CN110339828A - Using benzothiazolyl thiocarboxylic acid as chromatography media of functional ligand and preparation method thereof - Google Patents
Using benzothiazolyl thiocarboxylic acid as chromatography media of functional ligand and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a kind of using benzothiazolyl thiocarboxylic acid as chromatography media of functional ligand and preparation method thereof.Using hydrophilic porous microballoon as chromatography substrate, sequentially adds dimethyl sulfoxide and allyl bromide, bromoallylene is activated；The chromatography substrate of activation is reacted with N- bromo-succinimide, carries out bromo alcoholization；Chromatography substrate after bromo is refined is mixed with hexamethylene diamine solution, carries out amination；Amination matrix is further coupled benzothiazolyl thiocarboxylic acid aglucon；Finally unreacted amino is closed using sodium acetate and solution of acetic anhydride, is obtained using benzothiazolyl thiocarboxylic acid as the chromatography media of functional group.Novel chromatography media of the invention has the characteristics such as antibody adsorption capacity is big, salt tolerance is strong, and pH, which is adjusted to faintly acid, can be realized antibody high efficiente callback, and preparation process is easy, cheap, can be used for antibody separation.
Description
Technical field
The present invention relates to a kind of using benzothiazolyl thiocarboxylic acid as chromatography media of functional ligand and preparation method thereof, belongs to
Protein chromatographic isolation technics in biological chemical field.
Background technique
Often purity requirement is higher for antibody product, while must also keep bioactivity, therefore traditional separation process is past
It is past to be difficult to meet the requirements.Albumin A or protein g affinity chromatography medium can specifically bind antibody, but protide affinity ligand is easy
It falls off, it is low to reuse number, and medium is expensive, use cost is high, limits scale application.Traditional ion is handed over
Although change the methods of chromatography and hydrophobic interaction chromatography can binding antibody, it is specific and selectivity it is poor, separation step
Rapid more, purification effect is limited.Therefore, it is Research Emphasis that exploitation, which has the non-protein class aglucon of antibody selectivity,.
Mainly there are bionical affinity ligand and polypeptide aglucon etc. for the non-protein aglucon of antibody separation, these aglucons often mistake
In concern specificity and affinity, cause antibody elution condition more harshness or yield lower.Mixed-Modechromatography is a kind of
Novel bioseparation technology, aglucon have multiple functions group concurrently, can generate a variety of phases such as hydrophobic and electrostatic with target protein
Interaction.The ligand density of mixed mode medium is usually higher, and adsorption capacity is big, has salt tolerant characterization of adsorption, elution requirement temperature
With particularly suitable for isolating and purifying on a large scale, be applied in the isolating and purifying of the albumen such as antibody.Mixed-Modechromatography
Key be special designing, a variety of interactions of combination functional ligand.Patent CN101402671B, CN101899110B and
CN101948535A is reported to be situated between by the Mixed-Modechromatography of aglucon of mercapto ethylpyridine, thiopurine methyltransferase imidazoles and mercaptobenzimidazole
Matter separating immune globulin from livestock and poultry blood has the characteristics that step is simple, separative efficiency is high, low in cost.Patent
EP2459308A1 reports the preparation process of the Mixed-Modechromatography medium using benzothiazole compound as aglucon, realizes
Multiple protein isolates and purifies.Patent US6919436B2 and CN1972961B report benzothiazole compound as chromatography
The separation ligand of medium, is coupled to agarose matrix, for isolating and purifying for the albumen such as serum or blood plasma.Wang etc.
(J.Chromatogr.B, 2013,936:33) uses mixed mode medium Bestarose Diamond MMA, Bestarose
Diamond MMC, MEP HyperCel and PPA HyperCel medium separate IgG from simulation serum, wherein Bestarose
Diamond MMA medium adsorbs IgG and HSA simultaneously, and adsorptive selectivity is poor；Bestarose Diamond MMC and PPA
HyperCel medium is unfavorable for actual separation application there are antibody elution difficulty；MEP HyperCel medium primary attachment IgG,
But still with a small amount of HSA, the selectivity of IgG is general.Therefore, the mixed mode medium for developing more excellent performance, for antibody
Large-scale separation purifying has great significance.
It is separated for antibody, the small molecule compounds aglucon such as screening pyridines, imidazoles, thiazoles and multi-aromatic ring class
Library has carried out aglucon and IgG compatibility characterization, discovery benzothiazolyl thiocarboxylic acid class compound high, salt tolerant to IgG compatibility
Property it is strong, be that potential antibody separates novel mixed mode aglucon.
Summary of the invention
The object of the present invention is to provide a kind of using benzothiazolyl thiocarboxylic acid as the chromatography media of functional ligand and its system
Preparation Method.
It include chromatography substrate and aglucon, the chromatography by the chromatography media of functional ligand of benzothiazolyl thiocarboxylic acid
Matrix is the hydrophilic porous microballoon with hydroxyl, and aglucon is the thio carboxylic of benzothiazolyl being coupled after allyl bromide, bromoallylene activates
Acid, the benzothiazolyl thiocarboxylic acid are 3- (2-[4-morpholinodithio base is thio) propionic acid, 2- (benzothiazolyl is thio) acetic acid
Or one of 2- (2-[4-morpholinodithio base is thio) propionic acid.
When benzothiazolyl thiocarboxylic acid is 3- (2-[4-morpholinodithio base is thio) propionic acid, the structure composition of chromatography media
Are as follows:
When benzothiazolyl thiocarboxylic acid is 2- (benzothiazolyl is thio) acetic acid, the structure composition of chromatography media are as follows:
When benzothiazolyl thiocarboxylic acid is 2- (2-[4-morpholinodithio base is thio) propionic acid, the structure composition of chromatography media are as follows:
Chromatography substrate and a ligand molecule structure are only gived in the structure composition of the chromatography media, are only to show
Example explanation, the surface of chromatography substrate and internal channel surfaces have a large amount of ligand molecule.
The chromatography substrate is the Hydrophilic polymeric microspheres with porous structure and surface hydroxyl, and structural formula is as follows:Structural formula only provides-an OH, only exemplary illustration, and surface has a large amount of-OH.
The chromatography substrate is Ago-Gel or cellulose microsphere.
The ligand density of the chromatography media is 40-140 μm of ol/ml.
Described includes the following steps: by the preparation method of the chromatography media of functional ligand of benzothiazolyl thiocarboxylic acid
1) after draining chromatography substrate, 20% (v/v) dimethyl sulfoxide, the 0.1-1 of 0.2-1 times of chromatography substrate quality is added
Times allyl bromide, bromoallylene of chromatography substrate quality and the sodium hydroxide of 0.1-0.5 times of chromatography substrate quality, at 25 DEG C in 150rpm shaking table
Activation 8-48 hours filters, and is washed with deionized to obtain activation chromatography substrate；
2) the N- bromo-succinimide for activating chromatography substrate and 0.1-0.5 times of chromatography substrate quality is mixed and carries out bromo
Refine, reacted 1-5 hours in 150rpm shaking table at 25 DEG C, filter, be washed with deionized, obtains the matrix of bromo alcoholization；
3) by the hexamethylene diamine and 0.1-1 times of chromatography substrate matter of the matrix of bromo alcoholization and 0.1-1 times of chromatography substrate quality
The 0.5-1M sodium carbonate buffer of amount mixes, and the pH of sodium carbonate buffer is 10-12, reacts 8-48 in 150rpm shaking table at 25 DEG C
Hour, obtain amidized matrix；
4) by the benzothiazolyl thiocarboxylic acid of amidized matrix and 0.1-0.3 times of chromatography substrate quality, 0.4-2 times of layer
Analyse 2- (7- azo benzotriazole)-N, N, N' of matrix quality, N'- tetramethylurea hexafluorophosphoric acid ester, 0.8-4 times of chromatography substrate
The n,N-Dimethylformamide solution of the n,N-diisopropylethylamine of quality mixes, and reaction 8-48 is small in 150rpm shaking table at 25 DEG C
When, the medium after obtaining the coupling of benzothiazolyl thiocarboxylic acid；
5) medium after the coupling of benzothiazolyl thiocarboxylic acid is filtered, deionized water washing is added to sodium acetate and second
It in the mixed liquor of acid anhydrides, is reacted 2-8 hours in 150rpm shaking table at 25 DEG C, deionized water washing is obtained with benzothiazolyl sulphur
It is the chromatography media of functional ligand for carboxylic acid.
The present invention develop using benzothiazolyl thiocarboxylic acid as the chromatography media of functional ligand, can be used for mixed mode
Chromatography has the advantage that (1) antibody adsorption capacity is big, and processing capacity is strong, and hIgG static capacity is up to 140mg/
Ml humid medium；(2) salt tolerant strong adsorption, in wider conductivity range (0-100mS/cm), adsorption capacity is kept not substantially
Become；(3) elution is convenient, near eluent pH to 3.5, so that it may realize elution completely, avoid peracid, crosses alkali or with high salt etc. to albumen
Structure and activity have an adverse effect；(4) medium character is stablized, and aglucon is activated and be coupled using allyl bromide, bromoallylene, gained medium
Aglucon is stablized, and cleaning and regeneration is convenient.The novel Mixed-Modechromatography medium that the present invention is developed, with benzothiazolyl thiocarboxylic acid
For functional ligand, have the characteristics that significant mixed mode adhesion protein and chromatography, there is good adsorption capacity to antibody
And selectivity, it can be used for the prepare with scale of antibody.
Detailed description of the invention
Fig. 1 is the combination carrying capacity that the chromatography media absorption IgG of 3- (2-[4-morpholinodithio base is thio) propionic acid is coupled in embodiment 2
Distribution map.
Specific embodiment
The invention will be further described by the following examples:
Embodiment 1
It takes and drains Ago-Gel 10g, 2g 20% (v/v) dimethyl sulfoxide, 1g allyl bromide, bromoallylene and 1g hydroxide is added
Sodium is activated 48 hours in 150rpm shaking table at 25 DEG C, is filtered, is washed with deionized to obtain activated substrate；Then base will be activated
Matter, the mixing of 1g N- bromo-succinimide carry out bromhydrin, react 2 hours in 150rpm shaking table at 25 DEG C, filter, spend
Ion water washing；Then bromo matrix and 2g hexamethylene diamine and 1M sodium carbonate buffering (pH 12) are mixed, 150rpm shakes at 25 DEG C
It is reacted 48 hours in bed, obtains amino-reactive matrix；1g amino-reactive matrix is taken, the 2mL (2-[4-morpholinodithio of 3- containing 3g is added to
Base is thio) propionic acid, 60mg 2- (7- azo benzotriazole)-N, N, N', N'- tetramethylurea hexafluorophosphoric acid ester and 37.5 μ L N,
In the n,N-Dimethylformamide of N- diisopropylethylamine, reacted 8 hours in shaking bath at 25 DEG C；Finally medium is filtered,
It is washed with deionized, is added in the mixed liquor of sodium acetate and acetic anhydride, react 12 hours, go in 150rpm shaking table at 25 DEG C
Ion water washing is obtained using 3- (2-[4-morpholinodithio base is thio) propionic acid as the chromatography media of aglucon, and ligand density is 40 μm of ol/
ml。
Embodiment 2
It takes and drains Ago-Gel 10g, 10g 20% (v/v) dimethyl sulfoxide, 10g allyl bromide, bromoallylene and 5g hydroxide is added
Sodium is activated 48 hours in 150rpm shaking table at 25 DEG C, is filtered, is washed with deionized to obtain activated substrate；Then base will be activated
Matter, the mixing of 5g N- bromo-succinimide carry out bromhydrin, react 3 hours in 150rpm shaking table at 25 DEG C, filter, spend
Ion water washing；Then bromo matrix and 3g hexamethylene diamine and 1M sodium carbonate buffering (pH 12) are mixed, 150rpm shakes at 25 DEG C
It is reacted 24 hours in bed；Take 1g amino-reactive matrix, be added to 2mL 3- containing 10g (2-[4-morpholinodithio base is thio) propionic acid,
100mg2- (7- azo benzotriazole)-N, N, N', N'- tetramethylurea hexafluorophosphoric acid ester and 64 μ L N, N- diisopropylethylamine
N,N-Dimethylformamide in, reacted 8 hours in shaking bath at 25 DEG C；Finally medium is filtered, is washed with deionized,
It is added in the mixed liquor of sodium acetate and acetic anhydride, is reacted 48 hours in 150rpm shaking table at 25 DEG C, deionized water washing obtains
Using 3- (2-[4-morpholinodithio base is thio) propionic acid as the chromatography media of aglucon, ligand density is 140 μm of ol/ml.In different pH and
Under NaCl concentration, medium is determined to the combination carrying capacity of human immunoglobulin(HIg) IgG, carrying capacity distribution map is shown in Fig. 1, abscissa pH,
Ordinate is NaCl concentration, and color and numerical value, which represent, in figure combines carrying capacity, and carrying capacity unit is mg/ml medium, it can be found that in pH
Within the scope of 5.0~9.0 and 0~500mM NaCl concentration, IgG combination carrying capacity is both greater than 100mg/ml medium, up to
140mg/ml medium illustrates that adsorption capacity is big, and salinity is small to Adsorption Effect, and salt tolerant characterization of adsorption is obvious.
Embodiment 3
It takes and drains Ago-Gel 10g, 6g 20% (v/v) dimethyl sulfoxide, 6g allyl bromide, bromoallylene and 4g hydroxide is added
Sodium is activated 25 hours in 150rpm shaking table at 25 DEG C, is filtered, is washed with deionized to obtain activated substrate；Then base will be activated
Matter, the mixing of 3g N- bromo-succinimide carry out bromhydrin, react 3 hours in 150rpm shaking table at 25 DEG C, filter, spend
Ion water washing；Then bromo matrix and 1.8g hexamethylene diamine and 0.5M sodium carbonate buffering (pH 11) are mixed, at 25 DEG C
It is reacted 16 hours in 150rpm shaking table；1g amino-reactive matrix is taken, 2mL 3- containing 6g (2-[4-morpholinodithio base is thio) third is added to
Acid, 60mg 2- (7- azo benzotriazole)-N, N, N', N'- tetramethylurea hexafluorophosphoric acid ester and 38 μ L N, N- diisopropyls
In the n,N-Dimethylformamide of ethamine, reacted 15 hours in shaking bath at 25 DEG C；Finally medium is filtered, uses deionized water
Washing, is added in the mixed liquor of sodium acetate and acetic anhydride, reacts 7 hours in 150rpm shaking table at 25 DEG C, deionized water washing,
It obtains using 3- (2-[4-morpholinodithio base is thio) propionic acid as the chromatography media of aglucon, ligand density is 60 μm of ol/ml.
Embodiment 4
It takes and drains Ago-Gel 10g, 3g 20% (v/v) dimethyl sulfoxide, 2g allyl bromide, bromoallylene and 1g hydroxide is added
Sodium is activated 8 hours in 150rpm shaking table at 25 DEG C, is filtered, is washed with deionized to obtain activated substrate；Then base will be activated
Matter, the mixing of 1g N- bromo-succinimide carry out bromhydrin, react 2 hours in 150rpm shaking table at 25 DEG C, filter, spend
Ion water washing；Then bromo matrix and 1g hexamethylene diamine and 1M sodium carbonate buffering (pH 12) are mixed, 150rpm shakes at 25 DEG C
It is reacted 8 hours in bed；1g amino-reactive matrix is taken, 2mL 3- containing 2g (2-[4-morpholinodithio base is thio) propionic acid, 20mg 2- are added to
(7- azo benzotriazole)-N, N, N', the N, N- of N'- tetramethylurea hexafluorophosphoric acid ester and 12 μ L N, N- diisopropylethylamine
In dimethylformamide, reacted 8 hours in shaking bath at 25 DEG C；Finally medium is filtered, is washed with deionized, is added to
It in the mixed liquor of sodium acetate and acetic anhydride, is reacted 5 hours in 150rpm shaking table at 25 DEG C, deionized water washing is obtained with 3- (2-
Benzothiazolyl is thio) propionic acid be aglucon chromatography media, ligand density be 45 μm of ol/ml.
Embodiment 5
It takes and drains Ago-Gel 10g, 10g 20% (v/v) dimethyl sulfoxide, 10g allyl bromide, bromoallylene and 5g hydroxide is added
Sodium is activated 48 hours in 150rpm shaking table at 25 DEG C, is filtered, is washed with deionized to obtain activated substrate；Then base will be activated
Matter, the mixing of 5g N- bromo-succinimide carry out bromhydrin, react 3 hours in 150rpm shaking table at 25 DEG C, filter, spend
Ion water washing；Then bromo matrix and 3g hexamethylene diamine and 1M sodium carbonate buffering (pH 12) are mixed, 150rpm shakes at 25 DEG C
It is reacted 24 hours in bed；1g amino-reactive matrix is taken, 2mL 2- containing 10g (benzothiazolyl is thio) acetic acid, 100mg are added to
2- (7- azo benzotriazole)-N, N, N', the N of N'- tetramethylurea hexafluorophosphoric acid ester and 64 μ L N, N- diisopropylethylamine,
In dinethylformamide, reacted 8 hours in shaking bath at 25 DEG C；Finally medium is filtered, is washed with deionized, is added
It in the mixed liquor of sodium acetate and acetic anhydride, is reacted 48 hours in 150rpm shaking table at 25 DEG C, deionized water washing is obtained with 2-
(benzothiazolyl is thio) acetic acid is the chromatography media of aglucon, and ligand density is 140 μm of ol/ml.
Embodiment 6
It takes and drains Ago-Gel 10g, 2g 20% (v/v) dimethyl sulfoxide, 1g allyl bromide, bromoallylene and 1g hydroxide is added
Sodium is activated 48 hours in 150rpm shaking table at 25 DEG C, is filtered, is washed with deionized to obtain activated substrate；Then base will be activated
Matter, the mixing of 1g N- bromo-succinimide carry out bromhydrin, react 2 hours in 150rpm shaking table at 25 DEG C, filter, spend
Ion water washing；Then bromo matrix and 2g hexamethylene diamine and 1M sodium carbonate buffering (pH 12) are mixed, 150rpm shakes at 25 DEG C
It is reacted 48 hours in bed, obtains amino-reactive matrix；1g amino-reactive matrix is taken, the 2mL (benzothiazolyl of 2- containing 1g is added to
It is thio) acetic acid, 60mg 2- (7- azo benzotriazole)-N, N, N', N'- tetramethylurea hexafluorophosphoric acid ester and 37.5 μ L N, N-
In the n,N-Dimethylformamide of diisopropylethylamine, reacted 8 hours in shaking bath at 25 DEG C；Finally medium is filtered, is used
Deionized water washing, be added in the mixed liquor of sodium acetate and acetic anhydride, reacted 12 hours in 150rpm shaking table at 25 DEG C, go from
Sub- water washing is obtained using 2- (benzothiazolyl is thio) acetic acid as the chromatography media of aglucon, and ligand density is 40 μm of ol/ml.
Embodiment 7
It takes and drains Ago-Gel 10g, 3g 20% (v/v) dimethyl sulfoxide, 2g allyl bromide, bromoallylene and 1g hydroxide is added
Sodium is activated 8 hours in 150rpm shaking table at 25 DEG C, is filtered, is washed with deionized to obtain activated substrate；Then base will be activated
Matter, the mixing of 1g N- bromo-succinimide carry out bromhydrin, react 2 hours in 150rpm shaking table at 25 DEG C, filter, spend
Ion water washing；Then bromo matrix and 1g hexamethylene diamine and 1M sodium carbonate buffering (pH 12) are mixed, 150rpm shakes at 25 DEG C
It is reacted 8 hours in bed；1g amino-reactive matrix is taken, 2mL 2- containing 2g (benzothiazolyl is thio) acetic acid, 20mg 2- are added to
(7- azo benzotriazole)-N, N, N', the N, N- of N'- tetramethylurea hexafluorophosphoric acid ester and 12 μ L N, N- diisopropylethylamine
In dimethylformamide, reacted 8 hours in shaking bath at 25 DEG C；Finally medium is filtered, is washed with deionized, is added to
It in the mixed liquor of sodium acetate and acetic anhydride, is reacted 5 hours in 150rpm shaking table at 25 DEG C, deionized water washing is obtained with 2- (benzene
Benzothiazolyl is thio) acetic acid be aglucon chromatography media, ligand density be 45 μm of ol/ml.
Embodiment 8
It takes and drains Ago-Gel 10g, 10g 20% (v/v) dimethyl sulfoxide, 10g allyl bromide, bromoallylene and 5g hydroxide is added
Sodium is activated 48 hours in 150rpm shaking table at 25 DEG C, is filtered, is washed with deionized to obtain activated substrate；Then base will be activated
Matter, the mixing of 5g N- bromo-succinimide carry out bromhydrin, react 3 hours in 150rpm shaking table at 25 DEG C, filter, spend
Ion water washing；Then bromo matrix and 3g hexamethylene diamine and 1M sodium carbonate buffering (pH 12) are mixed, 150rpm shakes at 25 DEG C
It is reacted 24 hours in bed；Take 1g amino-reactive matrix, be added to 2mL 2- containing 10g (2-[4-morpholinodithio base is thio) propionic acid,
100mg2- (7- azo benzotriazole)-N, N, N', N'- tetramethylurea hexafluorophosphoric acid ester and 64 μ L N, N- diisopropylethylamine
N,N-Dimethylformamide in, reacted 8 hours in shaking bath at 25 DEG C；Finally medium is filtered, is washed with deionized,
It is added in the mixed liquor of sodium acetate and acetic anhydride, is reacted 48 hours in 150rpm shaking table at 25 DEG C, deionized water washing obtains
Using 2- (2-[4-morpholinodithio base is thio) propionic acid as the chromatography media of aglucon, ligand density is 140 μm of ol/ml.
Embodiment 9
It takes and drains Ago-Gel 10g, 3g 20% (v/v) dimethyl sulfoxide, 2g allyl bromide, bromoallylene and 1g hydroxide is added
Sodium is activated 8 hours in 150rpm shaking table at 25 DEG C, is filtered, is washed with deionized to obtain activated substrate；Then base will be activated
Matter, the mixing of 1g N- bromo-succinimide carry out bromhydrin, react 2 hours in 150rpm shaking table at 25 DEG C, filter, spend
Ion water washing；Then bromo matrix and 1g hexamethylene diamine and 1M sodium carbonate buffering (pH 12) are mixed, 150rpm shakes at 25 DEG C
It is reacted 8 hours in bed；1g amino-reactive matrix is taken, 2mL 2- containing 2g (2-[4-morpholinodithio base is thio) propionic acid, 20mg 2- are added to
(7- azo benzotriazole)-N, N, N', the N, N- of N'- tetramethylurea hexafluorophosphoric acid ester and 12 μ L N, N- diisopropylethylamine
In dimethylformamide, reacted 8 hours in shaking bath at 25 DEG C；Finally medium is filtered, is washed with deionized, is added to
It in the mixed liquor of sodium acetate and acetic anhydride, is reacted 5 hours in 150rpm shaking table at 25 DEG C, deionized water washing is obtained with 2- (2-
Benzothiazolyl is thio) propionic acid be aglucon chromatography media, ligand density be 45 μm of ol/ml.
Claims (5)
1. a kind of using benzothiazolyl thiocarboxylic acid base as the chromatography media of functional ligand, it is characterised in that including chromatography substrate and
Aglucon, the chromatography substrate are the hydrophilic porous microballoon with hydroxyl, and aglucon is the benzene being coupled after allyl bromide, bromoallylene activates
Benzothiazolyl thiocarboxylic acid, the benzothiazolyl thiocarboxylic acid are 3- (2-[4-morpholinodithio base is thio) propionic acid, 2- (benzo thiophene
Oxazolyl is thio) one of acetic acid or 2- (2-[4-morpholinodithio base is thio) propionic acid；
When benzothiazolyl thiocarboxylic acid is 3- (2-[4-morpholinodithio base is thio) propionic acid, the structure composition of chromatography media are as follows:
When benzothiazolyl thiocarboxylic acid is 2- (benzothiazolyl is thio) acetic acid, the structure composition of chromatography media are as follows:
When benzothiazolyl thiocarboxylic acid is 2- (2-[4-morpholinodithio base is thio) propionic acid, the structure composition of chromatography media are as follows:
2. according to claim 1 using benzothiazolyl thiocarboxylic acid as the chromatography media of functional ligand, it is characterised in that
The chromatography substrate is the Hydrophilic polymeric microspheres with porous structure and surface hydroxyl.
3. according to claim 1 using benzothiazolyl thiocarboxylic acid as the chromatography media of functional ligand, it is characterised in that
The chromatography substrate is Ago-Gel or cellulose microsphere.
4. according to claim 1 using benzothiazolyl thiocarboxylic acid as the chromatography media of functional ligand, it is characterised in that
The ligand density of the chromatography media is 40-140 μm of ol/ml.
5. one kind is as described in claim 1 using benzothiazolyl thiocarboxylic acid as the preparation side of the chromatography media of functional ligand
Method, it is characterised in that include the following steps:
1) after draining chromatography substrate, 20% (v/v) dimethyl sulfoxide, the 0.1-1 times of layer of 0.2-1 times of chromatography substrate quality is added
The allyl bromide, bromoallylene of matrix quality and the sodium hydroxide of 0.1-0.5 times of chromatography substrate quality are analysed, is activated in 150rpm shaking table at 25 DEG C
It 8-48 hours, filters, is washed with deionized to obtain activation chromatography substrate；
2) the N- bromo-succinimide for activating chromatography substrate and 0.1-0.5 times of chromatography substrate quality is mixed and carries out bromhydrin
Change, reacted 1-5 hours in 150rpm shaking table at 25 DEG C, filter, be washed with deionized, obtains the matrix of bromo alcoholization；
3) by the hexamethylene diamine of the matrix of bromo alcoholization and 0.1-1 times of chromatography substrate quality and 0.1-1 times of chromatography substrate quality
The mixing of 0.5-1 M sodium carbonate buffer, the pH of sodium carbonate buffer are 10-12, and reaction 8-48 is small in 150rpm shaking table at 25 DEG C
When, obtain amidized matrix；
4) the benzothiazolyl thiocarboxylic acid of amidized matrix and 0.1-0.3 times of chromatography substrate quality, 0.4-2 times are chromatographed into base
2- (7- azo benzotriazole)-N, N, N' of matter quality, N'- tetramethylurea hexafluorophosphoric acid ester, 0.8-4 times of chromatography substrate quality
N,N-diisopropylethylamine the mixing of n,N-Dimethylformamide solution, reacted 8-48 hours in 150rpm shaking table at 25 DEG C,
Medium after obtaining the coupling of benzothiazolyl thiocarboxylic acid；
5) medium after the coupling of benzothiazolyl thiocarboxylic acid is filtered, deionized water washing is added to sodium acetate and acetic anhydride
Mixed liquor in, reacted 2-8 hours in 150rpm shaking table at 25 DEG C, deionized water washing, obtain with the thio carboxylic of benzothiazolyl
Acid is the chromatography media of functional ligand.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101694485A (en) * | 2009-09-27 | 2010-04-14 | 上海交通大学 | Method for utilizing affinity column to analyze holoprotein |
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