CN1163515C - New affinity chromatography medium and purification method for cobra neurotoxin - Google Patents
New affinity chromatography medium and purification method for cobra neurotoxin Download PDFInfo
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Abstract
The present invention relates to new affinity chromatography media aiming at cobra neurotoxin and a preparing method thereof. The present invention also relates to a new method of using the new affinity chromatography media to produce cobra neurotoxin in a purifying mode.
Description
The present invention relates to biotechnology and biomedicine field, more specifically, the present invention relates to a class new at affinity chromatography medium of Cobratoxin and preparation method thereof.The invention still further relates to the novel method of producing Cobratoxin with the new affinity chromatography medium purifying of this class.
In the traditional medicine of China, elapid venom can be used for by numbness, analgesia.By numbness, analgesic effective ingredient is the neural snake venom toxin of Naja.This neurotoxin is the polypeptide that is shorter than 100 amino-acid residues, its combination and act on neuromuscular junction place N-type acetylcholine receptor (Nicotic acetylcholinereceptor, AchR).Acetylcholine receptor (AchR) is mediating the variation that chemical neurotransmitter is converted into current potential, causes the opening of ionic channel, the positively charged ion permeability is increased, and cause the motor end-plate depolarize, finally causes the contraction of muscle.The effect of Cobratoxin and acetylcholine receptor (AchR) has been checked it and has been combined with chemical neurotransmitter acetylcholine, causes property paralysis of flaccid muscles.
Snake venom is used for calmness, pain relieving and has had quite long history in Chinese medicine, wherein neurotoxin is main effective constituent.Chinese Academy of Sciences Kunming animal the Chinese patent (CN 93121051.8) of application in 1993 compound that the neural poison of snake venom and other material form has been described in the application aspect drug rehabilitation and the treatment senile dementia.In the world, the United States Patent (USP) (US 4341762, and US 4741902) of 1981 and 1988 has been described postsynaptic and the application of presynaptic neurotoxin aspect the treatment nervous system disorders in the snake venom.United States Patent (USP) in 1998 (US 5714468) has been described and has been utilized novain (Botulinum toxin), the application of esp meat toxin A (Botulinumtoxin A) aspect medical science.
Aspect medical, the analgesic effect of neurotoxin is obvious, does not have habituation, particularly patient with advanced cancer.At cancer of late stage, the Rapid Expansion of tumour causes that the intensity of pain is increasing.Use general anodyne, as morphine, because its habituation, used in amounts will get more and more, and DeGrain, is difficult to remove patient's misery.Neurotoxin is used for pain relieving does not then have corresponding shortcoming.Along with the improvement of medical condition, cancer patients's survival time is more and more longer, alleviates their misery, improves its quality of life and becomes considerable social needs.
In addition, expanding economy and growth in the living standard, the progress of global integration makes a part of person of weak will catch drug addiction.Drug abuse all has very big harm to social stability and people's health.Because the habituation after drugs are sucked makes the smoker be difficult to disconnected the ring.And all adopt so far with the methadone both at home and abroad is that a kind of medicine of representative replaces another kind of dependence producing drug, under doctor's control with the compulsory treatment scheme of mild toxicity for big poison.Though certain effect is arranged, because drug user's psychic dependence height, and there is certain habituation in alternative medicine itself, it is also high that the drug addict gives up the back relapse rate.Therefore, drug rehabilitation is society and the considerable challenge of some families.Neurotoxin is a kind of major ingredient (CN 93121051.8) of not habituation anti-additive medicament, experimental results show that effect is fine.Simultaneously, report has been arranged, the quantity that suppresses vagusstoff (Ach) can delay and treat senile dementia (Dtsch Med Wochenschr.1998 Nov 27; 123 (48 Suppl): 1-4).Cobratoxin can suppress the transmission of too much vagusstoff (Ach) signal, may have certain result of treatment to senile dementia.
Studies show that cobra venom contains tens of kinds of complicated albumen and polypeptide compositions.This class neurotoxin is to have 60 polypeptides matters to more than 80 amino-acid residues, accounts at 10～20% of cobra venom.And other composition such as phosphodiesterase then have haematolysis ability, are the materials that causes side effect.Should remove from the angle of medicine as far as possible.Because the Cobratoxin quite stable, therefore the main method of separation and purification is to repeat to use ion-exchange on laboratory scale, molecular sieve gel chromatography, and reversed phase chromatography (Karlsson, E.et al.Eur.J.Biochem.1971,21:1-16; Cooper, D.﹠amp; Reich, E.J BiolChem 1972,247:3008-13 Vogel, C.W., et al.J Immunol Methods.1984,73:203-20; Chang, L.S.et al.J Biochem (Tokyo), 1997,122:1252-9).Yet, usually still contain a spot of other composition with the neurotoxin of ion-exchange and gel permeation chromatography preparation.These compositions can seriously disturb pharmaceutical test.The high pressure reversed phase chromatography is to remove the indispensable means of these trace impurity compositions.In addition, use ion-exchange repeatedly, these operate molecular sieve gel chromatography and reversed phase chromatography, can reduce the rate of recovery of neurotoxin, increase production cost.Thereby reduction economy.These may be that Cobratoxin is failed the reason of scale operation so far.
Therefore, this area presses for easy extensive separation of exploitation high-level efficiency, low cost, step and or the production technology and the technology of producing Cobratoxin.
An object of the present invention is to provide the new affinity chromatography medium of a class, this affinity media can with single-minded the combining of Cobratoxin height, thereby can be used for the separation and purification of Cobratoxin.
Another object of the present invention provides the preparation method of this class affinity chromatography medium.
Another object of the present invention provides the affine technical matters of a kind of low cost, high-level efficiency, scale operation Cobratoxin that step is easy.
In a first aspect of the present invention, a kind of affinity chromatography medium is provided, it comprises: solid phase carrier and be coupled at the affinity ligand shown in the formula I on the solid phase carrier:
In the formula, R
1Be the basic group that is selected from down group: amino, single substituted-amino, two substituted-aminos, trisubstituted-amino, amidino groups;
R
2Be the acidic-group that is selected from down group: carboxyl, sulfonic group, phosphate;
M1 is 1 or 2;
M2 is 1 or 2;
N1 is 1,2,3, or 4
N2 is 1,2,3, or 4.
Preferably, the affinity ligand shown in the formula I is selected from down group: para-amino benzoic acid, gavaculine, Sulphanilic Acid, m-sulfanilic acid, 3-aminocyclohexane carboxylic acid, 4-aminocyclohexane carboxylic acid.
In a second aspect of the present invention, a kind of method of separation and purification Cobratoxin is provided, it comprises step:
(a) raw material that will contain Cobratoxin is splined on the affinity column that affinity media is housed, thus make Cobratoxin be adsorbed in affine in;
(b) impurity that do not adsorb of flush away affinity column;
(c) Cobratoxin that is adsorbed on the affinity chromatography medium is carried out wash-out, thereby obtain the Cobratoxin of purifying;
Wherein, contain an affinity chromatography medium in this affinity column, this affinity chromatography medium comprises solid phase carrier and is coupled at the affinity ligand shown in the formula I on the solid phase carrier:
In the formula, R
1Be the basic group that is selected from down group: amino, single substituted-amino, two substituted-aminos, trisubstituted-amino, amidino groups;
R
2Be the acidic-group that is selected from down group: carboxyl, sulfonic group, phosphate;
M1 is 1 or 2;
M2 is 1 or 2;
N1 is 1,2,3, or 4
N2 is 1,2,3, or 4.
Preferably, in step (a), the condition of affinity media absorption Cobratoxin is that salt concn is 0.005-0.03M, and the pH condition is pH5.0-9.0; In step (c), the elution requirement of Cobratoxin is that pH is the damping fluid of pH2.0-5.0 or pH9.5-11.0, and wherein salt concn is 0.005-2.0M.
In a preference, this method also comprises and also comprises step: (d) Cobratoxin to the purifying that obtains in the step (c) carries out molecular sieve gel filtration, thereby is further purified Cobratoxin.
In a preference of the present invention, the epoxy chloropropane that passes through of affinity ligand is fixed on the agarose chromatography medium, and in the chromatography column of packing into.The neurotoxin sample is handled through exchange buffering liquid, goes up in affinity column, through washing the not impurity of absorption, single-minded again wash-out Cobratoxin.This Cobratoxin also can pass through the molecular sieve gel chromatography, and when being exchanged for suitable damping fluid, Cobratoxin obtains further refining.
Inventive point of the present invention is, found a kind of Cobratoxin affinity chromatography medium of high-affinity of novelty, and, provide a kind of separation and purification production technique of new scale operation Cobratoxin based on the affinity chromatography of Cobratoxin.
The characteristics of this technology are that low cost, high-level efficiency and step are easy.This affine technical matters, cost and production efficiency are far above other conventional separation methods, and the purity of product reaches more than 95%, product recovery rate 90%.
In addition, affinity purification technology of the present invention has the characteristics quick, process stabilizing of producing, used affinity ligand can tolerate essential on-the-spot on-line cleaning and sterilization in the medicine production, satisfies pharmaceutical production management regulation (GMP, Good Manufacturing Practice) easily.
Affinity chromatography is called the biological selectivity adsorption chromatography again, has become the indispensable isolation technique of purifying biological macromole active substance.Along with increase to the purity and the demand of activeconstituents in the biological products, the reduction of foreign matter content, traditional isolation technique such as gel-filtration and ion-exchange chromatography can not satisfy the requirement of industrial production and academic research again.
Compare with other method, the affinity chromatography method has following tangible characteristics.The affinity chromatography medium permission is selectively adsorbed biomolecules and is dissociated, and can obtain very high purifying multiple, usually is more than 1000 times.In addition, albumen is not only concentrated in purge process, and in the time of on being attached to affinity ligand, proteic character is also more stable; Its result has improved the activity recovery of target product again.Therefore, it is big that affine isolation technique is highly suitable for handling volume, the biologically active substance that concentration is low.
In the purifying process of scale operation, adopt the affinity chromatography technology can significantly reduce the step of purge process, thereby reduced the production time and the cost of qualified product.When the downstream production cost account for total cost of production 80% the time seem more important.Any link in purge process can be used the affinity chromatography technology, but adopt more early, the economic benefit of acquisition is big more.J.Bonnerjea (Biotechnology, 4,954-958,1986) shows in all technologies of being investigated, in the unit operation of single step purification best results, have 45% to utilize affine step to obtain to the result of biological products production technique investigation.
The key of affine separating medium is affinity ligand (also can be described as " affinity ligand ").Affinity ligand must can be optionally and is reversibly adsorbed biomacromolecule.Traditional affinity ligand is the basis that the successful use of affinity media is established.Yet, in industrial production, natural affinity ligand as antibody, coenzyme, amino acid, polypeptide, albumen, lectin, has unsurmountable shortcoming: the most outstanding factor is the cost costliness, biological and chemical character instability, be difficult in the production keep in conjunction with active, can not stand online cleaning and disinfection, therefore, also may be by other material, as virus and contaminated with endotoxins.
At structure and the character according to Cobratoxin, the inventor screens and has found that a kind of cost is low, the affinity ligand that avidity is high.Such affinity ligand is fixed in the chromatography media that forms behind the solid phase carrier, not only can provide the result of purifying multiple, repeatability efficiently, and ligand comes off seldom.Be more suitable for industrialization, scale operation is medical and the reagent Cobratoxin.
The inventor is by methods such as the simulation of comprehensive utilization Computer-aided Molecular, molecular designing and organic syntheses, and designing and synthesized can single-minded small molecules affinity ligand in conjunction with Cobratoxin.In the Brookhaven albumen database, the structured data of Cobratoxin has several, and wherein (Yu, C.et al.Biochem.1993 32:2131) are the structure of Taiwan Cobratoxin to lcod.pdb, and be more approaching with Chinese South China.The primary structure of this neurotoxin is: Leu Glu Cys His Asn GlnGln Ser Ser Gln Thr Pro Thr Thr Thr Gly Cys Ser Gly Gly Glu Thr Asn CysTyr Lys Lys Arg Trp Arg Asp His Arg Gly Tyr Arg Thr Glu Arg Gly Cys GlyCys Pro Ser Val Lys Asn Gly Ile Glu Ile Asn Cys Cys Thr Thr Asp Arg CysAsn Asn.
Utilize the molecular simulation software SYBYL (version6.2) of TRIPOS company on SGI Indigo zl 4000 workstations, to carry out the space structure research of molecule, found that a more characteristic site: by amino-acid residue Lys27, Glu38, Tyr25 forms.The side-chain benzene ring group of Tyr25 is in the center of a depression, Lys27, and the charged side chain of Glu38 is positioned at the edge of this depression.When making the neurotoxin solvation as solvent, there is the position of a benzene molecular just in time can put into the depression in this site with benzene.Therefore, the design of subsequently affinity ligand mainly with this phenyl ring as starting point, be combining site with this sunk area.
By taking all factors into consideration the factor of following several respects, the inventor has designed the affinity ligand shown in the formula I: spatial surface, hydrophobic surface, the cooperation and the complementation of electric charge and the several aspects of hydrogen bond.Particularly, the centre portions of the combining site of selection is the side-chain benzene ring group of Tyr25, and this group is hydrophobic.The phenyl ring of putting into this position during with the phenyl ring solvation also is hydrophobic.From hydrophobic angle, these two hydrophobic groupings match.From the angle of electric charge, the side chain carboxyl group of the amino and Glu38 of the side chain of Lys27 is arranged as the phenyl ring paripheral zone electric group of solvent.Therefore, the side chain amino that can directly meet an electronegative carboxyl and positively charged Lys27 on phenyl ring matches; The side chain carboxyl group that meets positively charged amino and electronegative Glu38 matches; This amino simultaneously can also being used for fixing.Because the side chain of Lys27 side chain carboxyl group amino and Glu38 all is in the surface of molecule, these two groups are not immobilized under solution environmental, but have certain scope of activity.Therefore, according to amino and carboxyl may distribute on phenyl ring, design the affinity ligand shown in the formula I
In the formula, R
1Be the basic group that is selected from down group: amino, single substituted-amino, two substituted-aminos, trisubstituted-amino, amidino groups;
R
2Be the acidic-group that is selected from down group: carboxyl, sulfonic group, phosphate;
M1 is 1 or 2;
M2 is 1 or 2;
N1 is 1,2,3, or 4
N2 is 1,2,3, or 4.
Structurally, formula I compound has following feature: around ring center, alkalescence and acidic-group are connected on the center ring with a position or contraposition.R
1Can be any basic group, be preferably amino, single substituted-amino, two substituted-aminos, trisubstituted-amino, amidino groups etc.; R
2Can be any acidic-group, as carboxyl, sulfonic group, phosphate etc.Preferably, this center ring is five-ring or six-ring, and the atom of the ring of formation is selected from down group: carbon atom, nitrogen-atoms, Sauerstoffatom and sulphur atom.More preferably, this center ring is selected from: phenyl ring and cyclohexane ring.
Some are representational to can be used for affinity ligand example of the present invention and comprises (but being not limited to): para-amino benzoic acid, gavaculine, Sulphanilic Acid, m-sulfanilic acid, 3-aminocyclohexane carboxylic acid, 4-aminocyclohexane carboxylic acid.They are compound known mostly, and available ordinary method is synthesized or buied.
Can be used for solid phase carrier of the present invention can be any solid phase carrier with active linking group (as hydroxyl, amino, carboxyl, epoxy group(ing), halogen etc.) in the field of affinity chromatography.The affinity chromatography ligand of novelty of the present invention also can be connected on the solid phase carrier commonly used by bridging molecule.Representational solid phase carrier comprises (but being not limited to): dextrane gel such as Sephadex, crosslinked dextran bead such as PDX, allyl group dextran and N, cross-linking copolymer such as the Sephacryl of N '-methylene-bis (acrylamide), sepharose such as Sepharose, Sepharose CL, Sepharose FF, the cross-linking copolymer of agarose and dextran such as Superdex, highly cross-linked agarose and dextran such as Superose, the multipolymer pearl such as the Trisacryl of N-three [methylol] Methacrylamide and hydroxylation cross linker, Trisacryl plus, sepharose 4B such as Ultrogel A, polyacrylamide/agarose mixture pearl such as Ultrogel AcA, cellulose bead such as highly porous regenerated cellulose pearl, scribble the silica gel of polymkeric substance, or their mixture.
The method that affinity ligand of the present invention is coupled to or is fixed in solid phase carrier can be selected the method for this area routine for use, and this depends on used solid phase carrier kind.For example carry out coupling as linking agent or maleic anhydride etc. with epoxy chloropropane.To the general summary of immobilization technology, can be referring to Turkova, J. (1993) " Bioaffinity Chromatography ", Elsevier Science, London, U.K (document is all quoted as a reference at this).
In novel process of the present invention, the raw material that contains Cobratoxin of available the inventive method separation and purification can be any raw material that contains Cobratoxin, for example the thick poison of the Naja of Cai Jiing, gene engineering expression product.
Usually, can earlier the raw material that contains Cobratoxin be done suitably dilution, for example be made into the solution of 1-12% (preferably 2-10%, 4-8% best).
In order to improve the efficient of affinity chromatography, can change the damping fluid processing to raw material or its diluent that contains Cobratoxin.For example passing through Sephadex G-25 gel permeation chromatography post, is binding buffer liquid with buffer-exchanged, as phosphoric acid buffer (20mM, pH6.0).
Afterwards, for handling sample afterwards, carry out affinity chromatography, thereby obtain the Cobratoxin of purifying through changing damping fluid.Particularly, the affinity chromatography of Cobratoxin can comprise sample (absorption), washing and elution step.In addition, for the Cobratoxin elutriant after the affinity chromatography, also available anionresin, cationic exchange or methods such as gel permeation chromatography and ultrafiltration are further purified Cobratoxin.
In the affinity chromatography step of Cobratoxin, a kind ofly be to use following binding buffer liquid in conjunction with condition: the salt concn of damping fluid is 0.005-0.03M, pH5.0-9.0; More preferably, 0.01-0.02M, pH5.5-7.0; Best, be citric acid (5 mM), Na
2HPO
4(20mM) damping fluid (pH6.0-7.2).A kind of salt commonly used is sodium-chlor, Repone K.
A kind of elution requirement is to use following elution buffer: the salt concn of damping fluid is 0.005-2.0M, pH2.0-5.0 or 9.5-11.0; Preferably, salt concn is 0.01-0.50M, pH3.5-4.8; Be that (50mM pH9.6) (if need Cobratoxin is carried out repurity, so just needn't change damping fluid) again for 15mM acetate buffer solution (pH4.6) or the glycine buffer that contains NaCl0.5M best.
In a concrete preference of the present invention, with the raw material affinity column that affinity chromatography medium is housed, at citric acid (5mM), Na
2HPO
4(20mM) damping fluid (pH6.5-7.2) adsorbs Cobratoxin under the condition of NaCl (0.01-0.03M).Citric acid (5mM), Na with q.s
2HPO
4(20mM) damping fluid (pH6.5-7.2) behind the impurity that NaCl (0.01-0.03M) flush away does not adsorb, contains glycine buffer (50mM, pH9.6) the single-minded wash-out Cobratoxin of NaCl0.5M again.Change the damping fluid of Cobratoxin into the directly physiological saline of preparation again with Sephadex G-25 gel permeation chromatography post again.The half-finished purity of the Cobratoxin of this prepared is greater than 96%.Product recovery rate is greater than 85%.
In Figure of description,
Fig. 1 is the electrophorogram with Cobratoxin after 12%SDS-PAGE (Tricine-Gly Laemmli buffer system Laemmli) the detection affinity chromatography.Among the figure, the left side is the standard molecular weight protein marker; Intermediary swimming lane NT is the Cobratoxin through affinitive layer purification; The swimming lane SPl on right side is the thick malicious sample of Naja.
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to normal condition, people such as Sambrook for example, molecular cloning: laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.
Embodiment 1
Preparation affinity chromatography medium SK1
30ml Sepharose 6B is used distilled water (200ml) thorough washing in sintered glass funnel and under the suction filtration of vacuum pump, add NaOH (4N then, 8ml), epoxy chloropropane (0.5ml) and 2-oxygen six rings (5ml), 60 ℃ of shakes take out after 2 hours with distilled water (200ml) thorough washing, in the screw socket of packing into the test tube with cover.Take by weighing anthranilic acid 0.3g again and be dissolved in 10ml Na
2CO
3(0.5M, pH11) in, add again and be equipped with in the test tube of activatory Sepharose 6B, 60 ℃ of shakes spend the night.Before in the end stopping, adding the thanomin of 0.2ml in every pipe, be incubated 2 hours.Sepharose 6B is through 0.5M acetic acid (50ml) in the taking-up test tube, and 0.1MNaOH (50ml) and distilled water (200ml) obtain affinity chromatography medium after washing respectively, and called after SK1 adds 20% ethanol and stores stand-by.
Embodiment 2
Preparation affinity chromatography medium SK2
Compound gavaculine (20g) is dissolved in 50-400ml Na
2CO
3(0.2-0.8M pH8.0-12.0), joins in the salable bottle of containing epoxy group(ing) cellulose pearls (being called cellulose bead again) (about 1000ml), and 40-60 ℃ of shake spends the night.Before stopping, adding the thanomin of 10ml, insulation vibration 2 hours.Take out cellulose pearls in the reaction flask at last,, after 0.1MNaOH (1000ml) and distilled water (1000ml * 5) wash respectively, obtain the affinity chromatography medium SK2 of required cellulose matrix, add with the storage of 20% ethanol stand-by through 0.5M acetic acid (1000ml).
Embodiment 3
Preparation affinity chromatography medium SK3
With compound m-sulfanilic acid (18g), be dissolved in 50-400ml Na
2CO
3(0.2-0.8M pH8.0-12.0), joins in the salable bottle of containing epoxy group(ing) Trisacryl (about 1000ml), and 40-60 ℃ of shake spends the night.Before stopping, adding the thanomin of 10ml, insulation vibration 2 hours.Take out Trisacryl in the reaction flask at last, through 0.5M acetic acid (1000ml), 0.1M after NaOH (1000ml) and distilled water (1000ml * 5) wash respectively, obtain the affinity chromatography medium SK3 of required Trisacryl matrix, add with the storage of 20% ethanol stand-by.
Embodiment 4
(1) changing damping fluid handles
Get 5g Cobratoxin dissolving crude product in 20ml, remove by filter precipitation.Last sample is to using citric acid (5mM), Na
2HPO
4(20mM) damping fluid (pH6.5-7.2) equilibrated Sephadex G-25 post (on 5 * 20cm), continues with phosphoric acid buffer (20mM, pH6.0) wash-out.Collect the absorption peak of 280nm, obtain the 30ml sample.
(2) affinity chromatography
Get affinity chromatography medium SK1 (1ml) and pack disposable polypropylene chromatography post into (in 0.5 * 6.0cm), with 15ml citric acid (5mM), Na
2HPO
4(20mM) damping fluid (pH6.5-7.2), behind NaCl (0.01-0.03M) balance columns, add the thick poison of 0.2ml Naja (10mg/ml) sample, behind the material that does not adsorb with 20ml balance liquid flush away, again with the glycine buffer (50mM that contains NaCl 0.5M, pH9.6) the neurotoxin composition of the absorption on the 10ml elution chromatography post is used the SDS-PAGE detected result then.
The result shows that the neurotoxin rate of recovery reaches 25% of the thick poison of Naja.The neurotoxin rate of recovery of one step affinity purification can reach more than 94%, and purity is 90.4%, and can reach 95.6% again after a step Superdex molecular sieve is refining.Neurotoxin iso-electric point pI value behind the mensuration purifying is 9.468, is consistent with literature value (9.5); The molecular weight that records with mass spectrum (ESI) is between the 6700-6800, is short-chain neurotoxin.
Embodiment 5
Affinity chromatography
In this embodiment, recombinant expressed Cobratoxin is separated.
Get affinity chromatography medium SK
1(1ml) pack disposable polypropylene chromatography post into (in 0.5 * 6.0cm), with 15ml citric acid (5mM) Na
2HPO
4(20mM) damping fluid (pH6.5-7.2), behind NaCl (0.01-0.03M) balance columns, bromize fluoride crack liquid (20mg/ml) sample of the Cobratoxin of adding 0.2ml escherichia coli expression and the fusion rotein of albumin A, behind the material that 20ml balance liquid flush away does not adsorb, again with the glycine buffer (50mM that contains NaCl0.5M, pH9.6), absorption composition on the elution chromatography post and SDS-PAGE detected result.The neurotoxin rate of recovery of a step affinity purification can reach more than 80% as a result, and purity is 92%, and again behind a step Superdex molecular sieve, purity can reach 97%.
Embodiment 6
To in the affinity chromatography in conjunction with the optimization of condition and elution requirement
In this embodiment, respectively to being optimized in conjunction with condition and elution requirement (salt concn and pH value) in the affinity chromatography.Select embodiment 1 preparation SK1 affinity chromatography medium for use, under neutrallty condition, carry out salt concn optimization → carrying out combined acid basicity condition optimizing → carrying out wash-out potential of hydrogen condition optimizing → under the constant situation of damping fluid specific conductivity, carry out wash-out salt concentration conditions optimization under the constant situation of damping fluid specific conductivity under the constant situation of damping fluid specific conductivity in conjunction with condition.
The result shows, is preferably 0.005-0.03M in the bonded salt concn of Cobratoxin, 0.01-0.02M more preferably, and the pH condition is more preferably pH5.5-7.0 of pH5.0-9.0.
Elution requirement is that the salt concn of elution buffer is 0.005-2.0M, and preferably, salt concn is 0.01-0.50M.The pH condition is pH2.0-5.0 or pH9.5-11.0, preferably, and pH3.5-4.8.
Embodiment 7
In this embodiment, the affinity chromatography medium SK2 with preparation among the embodiment 2 carries out separation and purification to Cobratoxin.
As embodiment 4, with the raw material affinity column that affinity chromatography medium SK2 is housed, at citric acid (5mM), Na
2HPO
4(20mM) damping fluid (pH6.5-7.2) adsorbs Cobratoxin under the condition of NaCl (0.01-0.03M).Citric acid (5mM), Na with q.s
2HPO
4(20mM) damping fluid (pH6.5-7.2) is behind the impurity that NaCl (0.01-0.03M) flush away does not adsorb, again with the glycine buffer (50mM, pH9.6) the single-minded wash-out Cobratoxin that contain NaCl 0.5M.Change the damping fluid of Cobratoxin into the directly physiological saline of preparation again with Sephadex G-25 gel permeation chromatography post again.The half-finished purity of the Cobratoxin of this prepared is greater than 96%.Product recovery rate is greater than 85%.
All quote in this application as a reference at all documents that the present invention mentions, just quoted as a reference separately as each piece document.Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims institute restricted portion equally.
Claims (8)
1. affinity chromatography medium is characterized in that it comprises: solid phase carrier and be coupled at the affinity ligand shown in the formula I on the solid phase carrier:
In the formula, R
1Be the basic group that is selected from down group: amino, single substituted-amino, two substituted-aminos, trisubstituted-amino, amidino groups;
R
2Be the acidic-group that is selected from down group: carboxyl, sulfonic group, phosphate;
M1 is 1 or 2;
M2 is 1 or 2;
N1 is 1,2, or 3,
N2 is 1,2, or 3,
And center ring structure is selected from: phenyl ring or cyclohexane ring.
2. affinity chromatography medium as claimed in claim 1, it is characterized in that, described solid phase carrier is selected from: dextrane gel, crosslinked dextran bead, allyl group dextran and N, the multipolymer pearl of the cross-linking copolymer of cross-linking copolymer, sepharose, agarose and the dextran of N '-methylene-bis (acrylamide), highly cross-linked agarose and dextran, N-three [methylol] Methacrylamide and hydroxylation cross linker, sepharose 4B, polyacrylamide/agarose mixture pearl, cellulose bead and scribble the silica gel of polymkeric substance.
3. affinity chromatography medium as claimed in claim 1, it is characterized in that described solid phase carrier is selected from: Sephadex, PDX, Sephacryl, Sepharose, Sepharose CL, Sepharose FF, Superdex, Superose, Trisacryl, Trisacryl plus, Ultrogel A, Ultrogel AcA, highly porous regenerated cellulose pearl.
4. affinity chromatography medium as claimed in claim 1, it is characterized in that the affinity ligand shown in the formula I is selected from down group: para-amino benzoic acid, gavaculine, Sulphanilic Acid, m-sulfanilic acid, 3-aminocyclohexane carboxylic acid, 4-aminocyclohexane carboxylic acid.
5. the method for a separation and purification Cobratoxin, it comprises step:
(a) raw material that will contain Cobratoxin is splined on the affinity column that affinity media is housed, thus make Cobratoxin be adsorbed in affine in;
(b) impurity that do not adsorb of flush away affinity column;
(c) Cobratoxin that is adsorbed on the affinity chromatography medium is carried out wash-out, thereby obtain the Cobratoxin of purifying;
It is characterized in that, contain an affinity chromatography medium in this affinity column, this affinity chromatography medium comprises solid phase carrier and is coupled at the affinity ligand shown in the formula I on the solid phase carrier:
In the formula, R
1Be the basic group that is selected from down group: amino, single substituted-amino, two substituted-aminos, trisubstituted-amino, amidino groups;
R
2Be the acidic-group that is selected from down group: carboxyl, sulfonic group, phosphate;
M1 is 1 or 2;
M2 is 1 or 2;
N1 is 1,2, or 3,
N2 is 1,2, or 3,
And center ring structure is selected from: phenyl ring or cyclohexane ring.
6. method as claimed in claim 5 is characterized in that, in step (a), the condition of affinity media absorption Cobratoxin is that salt concn is 0.005-0.03M, and the pH condition is pH5.0-9.0;
In step (c), the elution requirement of Cobratoxin is that pH is the damping fluid of pH2.0-5.0 or pH9.5-11.0, and wherein salt concn is 0.005-2.0M.
7. method as claimed in claim 5 is characterized in that, this method also comprises step:
(d) Cobratoxin to the purifying of acquisition in the step (c) carries out molecular sieve gel filtration, thereby is further purified Cobratoxin.
8. method as claimed in claim 5 is characterized in that, the affinity ligand shown in the formula I is selected from down group: para-amino benzoic acid, gavaculine, Sulphanilic Acid, m-sulfanilic acid, 3-aminocyclohexane carboxylic acid, 4-aminocyclohexane carboxylic acid.
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| CNB001154028A CN1163515C (en) | 2000-04-17 | 2000-04-17 | New affinity chromatography medium and purification method for cobra neurotoxin |
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| CNB001154028A CN1163515C (en) | 2000-04-17 | 2000-04-17 | New affinity chromatography medium and purification method for cobra neurotoxin |
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| CN1318553A CN1318553A (en) | 2001-10-24 |
| CN1163515C true CN1163515C (en) | 2004-08-25 |
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| CN101845089B (en) * | 2010-01-15 | 2014-01-15 | 中鑫东泰(莱阳)纳米基因生物技术有限公司 | Method for large-scale production of neurotoxin in cobra venin and reduction of neurotoxicity |
| CN106622159B (en) * | 2016-12-22 | 2019-06-14 | 苏州楚博生物技术有限公司 | Affinity chromatography medium for protein drug purifying |
| WO2021068432A1 (en) * | 2019-10-11 | 2021-04-15 | 祁展楷 | Application of elapidae snake postsynaptic neurotoxin monomer molecule in treatment of alzheimer's disease |
| CN112094335A (en) * | 2020-09-11 | 2020-12-18 | 广西中恒创新医药研究有限公司 | Simple and efficient cobratide preparation method |
| CN114018906B (en) * | 2021-09-28 | 2024-03-12 | 神美科技有限公司 | Test paper for detecting nitrite content and preparation method thereof |
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