionized water of rear employing and cleans medium.Obtained glucan graft type medium, its beta-dextran content is 10mg/mL glue.
Re-activation: get 10g glucan graft type medium take allyl glycidyl ether as activator, and obtained pi-allyl activated dextran graft type medium, its pi-allyl density is that 220 μm of ol/g drain wet glue.
Secondary bromination: add water 10g pi-allyl activated media 20mL, sodium acetate 1.2g and excessive bromine water, reacts 1h under room temperature, continues to add a certain amount of sodium formate colourless to system.Reaction terminates rear massive laundering and washs medium.Obtain bromination glucan graft type medium.
Coupling-chelating agent: get 10g bromination glucan graft type medium, adds the aqueous solution that 20mL dissolves 5g iminodiacetic acid, in 50 DEG C of reaction 24h.After reaction terminates, with a large amount of deionized water cyclic washing medium.
Chelating Ni2+: by the glucan graft type medium of coupling-chelating agent dress post, with the 0.05M nickel nitrate solution chelated metal ions of 5 times of column volumes, continues to use the same terms chelating once afterwards again.Chelating process terminates a large amount of deionized water washing medium of rear use.Obtained high carrying capacity metal-chelating medium, recording Ni2+ density is that 140 μm of ol/g drain wet glue.
After measured, this medium is 37mg/mL glue to the carrying capacity of His-tagged lactic dehydrogenase.
Embodiment 2
Once activate: accurately take the crosslinked Archon that 10g is prepared through 2% agarose solution concentration, all the other are with embodiment 1.Obtained pi-allyl activated media, its pi-allyl density is that 10 μm of ol/g drain wet glue.
A bromination: add water 10g pi-allyl activated media 20mL, and sodium acetate 1.2g, bromine water 1mL, react 1h under room temperature.Reaction terminates rear massive laundering and washs medium.Obtain part bromination agarose medium, its pi-allyl density is that 1 μm of ol/g drains wet glue.
Grafting glucan: get 10g part bromination agarose medium, add 15mL0.6g/ml dextran solution (glucan T2, molecular weight 2kDa), 7mL water, 10mL1MNaOH solution (3.6mg sodium borohydride), at 30 DEG C, react 18h, reaction terminates a large amount of deionized water of rear employing and cleans medium.Obtained glucan graft type medium, its beta-dextran content is 0.1mg/mL glue.
Re-activation: get 10g glucan graft type medium take allyl glycidyl ether as activator, and obtained pi-allyl activated dextran graft type medium, its pi-allyl density is that 50 μm of ol/g drain wet glue.
Secondary bromination: add water 10g pi-allyl activated media 20mL, sodium acetate 1.2g and excessive bromine water, reacts 1h under room temperature, continues to add a certain amount of sodium formate colourless to system.Reaction terminates rear massive laundering and washs medium.Obtain bromination glucan graft type medium.
Coupling-chelating agent: get 10g bromination glucan graft type medium, adds the aqueous solution that 20mL dissolves 5g iminodiacetic acid, in 50 DEG C of reaction 24h.After reaction terminates, with a large amount of deionized water cyclic washing medium.
Chelating Cu2+: by the glucan graft type medium of coupling-chelating agent dress post, with the 0.05M nickel nitrate solution chelated metal ions of 5 times of column volumes, continues to use the same terms chelating once afterwards again.Chelating process terminates a large amount of deionized water washing medium of rear use.Obtained high carrying capacity metal-chelating medium, recording Ni2+ density is that 1 μm of ol/g drains wet glue.
After measured, this medium is 28mg/mL glue to the carrying capacity of His-tagged lactic dehydrogenase.
Embodiment 3
Once activate: accurately take the crosslinked Archon that 10g is prepared through 15% agarose solution concentration, take allyl bromide, bromoallylene as activator, add 0.01g sodium borohydride successively, 1g anhydrous sodium sulfate, 2mL30%NaOH solution, 3mL allyl glycidyl ether and 3mL water, system is in 50 DEG C of reaction 12h.Obtained pi-allyl activated media, its pi-allyl density is that 2000 μm of ol/g drain wet glue.
A bromination: add water 10g pi-allyl activated media 20mL, and sodium acetate 1.2g, bromine water 3mL, react 1h under room temperature.Reaction terminates rear massive laundering and washs medium.Obtain part bromination agarose medium, its pi-allyl density is that 1900 μm of ol/g drain wet glue.
Grafting glucan: get 10g part bromination agarose medium, add 15mL0.6g/ml dextran solution (glucan T150, molecular weight 150kDa), 7mL water, 20mL1MNaOH solution (3.6mg sodium borohydride), at 30 DEG C, react 18h, reaction terminates a large amount of deionized water of rear employing and cleans medium.Obtained glucan graft type medium, its beta-dextran content is 600mg/mL glue.
Re-activation: get 10g glucan graft type medium take epoxychloropropane as activator.Add 0.01g sodium borohydride successively, 5mL10%NaOH solution, 5mL epoxychloropropane, system is in 30 DEG C of reaction 5h.Reaction adopts a large amount of deionized water cyclic washing medium after stopping.Obtained epoxy-activated medium, its epoxy radicals density is that 250 μm of ol/g drain wet glue.
Coupling-chelating agent: get 10g bromination glucan graft type medium, adds the aqueous solution that 20mL dissolves 5g carboxymethyl asparagic acid, in 50 DEG C of reaction 24h.After reaction terminates, with a large amount of deionized water cyclic washing medium.
Chelating Co2+: by the glucan graft type medium of coupling-chelating agent dress post, with the 0.05M cobalt sulfate solution chelated metal ions of 5 times of column volumes, continues to use the same terms chelating once afterwards again.Chelating process terminates a large amount of deionized water washing medium of rear use.Obtained high carrying capacity metal-chelating medium, recording Co2+ density is that 2000 μm of ol/g drain wet glue.
After measured, this medium is 38mg/mL glue to the carrying capacity of His-tagged lactic dehydrogenase.
Embodiment 4
Once activate: accurately take 10gSepharose6B (uncrosslinked matrix), with the mixture (volume ratio 1:1) of 1mL allyl glycidyl ether and allyl bromide, bromoallylene, all the other are with pi-allyl activated media.Obtained pi-allyl activated media, its pi-allyl density is that 160 μm of ol/g drain wet glue.
A bromination: with embodiment 1.Obtain part bromination agarose medium, its pi-allyl density is that 140 μm of ol/g drain wet glue.
Grafting glucan: with embodiment 1.Obtained glucan graft type medium, its beta-dextran content is 10mg/mL glue.
Re-activation: get 10g glucan graft type medium take allyl glycidyl ether as activator, and obtained pi-allyl activated dextran graft type medium, its pi-allyl density is that 240 μm of ol/g drain wet glue.
Secondary bromination: with embodiment 1.Obtain bromination glucan graft type medium.
Coupling-chelating agent: get 10g bromination glucan graft type medium, adds the aqueous solution that 20mL dissolves 5g nitrilotriacetic acid, in 50 DEG C of reaction 24h.After reaction terminates, with a large amount of deionized water cyclic washing medium.
Chelating Fe2+: by the glucan graft type medium of coupling-chelating agent dress post, with the 0.05M ferrum sulfuricum oxydatum solutum chelated metal ions of 5 times of column volumes, continues to use the same terms chelating once afterwards again.Chelating process terminates a large amount of deionized water washing medium of rear use.Obtained high carrying capacity metal-chelating medium, recording Fe2+ density is that 150 μm of ol/g drain wet glue.
After measured, this medium is 34mg/mL glue to the carrying capacity of His-tagged lactic dehydrogenase.
Comparative example 1
Once activate: accurately take 10gSepharose6FF, add in three mouthfuls of stills, add 0.01g sodium borohydride successively, 1g anhydrous sodium sulfate, 2mL30%NaOH solution, 1mL allyl glycidyl ether and 3mL water, system is in 50 DEG C of reaction 18h.Reaction adopts a large amount of deionized water cyclic washing medium after stopping.Obtained pi-allyl activated media, its pi-allyl density is that 150 μm of ol/g drain wet glue.
A bromination (complete bromination): 10g pi-allyl activated media is added 20mL water, and 1.2g sodium acetate and excessive bromine water, react 1h under room temperature, continues to add a certain amount of sodium formate colourless to system.Reaction terminates rear massive laundering and washs medium.Obtain whole bromination agarose medium, its pi-allyl density is 0.
Grafting glucan: get the complete bromination agarose medium of 10g, add 15mL0.6g/ml dextran solution (Dextran T 40), 7mL water, 15mL1MNaOH solution (3.6mg sodium borohydride), at 30 DEG C, react 18h, reaction terminates a large amount of deionized water of rear employing and cleans medium.Obtained glucan graft type medium, its beta-dextran content is 15mg/mL glue.
Re-activation: get 10g glucan graft type medium, continues activation according to the method once activated, and obtained pi-allyl activated dextran graft type medium, its pi-allyl density is that 200 μm of ol/g drain wet glue.
Secondary bromination: 10g pi-allyl activated media is added 20mL water, 1.2g sodium acetate and excessive bromine water, react 1h under room temperature, continues to add a certain amount of sodium formate colourless to system.Reaction terminates rear massive laundering and washs medium.Obtain bromination glucan graft type medium.
Coupling-chelating agent: get 10g bromination glucan graft type medium, adds the aqueous solution that 20mL dissolves 5g iminodiacetic acid, in 50 DEG C of reaction 24h.After reaction terminates, with a large amount of deionized water cyclic washing medium.
Chelating Ni
2+: by the glucan graft type medium of coupling-chelating agent dress post, with the 0.05M nickel nitrate solution chelated metal ions of 5 times of column volumes, continue again afterwards to use the same terms chelating once.Chelating process terminates a large amount of deionized water washing medium of rear use.Obtained high carrying capacity metal-chelating medium, records Ni
2+density is that 100 μm of ol/g drain wet glue.
After measured, this medium is 22mg/mL glue to the carrying capacity of His-tagged lactic dehydrogenase.
Comparative example 2
Once activate: accurately take 10gSepharose6FF, add in three mouthfuls of stills, add 0.01g sodium borohydride successively, 1g anhydrous sodium sulfate, 2mL30%NaOH solution, 1mL allyl glycidyl ether and 3mL water, system is in 50 DEG C of reaction 18h.Reaction adopts a large amount of deionized water cyclic washing medium after stopping.Obtained pi-allyl activated media, its pi-allyl density is that 150 μm of ol/g drain wet glue.
A bromination: 10g pi-allyl activated media is added 20mL water, 1.2g sodium acetate, 2mL bromine water, react 1h under room temperature.Reaction terminates rear massive laundering and washs medium.Obtain part bromination agarose medium, its pi-allyl density is that 105 μm of ol/g drain wet glue.
Grafting glucan: get the complete bromination agarose medium of 10g, add 15mL0.6g/ml dextran solution (Dextran T 40), 7mL water, 15mL1MNaOH solution (3.6mg sodium borohydride), at 30 DEG C, react 18h, reaction terminates a large amount of deionized water of rear employing and cleans medium.Obtained glucan graft type medium, its beta-dextran content is 10mg/mL glue.
Re-activation: get 10g glucan graft type medium, continues activation according to the method once activated, and obtained pi-allyl activated dextran graft type medium, its pi-allyl density is that 220 μm of ol/g drain wet glue.
Secondary bromination: 10g pi-allyl activated media is added 20mL water, 1.2g sodium acetate, 2mL bromine water, react 1h under room temperature.Reaction terminates rear massive laundering and washs medium.Obtain part bromination glucan graft type medium, its pi-allyl density is that 170 μm of ol/g drain wet glue.
Coupling-chelating agent: get 10g bromination glucan graft type medium, adds the aqueous solution that 20mL dissolves 5g iminodiacetic acid, in 50 DEG C of reaction 24h.After reaction terminates, with a large amount of deionized water cyclic washing medium.
Chelating Ni
2+: by the glucan graft type medium of coupling-chelating agent dress post, with the 0.05M nickel nitrate solution chelated metal ions of 5 times of column volumes, continue again afterwards to use the same terms chelating once.Chelating process terminates a large amount of deionized water washing medium of rear use.Obtained high carrying capacity metal-chelating medium, records Ni
2+density is that 30 μm of ol/g drain wet glue.
After measured, this medium is 16mg/mL glue to the carrying capacity of His-tagged lactic dehydrogenase.
Comparative example 3
Measuring NiSepharoseFF (GEHealthcare) is 14mg/mL glue to the carrying capacity of His-tagged lactic dehydrogenase.
Analytical method
(1) pi-allyl density measurement
According to the pi-allyl density of potassium bromate standard liquid titration measuring microballoon.
(2) beta-dextran content measures
The beta-dextran content of microballoon is measured according to dry weight-loss method.
(3) metal ion density measures
According to the metal ion density of precipitation by metallic ion titration measuring microballoon.
(4) carrying capacity measures
Medium is filled post (
c.V.=10mL), be connected to AKTAPURIFIER10 tomographic system, level pad is 0.02MPB-0.15MNaCl-5mM imidazoles (pH7.4) solution, with histidine mark (His-tagged) lactic dehydrogenase loading, flow velocity 0.4mL/min, stream is worn after liquid reaches capacity and is stopped loading, with 0.02MPB-0.15MNaCl-0.5M imidazoles (pH7.4) eluant solution, collect eluting peak, detect A280, measure efflux and eluent protein concentration (Fig. 2) respectively.Media charge calculates according to the following formula:
Q＝C·V/Vgel
In formula, Q is media charge (mg), C be eluent protein concentration (mg/mL), V be effluent volume (mL), Vgel is medium volume (mL).
The above is only the preferred embodiment of the present invention; be not limited to the present invention; should be understood that; for those skilled in the art; under the prerequisite not departing from the technology of the present invention principle; can also make some improvement and modification, these improve and modification also should be considered as protection scope of the present invention.
Claims (10)
1. a high carrying capacity agarose chromatography media, is characterized in that:
This medium is that a kind of shell/core of matrix is composite structured with agarose, wherein shell through chemical bonding glucan, kernel is not containing glucan.The shell of medium and kernel equal chelating aglucon, shell and this two-part ligand density differentiation of kernel.
2. a preparation method for high carrying capacity agarose chromatography media, is characterized in that, comprise following step:
S1: the Heterobifunctional group activator activated agarose matrix adopting active one end, one end inertia, makes agarose matrix is connected with inertia group;
S2: activation inertia group, by regulating and controlling the activation degree of inertia group, the access degree of regulation and control glucan, forms a kind of shell/shell-core composite structure being connected with glucan, and shell thickness and kernel size all controlled;
S3: with this shell/shell-core composite structure being connected with glucan for matrix, carry out successively activating and coupling reaction, prepare high carrying capacity chromatography media.Activate this shell/shell-core composite structure being connected with glucan, then react with chelating agent successively, chelated metal ions, prepare metal-chelating medium.
3. method as claimed in claim 2, is characterized in that: in described step S1, described agarose matrix, its agarose concentration scope 2%-15%.
4. method as claimed in claim 2, it is characterized in that: in described step S1, described agarose matrix can be crosslinked matrix, also can be uncrosslinked matrix.
5. method as claimed in claim 2, is characterized in that: in described step S1, and activator system of described Heterobifunctional group is containing allyl compound, and described is one in allyl glycidyl ether and allyl bromide, bromoallylene or mixture containing allyl compound.
6. method as claimed in claim 2, is characterized in that: in described step S2, the access content range 0.1-600mg/ml of described regulation and control glucan.
7. method as claimed in claim 2, is characterized in that: in described step S2, described composite construction molecular weight ranges 2kDa-150kDa.
8. method as claimed in claim 2, it is characterized in that: in described step S3, described chelating agent is selected from the one in iminodiacetic acid, carboxymethyl asparagic acid, nitrilotriacetic acid.
9. method as claimed in claim 2, it is characterized in that: in described step S3, described metal ion is selected from the one of Ni2+, Cu2+, Co2+, Fe2+.
10. method as claimed in claim 2, it is characterized in that: in described step S3, except metal ion, can also be ion-exchange group, hydrophobic grouping, albumen etc.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105944686A (en) * | 2016-05-18 | 2016-09-21 | 绿麒（厦门）海洋生物科技有限公司 | Agarose gel microspheres containing glucosamine group and preparation method of agarose gel microspheres |
| CN107267475A (en) * | 2017-08-11 | 2017-10-20 | 浙江福斯特新材料研究院有限公司 | A kind of method that metal chelate affinity chromatography purifies thioredoxin |
| CN107674112A (en) * | 2017-10-31 | 2018-02-09 | 苏州博进生物技术有限公司 | A kind of affinity chromatography medium using heparin as part |
| CN107754767A (en) * | 2017-10-31 | 2018-03-06 | 苏州博进生物技术有限公司 | A kind of high stability immobilization metal chelates affinity chromatography medium |
| CN109647361A (en) * | 2019-02-22 | 2019-04-19 | 北京石油化工学院 | A kind of preparation method of composite polymer chromatography media |
| CN109806916A (en) * | 2019-03-15 | 2019-05-28 | 中科森辉微球技术（苏州）有限公司 | High performance anion exchange media and preparation method thereof |
| CN112980037A (en) * | 2021-03-01 | 2021-06-18 | 苏州星谱生物科技有限公司 | Hydrophobic medium and preparation method thereof |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101003017A (en) * | 2006-01-20 | 2007-07-25 | 中国科学院过程工程研究所 | Agarose hydrophobic chromatoghaphy medium and its application in purifying yeast expression HBsAg |
| CN101602805A (en) * | 2009-07-08 | 2009-12-16 | 中国科学院过程工程研究所 | Be used for agarose compatible medium of purifying hand foot mouth disease immunoglobulin (Ig) and preparation method thereof |
| CN101765458A (en) * | 2007-07-25 | 2010-06-30 | 通用电气健康护理生物科学股份公司 | separation matrix |
| CN101948529A (en) * | 2010-08-13 | 2011-01-19 | 中国科学院过程工程研究所 | Hydrophobic chromatography separating and purifying method for efficiently purifying metal ion binding proteins |
-
2015
- 2015-10-21 CN CN201510685375.0A patent/CN105498701B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101003017A (en) * | 2006-01-20 | 2007-07-25 | 中国科学院过程工程研究所 | Agarose hydrophobic chromatoghaphy medium and its application in purifying yeast expression HBsAg |
| CN101765458A (en) * | 2007-07-25 | 2010-06-30 | 通用电气健康护理生物科学股份公司 | separation matrix |
| CN101602805A (en) * | 2009-07-08 | 2009-12-16 | 中国科学院过程工程研究所 | Be used for agarose compatible medium of purifying hand foot mouth disease immunoglobulin (Ig) and preparation method thereof |
| CN101948529A (en) * | 2010-08-13 | 2011-01-19 | 中国科学院过程工程研究所 | Hydrophobic chromatography separating and purifying method for efficiently purifying metal ion binding proteins |
Non-Patent Citations (1)
| Title |
|---|
| 张静飞等: ""葡聚糖接枝型高载量金属螯合介质的制备与性能"", 《过程工程学报》 * |
Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105944686A (en) * | 2016-05-18 | 2016-09-21 | 绿麒（厦门）海洋生物科技有限公司 | Agarose gel microspheres containing glucosamine group and preparation method of agarose gel microspheres |
| CN105944686B (en) * | 2016-05-18 | 2018-04-20 | 绿麒（厦门）海洋生物科技有限公司 | A kind of agarose gel microsphere of the glycosyl containing aminoglucose and preparation method thereof |
| CN107267475A (en) * | 2017-08-11 | 2017-10-20 | 浙江福斯特新材料研究院有限公司 | A kind of method that metal chelate affinity chromatography purifies thioredoxin |
| CN107674112A (en) * | 2017-10-31 | 2018-02-09 | 苏州博进生物技术有限公司 | A kind of affinity chromatography medium using heparin as part |
| CN107754767A (en) * | 2017-10-31 | 2018-03-06 | 苏州博进生物技术有限公司 | A kind of high stability immobilization metal chelates affinity chromatography medium |
| CN107754767B (en) * | 2017-10-31 | 2020-04-21 | 苏州博进生物技术有限公司 | High-stability immobilized metal chelating affinity chromatography medium |
| CN109647361A (en) * | 2019-02-22 | 2019-04-19 | 北京石油化工学院 | A kind of preparation method of composite polymer chromatography media |
| CN109806916A (en) * | 2019-03-15 | 2019-05-28 | 中科森辉微球技术（苏州）有限公司 | High performance anion exchange media and preparation method thereof |
| CN109806916B (en) * | 2019-03-15 | 2021-12-21 | 中科森辉微球技术（苏州）有限公司 | High-performance anion exchange medium and preparation method thereof |
| CN112980037A (en) * | 2021-03-01 | 2021-06-18 | 苏州星谱生物科技有限公司 | Hydrophobic medium and preparation method thereof |
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