CN100335623C - Bionic affinity purification method of plasminogen activator - Google Patents
Bionic affinity purification method of plasminogen activator Download PDFInfo
- Publication number
- CN100335623C CN100335623C CNB2005100306569A CN200510030656A CN100335623C CN 100335623 C CN100335623 C CN 100335623C CN B2005100306569 A CNB2005100306569 A CN B2005100306569A CN 200510030656 A CN200510030656 A CN 200510030656A CN 100335623 C CN100335623 C CN 100335623C
- Authority
- CN
- China
- Prior art keywords
- plasminogen activator
- amino
- affinity
- natural
- type plasminogen
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
Landscapes
- Enzymes And Modification Thereof (AREA)
Abstract
The present invention relates to a bionic affinity purification method for a plasminogen activator, which belongs to the technical field of biology. The present invention comprises the following steps: (1) after reacting with tri-chlorinetri-nitrogen zin to be activated, a basal chromatography medium reacts with p-aminobenzamidine to synthesize a bionic affinity separation material; (2) an affinity chromatography column is prepared from the synthesized bionic affinity separation material, and a sample of a human tissue-type plasminogen activator containing a natural or modified structure flows through the affinity chromatography column; the human tissue-type plasminogen activator containing a natural or modified structure is absorbed on the affinity chromatography, and the affinity chromatography column is washed; then the condition of buffer solution is changed, and the affinity chromatography column is washed again; the combined human tissue-type plasminogen activator containing a natural or modified structure is eluted to obtain a purified human tissue-type plasminogen activator containing a natural or modified structure. The present invention has the advantages of few purification steps, low cost, high efficiency, high speed and convenience. By using the method of the present invention, a human tissue-type plasminogen activator containing a natural or modified structure and a urea-type plasminogen activator can be purified in large batches.
Description
Technical field
What the present invention relates to is a kind of method of biological technical field, specifically, is a kind of bio-affinity purifying method of plasminogen activator.
Background technology
Tissue-type plasminogen activator is a kind of serine protease, has very high affinity with scleroproein, show activity in the recycle system with after scleroproein combines, but plasminogen activation becomes plasmin, thrombus.There is natural form in tissue-type plasminogen activator, the form that the part-structure territory disappearance of artificial design is also arranged, as lack scleroproein finger ring district, Urogastron (E) district and/or Kringle-1 district change the configuration formula, with the transformation period in the extension body, quickening diffuses into the speed with the lysed blood grumeleuse.Tissue-type plasminogen activator is efficient thrombolytic agent, is one of six big genoid reconstituted drugs on the American market.The purification process that plasminogen activator is commonly used has ion exchange chromatography, hydrophobic chromatography, reversed phase chromatography, and metal-chelating, Methionin, the method for affinity chromatographys such as benzene carbon amidine.For the plasminogen activator purifying, these methods, specificity is poor, need the several different methods combination in the production, so production stage is many, the production cost height; Though utilize the affinity chromatography method of natural macromolecular part such as proteinase inhibitor, antibody and lectin to overcome to plasminogen activator specificity difference and inefficient shortcoming, but these bio-ligands preparation difficulty itself, cost costliness, character instability are not suitable for scale operation and use.
Find by prior art documents, " with transition state analog affinity purification tissue-type plasminogen activator " that Patel A delivers on " chromatography periodical " (Affinity purification of tissueplasminogen activator using transition-state analogues J.Chromatogr.1990,510:83-93.).This paper contains arginic tripeptides by solid phase synthesis on agarose, with urokinase and tissue-type plasminogen activator, finds effectively purifying tissue-type plasminogen activator of D-Phe-D-Phe-Argal.But the D-Phe-D-Phe-Argal material that this method is utilized need be synthetic with solid-phase polypeptide synthetic method, and preparation process is loaded down with trivial details, the cost height; D-Phe-D-Phe-Argal contains a plurality of polypeptide and connects in addition, and the character instability can not tolerate the severe condition of on-line cleaning.Though complicated purifying process is simplified, above drawbacks limit large-scale application.Therefore, be necessary to develop high-level efficiency, low cost, scale operation that step is easy is natural or changes the method and the material of structure human histiotype plasminogen activator, promote the industrialization paces.
Summary of the invention
The objective of the invention is to overcome the deficiencies in the prior art, a kind of bio-affinity purifying method of plasminogen activator is provided, make its purification step few, cost is low, the efficient height, quick, easy, purifying natural or change the structure human histiotype plasminogen activator and urine type plasminogen activator low-costly and in high volume.
The present invention is achieved by the following technical solutions, and the present invention is the affine parting material of raw material synthesizing bionic with basic chromatography media, with the bionical affine parting material purifying natural of preparation or change the human histiotype plasminogen activator and the fragment of structure.
The described human histiotype plasminogen activator that changes structure, be meant by artificial design, keep catalyst structure domain and tissue-type plasminogen activator's form of scleroproein finger ring district, the part or all of disappearance in Urogastron (E) district and/or Kringle-1 district.
The present invention includes following steps:
(1) after basic chromatography media and the trichloride and triazine reaction activation, with p-Aminobenzamidine reaction, the affine parting material of synthesizing bionic;
Described step (1) is meant with amino basic chromatography media, activates with the trichloride and triazine reaction; Perhaps with the conventional chemical method to not activating with amino basic chromatography media, generate amino with ammoniacal liquor or aminocompound reaction after, again with trichloride and triazine reaction activation;
Describedly do not comprise: dextrane gel, crosslinked dextran bead, allyl group dextran and N, the cross-linking copolymer of the cross-linking copolymer of N '-methylene-bisacrylamide, sepharose, agarose and dextran, polyacrylamide/agarose mixture pearl, cellulose bead and scribble silica gel, sintered glass and the pottery of chemical active radical with amino basic chromatography media.
Described conventional chemical method refers to that C.R.Lowe is at " affinity chromatography introduction " (Lip river (C.R.Lowe) work; Liu Yuxiu translates. Science Press, May nineteen eighty-three the 1st edition, 61-84 page or leaf) in matrix activation and the functional method mentioned, other matrix as polysaccharide base and hydroxyl, available halogen cyan, triazine (all dichlorotriazine or three chlorotriazines), periodate oxidation, oxyethane (1,4-dihydroxyl normal butane bisglycidyl ether), epoxy chloropropionate alcohol, the epoxy bromopropyl alcohol, two aziridine, divinyl sulfone, benzoquinone compound such as quinone, bromoacetyl bromide activate and functionalization; Polyacrylamide matrix can be used anhydrous ethylenediamine, the hydrazides posthydrolysis, and directly basic hydrolysis activates and functionalization; Silica gel, sintered glass and ceramic available silylating reagent activate and functionalization; Thereby on basic chromatography media, introduce the group that needs.
Described bionical affine parting material contains the structure of two 4-amino-benzene carbonamidines.
(2) be prepared into affinity column with the affine parting material of above-mentioned synthetic, to contain sample flow natural or that change the human histiotype plasminogen activator of structure and cross this affinity column, human histiotype plasminogen activator natural or that change structure is adsorbed on the affinity column, the flushing affinity column, the foreign protein that is not adsorbed on the affinity column is removed, conversion buffer conditions then, wash affinity column again, bonded is natural or change the structure human histiotype plasminogen activator and elute, obtain the natural of purifying or change the human histiotype plasminogen activator of structure.
In the described step (2), the condition of affine parting material absorption plasminogen activator is pH5-8, and ionic strength is the damping fluid of 0.05-0.2M; Elution requirement is pH1.0-12, and ionic strength is the damping fluid of 0.05-0.5M.
It is many that the present invention had both overcome affine parting material synthesis step, complicated operation, cost height, the character instability, shortcoming, reduced the step of plasminogen activator large scale purification again, reduced production cost, can be quick, easy, purifying plasminogen activator low-costly and in high volume.Sample improves more than 5 times than vigor, and the rate of recovery is more than 80%.
Embodiment
Embodiment 1
One, preparation parting material
Get NH
2-Sepharose (250ml), with the NaCl washing of the 1M of 5 times of volumes, use the deionized water thorough washing again after, take out branchs that anhydrate, pour in the reaction vessels, add the 200ml deionized water, be put in precooling in the cryosel bath.Treat that temperature reduces to 5 ℃, begin to stir.Add in the reaction vessel with precooling acetone solution three nitrogen piperazines (45g) back.Use saturated NaHCO
3The pH of solution is remained between 6～7, and 5 ℃ are reacted 3h down, take out, the water/acetone of 3 * 10 times of volumes (1: 1,1: 3,0: 1,1: 1,3: 1,1: 0) washing successively, obtain 1-amino-Sepharose-3,5-two chloro-2,4,6-three nitrogen piperazines (190ml).
Get 1-amino-Sepharose-3,5-two chloro-2,4,6-three nitrogen piperazines (200ml) take by weighing amino-benzene carbonamidine (20g) and dissolve with deionized water (150ml), with 1-amino-Sepharose-3,5-two chloro-2,4,6-three nitrogen piperazine mixings, placed 50 ℃ of stirring reactions of temperature 24 hours, and be warming up to then to stir in 95 ℃ and reacted again 24 hours.After reaction finishes, take out reactant, use the deionized water wash of 12 times of volumes then.Obtain the amino Sepharose-3-(4-amino-benzene carbonamidine) of 1--5-(4-amino-benzene carbonamidine)-2,4,6-three nitrogen piperazines (180ml) store stand-by with 20% ethanol.
Two, change structure human histiotype plasminogen activator (N-PA) with affinity purification
With the amino Sepharose-3-(4-amino-benzene carbonamidine) of 1--5-(4-amino-benzene carbonamidine)-2,4, the 6-affine parting materials of three nitrogen piperazines (5ml), chromatography column pack into (in 1.5 * 5.0cm), with 50ml Tris-HCl balanced solution (50mM Tris-HCl, 0.25M NaCl, 0.05%Tween-80, pH7.5) after the balance, being dissolved in 10ml Tris-HCl balanced solution (50mM Tris-HCl, 0.25M NaCl, 0.05%Tween-80, pH7.5) 0.3g in the crude product that changes structure tissue-type plasminogen activator enzyme is added on the post.With Tris-HCl balanced solution (50mM Tris-HCl, 0.25M NaCl, 0.05%Tween-80, pH7.5) behind the material that flush away does not adsorb, wash the bonded foreign protein with 50ml 50mM ammonium acetate buffer (pH5.0), use 50ml 0.5M ammonium acetate buffer (pH3.4) to carry out wash-out afterwards, use 50ml 0.1M acetum wash-out again, substep is collected elution fraction.Measure protein concentration with the lowery method, chromophoric substrate is measured the vigor of tissue-type plasminogen activator's enzyme, and sample improves 10 times than vigor, the rate of recovery 100%.12% reduction SDS-polyacrylamide gel electrophoresis analysis purity, the not foreign protein of pbz polymer amount.
Embodiment 2
One, preparation parting material
Get NH
2-Sepharose (250ml), with the NaCl washing of the 1M of 5 times of volumes, use the deionized water thorough washing again after, take out branchs that anhydrate, pour in the reaction vessels, add the 200ml deionized water, be put in precooling in the cryosel bath.Treat that temperature reduces to 5 ℃, begin to stir.Add in the reaction vessel with precooling acetone solution three nitrogen piperazines (45g) back.Use saturated NaHCO
3The pH of solution is remained between 6～7, and 5 ℃ are reacted 3h down, take out, the water/acetone of 3 * 10 times of volumes (1: 1,1: 3,0: 1,1: 1,3: 1,1: 0) washing successively, obtain 1-amino-Sepharose-3,5-two chloro-2,4,6-three nitrogen piperazines (190ml).
Get 1-amino-Sepharose-3,5-two chloro-2,4,6-three nitrogen piperazines (200ml) take by weighing amino-benzene carbonamidine (20g) and dissolve with deionized water (150ml), with 1-amino-Sepharose-3,5-two chloro-2,4,6-three nitrogen piperazine mixings, placed 50 ℃ of stirring reactions of temperature 24 hours, and be warming up to then to stir in 95 ℃ and reacted again 24 hours.After reaction finishes, take out reactant, use the deionized water wash of 12 times of volumes then.Obtain the amino Sepharose-3-(4-amino-benzene carbonamidine) of 1--5-(4-amino-benzene carbonamidine)-2,4,6-three nitrogen piperazines (180ml) store stand-by with 20% ethanol.
Two, affinity purification tissue-type plasminogen activator enzyme
With the amino Sepharose-3-(4-amino-benzene carbonamidine) of 1--5-(4-amino-benzene carbonamidine)-2,4, the 6-affine parting materials of three nitrogen piperazines (5ml), chromatography column pack into (in 1.5 * 5.0cm), with 50ml Tris-HCl balanced solution (50mM Tris-HCl, 0.1M NaCl, 0.05%Tween-80, pH5.0) after the balance, being dissolved in 10ml Tris-HCl balanced solution (50mM Tris-HCl, 0.1M NaCl, 0.05%Tween-80, pH5.0) crude product of the 0.25 gram tissue-type plasminogen activator enzyme in is added on the post.With Tris-HCl balanced solution (50mM Tris-HCl, 0.1M NaCl, 0.05%Tween-80, pH5.0) behind the material that flush away does not adsorb, wash the bonded foreign protein with 50ml 50mM ammonium acetate buffer (pH5.0), use 50ml 0.5M ammonium acetate buffer (pH2.0) to carry out wash-out afterwards, substep is collected elution fraction.Measure protein concentration with the lowery method, chromophoric substrate is measured the vigor of tissue-type plasminogen activator's enzyme, and sample improves 5 times than vigor, and the rate of recovery is greater than 70%.
Embodiment 3
One, preparation parting material
Get NH
2-Sepharose (250ml), with the NaCl washing of the 1M of 5 times of volumes, use the deionized water thorough washing again after, take out branchs that anhydrate, pour in the reaction vessels, add the 200ml deionized water, be put in precooling in the cryosel bath.Treat that temperature reduces to 5 ℃, begin to stir.Add in the reaction vessel with precooling acetone solution three nitrogen piperazines (45g) back.Use saturated NaHCO
3The pH of solution is remained between 6～7, and 5 ℃ are reacted 3h down, take out, the water/acetone of 3 * 10 times of volumes (1: 1,1: 3,0: 1,1: 1,3: 1,1: 0) washing successively, obtain 1-amino-Sepharose-3,5-two chloro-2,4,6-three nitrogen piperazines (190ml).
Get 1-amino-Sepharose-3,5-two chloro-2,4,6-three nitrogen piperazines (200ml) take by weighing amino-benzene carbonamidine (20g) and dissolve with deionized water (150ml), with 1-amino-Sepharose-3,5-two chloro-2,4,6-three nitrogen piperazine mixings, placed 50 ℃ of stirring reactions of temperature 24 hours, and be warming up to then to stir in 95 ℃ and reacted again 24 hours.After reaction finishes, take out reactant, use the deionized water wash of 12 times of volumes then.Obtain the amino Sepharose-3-(4-amino-benzene carbonamidine) of 1--5-(4-amino-benzene carbonamidine)-2,4,6-three nitrogen piperazines (180ml) store stand-by with 20% ethanol.
Two, affinity purification tissue-type plasminogen activator enzyme
With the amino Sepharose-3-(4-amino-benzene carbonamidine) of 1--5-(4-amino-benzene carbonamidine)-2,4, the 6-affine parting materials of three nitrogen piperazines (5ml), chromatography column pack into (in 1.5 * 5.0cm), with 50ml Tris-HCl balanced solution (50mM Tris-HCl, 0.5M NaCl, 0.05%Tween-80, pH9.0) after the balance, being dissolved in 10ml Tris-HCl balanced solution (50mM Tris-HCl, 0.5M NaCl, 0.05%Tween-80, pH9.0) crude product of the 0.2g tissue-type plasminogen activator enzyme in is added on the post.With Tris-HCl balanced solution (50mM Tris-HCl, 0.5M NaCl, 0.05%Tween-80, pH9.0) behind the material that flush away does not adsorb, wash the bonded foreign protein with 50ml 50mM ammonium acetate buffer (pH5.0), use 50ml 0.5M ammonium acetate buffer (pH2.4) to carry out wash-out afterwards, substep is collected elution fraction.Measure protein concentration with the lowery method, chromophoric substrate is measured the vigor of tissue-type plasminogen activator's enzyme, and sample improves 7 times than vigor, and the rate of recovery is greater than 80%.
Embodiment 4
One, preparation parting material
Get NH
2-Sepharose (250ml), with the NaCl washing of the 1M of 5 times of volumes, use the deionized water thorough washing again after, take out branchs that anhydrate, pour in the reaction vessels, add the 200ml deionized water, be put in precooling in the cryosel bath.Treat that temperature reduces to 5 ℃, begin to stir.Add in the reaction vessel with precooling acetone solution three nitrogen piperazines (45g) back.Use saturated NaHCO
3The pH of solution is remained between 6～7, and 5 ℃ are reacted 3h down, take out, the water/acetone of 3 * 10 times of volumes (1: 1,1: 3,0: 1,1: 1,3: 1,1: 0) washing successively, obtain 1-amino-Sepharose-3,5-two chloro-2,4,6-three nitrogen piperazines (190ml).
Get 1-amino-Sepharose-3,5-two chloro-2,4,6-three nitrogen piperazines (200ml) take by weighing amino-benzene carbonamidine (20g) and dissolve with deionized water (150ml), with 1-amino-Sepharose-3,5-two chloro-2,4,6-three nitrogen piperazine mixings, placed 50 ℃ of stirring reactions of temperature 24 hours, and be warming up to then to stir in 95 ℃ and reacted again 24 hours.After reaction finishes, take out reactant, use the deionized water wash of 12 times of volumes then.Obtain the amino Sepharose-3-(4-amino-benzene carbonamidine) of 1--5-(4-amino-benzene carbonamidine)-2,4,6-three nitrogen piperazines (180ml) store stand-by with 20% ethanol.
Two, affinity purification urine plasminogen activator (urokinase)
With the amino Sepharose-3-(4-amino-benzene carbonamidine) of 1--5-(4-amino-benzene carbonamidine)-2,4, the 6-affine parting materials of three nitrogen piperazines (5ml), chromatography column pack into (in 1.5 * 5.0cm), after 50ml Tris-HCl balanced solution (50mM Tris-HCl 0.25M NaCl 0.05%Tween-80 pH7.5) balance, the crude product that is dissolved in urokinase 25,0000 unit of activity (about 60mg) in the 10ml Tris-HCl balanced solution (50mM Tris-HCl 0.25M NaCl 0.05%Tween-80 pH7.5) is added on the post.Behind the material that does not adsorb with Tris-HCl balanced solution (50mM Tris-HCl 0.25M NaCl 0.05%Tween-80 pH7.5) flush away, wash the bonded foreign protein with 50ml 50mM ammonium acetate buffer (pH5.0), use 50ml 0.5M ammonium acetate buffer (pH3.4) wash-out urokinase afterwards, substep is collected elution fraction.Measure protein concentration with the lowery method, chromophoric substrate is measured the urokinase vigor, and sample improves 7 times than vigor, and the rate of recovery is greater than 80%.Electrophoretic analysis shows and has been purified into low molecular weight urokinase (33KD).The urokinase sample improves 43 times than vigor, the total activity rate of recovery 100%.
Embodiment 5
One, preparation parting material
Get NH
2-Sepharose (250ml), with the NaCl washing of the 1M of 5 times of volumes, use the deionized water thorough washing again after, take out branchs that anhydrate, pour in the reaction vessels, add the 200ml deionized water, be put in precooling in the cryosel bath.Treat that temperature reduces to 5 ℃, begin to stir.Add in the reaction vessel with precooling acetone solution three nitrogen piperazines (45g) back.Use saturated NaHCO
3The pH of solution is remained between 6～7, and 5 ℃ are reacted 3h down, take out, the water/acetone of 3 * 10 times of volumes (1: 1,1: 3,0: 1,1: 1,3: 1,1: 0) washing successively, obtain 1-amino-Sepharose-3,5-two chloro-2,4,6-three nitrogen piperazines (190ml).
Get 1-amino-Sepharose-3,5-two chloro-2,4,6-three nitrogen piperazines (200ml) take by weighing amino-benzene carbonamidine (20g) and dissolve with deionized water (150ml), with 1-amino-Sepharose-3,5-two chloro-2,4,6-three nitrogen piperazine mixings, placed 50 ℃ of stirring reactions of temperature 24 hours, and be warming up to then to stir in 95 ℃ and reacted again 24 hours.After reaction finishes, take out reactant, use the deionized water wash of 12 times of volumes then.Obtain the amino Sepharose-3-(4-amino-benzene carbonamidine) of 1--5-(4-amino-benzene carbonamidine)-2,4,6-three nitrogen piperazines (180ml) store stand-by with 20% ethanol.
Two, affinity purification tissue-type plasminogen activator enzyme
With the amino Sepharose-3-(4-amino-benzene carbonamidine) of 1--5-(4-amino-benzene carbonamidine)-2,4, the 6-affine parting materials of three nitrogen piperazines (5ml), chromatography column pack into (in 1.5 * 5.0cm), with 50ml Tris-HCl balanced solution (50mM Tris-HCl, 0.25M NaCl, 0.05% Tween-80, pH5.0) after the balance, being dissolved in 10ml Tris-HCl balanced solution (50mM Tris-HCl, 0.25M NaCl, 0.05%Tween-80, pH5.0) 0.3g in the crude product that changes structure tissue-type plasminogen activator enzyme is added on the post.With Tris-HCl balanced solution (50mM Tris-HCl, 0.25M NaCl, 0.05% Tween-80, pH5.0) behind the material that flush away does not adsorb, wash the bonded foreign protein with 50ml 50mM ammonium acetate buffer (pH5.0), use 50ml 0.5M glycine-hydrochloric acid (pH1.0) to carry out wash-out afterwards, substep is collected elution fraction.Measure protein concentration with the lowery method, chromophoric substrate is measured the vigor of tissue-type plasminogen activator's enzyme, and sample improves 7.5 times than vigor, the rate of recovery 80%.
Embodiment 6
One, preparation parting material
Get NH
2-Sepharose (250ml), with the NaCl washing of the 1M of 5 times of volumes, use the deionized water thorough washing again after, take out branchs that anhydrate, pour in the reaction vessels, add the 200ml deionized water, be put in precooling in the cryosel bath.Treat that temperature reduces to 5 ℃, begin to stir.Add in the reaction vessel with precooling acetone solution three nitrogen piperazines (45g) back.Use saturated NaHCO
3The pH of solution is remained between 6～7, and 5 ℃ are reacted 3h down, take out, the water/acetone of 3 * 10 times of volumes (1: 1,1: 3,0: 1,1: 1,3: 1,1: 0) washing successively, obtain 1-amino-Sepharose-3,5-two chloro-2,4,6-three nitrogen piperazines (190ml).
Get 1-amino-Sepharose-3,5-two chloro-2,4,6-three nitrogen piperazines (200ml) take by weighing amino-benzene carbonamidine (20g) and dissolve with deionized water (150ml), with 1-amino-Sepharose-3,5-two chloro-2,4,6-three nitrogen piperazine mixings, placed 50 ℃ of stirring reactions of temperature 24 hours, and be warming up to then to stir in 95 ℃ and reacted again 24 hours.After reaction finishes, take out reactant, use the deionized water wash of 12 times of volumes then.Obtain the amino Sepharose-3-(4-amino-benzene carbonamidine) of 1--5-(4-amino-benzene carbonamidine)-2,4,6-three nitrogen piperazines (180ml) store stand-by with 20% ethanol.
Two, affinity purification tissue-type plasminogen activator enzyme
With the amino Sepharose-3-(4-amino-benzene carbonamidine) of 1--5-(4-amino-benzene carbonamidine)-2,4, the 6-affine parting materials of three nitrogen piperazines (5ml), chromatography column pack into (in 1.5 * 5.0cm), with 50ml Tris-HCl balanced solution (50mM Tris-HCl, 0.25M NaCl, 0.05% Tween-80, pH8.0) after the balance, being dissolved in 10ml Tris-HCl balanced solution (50mM Tris-HCl, 0.25M NaCl, 0.05%Tween-80, pH8.0) 0.3g in the crude product that changes structure tissue-type plasminogen activator enzyme is added on the post.(0.05% Tween-80 pH8.0) behind the material that flush away does not adsorb, carries out wash-out with 50ml 0.05M glycine-sodium hydroxide (pH12.0) for 50mM Tris-HCl, 0.25M NaCl, and substep is collected elution fraction with the Tris-HCl balanced solution.Measure protein concentration with the lowery method, chromophoric substrate is measured the vigor of tissue-type plasminogen activator's enzyme, and sample improves 8.5 times than vigor, the rate of recovery 85%.
Claims (6)
1. the bio-affinity purifying method of a plasminogen activator is characterized in that, is the affine parting material of raw material synthesizing bionic with basic chromatography media, with the preparation bionical affine parting material purifying natural or change the structure human histiotype plasminogen activator; May further comprise the steps:
(1) after basic chromatography media and the trichloride and triazine reaction activation, with the p-Aminobenzamidine reaction, the synthetic bionical affine parting material that contains the structure of two 4-amino-benzene carbonamidines;
(2) be prepared into affinity column with the affine parting material of above-mentioned synthetic, to contain sample flow natural or that change the human histiotype plasminogen activator of structure and cross this affinity column, human histiotype plasminogen activator natural or that change structure is adsorbed on the affinity column, the flushing affinity column, the foreign protein that is not adsorbed on the affinity column is removed, conversion buffer conditions then, wash affinity column again, bonded is natural or change the structure human histiotype plasminogen activator and elute, obtain the natural of purifying or change the human histiotype plasminogen activator of structure.
2. the bio-affinity purifying method of plasminogen activator according to claim 1 is characterized in that, in the described step (1), with amino basic chromatography media, activates with the trichloride and triazine reaction.
3. the bio-affinity purifying method of plasminogen activator according to claim 1, it is characterized in that, in the described step (1), with the conventional chemical method to not activating with amino basic chromatography media, behind ammoniacal liquor or aminocompound reaction generation amino, again with trichloride and triazine reaction activation; Describedly do not comprise with amino basic chromatography media: dextrane gel, crosslinked dextran bead, allyl group dextran and N, the cross-linking copolymer of the cross-linking copolymer of N '-methylene-bisacrylamide, sepharose, agarose and dextran, polyacrylamide/agarose mixture pearl, cellulose bead and scribble chemical active radical silica gel, scribble the sintered glass of chemical active radical and scribble the pottery of chemical active radical.
4. the bio-affinity purifying method of plasminogen activator according to claim 1 is characterized in that, described bionical affine parting material contains the structure of two 4-amino-benzene carbonamidines.
5. the bio-affinity purifying method of plasminogen activator according to claim 1 is characterized in that, in the described step (2), the condition of affine parting material absorption plasminogen activator is pH5-8, and ionic strength is the damping fluid of 0.05-0.2M.
6. the bio-affinity purifying method of plasminogen activator according to claim 1 is characterized in that, in the described step (2), elution requirement is pH1.0-12, and ionic strength is the damping fluid of 0.05-0.5M.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2005100306569A CN100335623C (en) | 2005-10-20 | 2005-10-20 | Bionic affinity purification method of plasminogen activator |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2005100306569A CN100335623C (en) | 2005-10-20 | 2005-10-20 | Bionic affinity purification method of plasminogen activator |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1786161A CN1786161A (en) | 2006-06-14 |
| CN100335623C true CN100335623C (en) | 2007-09-05 |
Family
ID=36783774
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNB2005100306569A Expired - Fee Related CN100335623C (en) | 2005-10-20 | 2005-10-20 | Bionic affinity purification method of plasminogen activator |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN100335623C (en) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1970745B (en) * | 2006-12-07 | 2010-05-12 | 上海交通大学 | Bio-affinity purifying method for lipase |
| CN101067131B (en) * | 2007-04-30 | 2011-08-17 | 华南理工大学 | Affinity chromatographic process of separating and purifying plasmin |
| CN111760556B (en) * | 2020-07-09 | 2023-06-30 | 江苏尤里卡生物科技有限公司 | Urokinase adsorbent and preparation method and application thereof |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0302503A2 (en) * | 1987-08-05 | 1989-02-08 | Roche Diagnostics GmbH | Method for the separation and purification of t-PA |
-
2005
- 2005-10-20 CN CNB2005100306569A patent/CN100335623C/en not_active Expired - Fee Related
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0302503A2 (en) * | 1987-08-05 | 1989-02-08 | Roche Diagnostics GmbH | Method for the separation and purification of t-PA |
Non-Patent Citations (3)
| Title |
|---|
| Affinity purification of tissue plasminogen activator usingtransition-state analogues A.PATEL et al,Journal of Chromatography,Vol.510 1990 * |
| 激肽释放酶的仿生亲和纯化研究 刘宏亮，大学硕士学位论文 2004 * |
| 生物药物规模化制备技术平台 李荣秀，2004年全国生化与生物技术药物学术年会论文集 2004 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN1786161A (en) | 2006-06-14 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Ma et al. | Monolith‐based immobilized enzyme reactors: recent developments and applications for proteome analysis | |
| CN104645949B (en) | Affinity chromatography medium employing tetrapeptide as functional ligand and preparation method of affinity chromatography medium | |
| Dartiguenave et al. | Immobilization of trypsin onto 1, 4-diisothiocyanatobenzene-activated porous glass for microreactor-based peptide mapping by capillary electrophoresis: Effect of calcium ions on the immobilization procedure | |
| CN100335623C (en) | Bionic affinity purification method of plasminogen activator | |
| CN113512545A (en) | Immobilized lipase synthesis and new method for splitting mandelic acid enantiomer | |
| JPH0427504B2 (en) | ||
| Zhong et al. | Self-assembly synthes is of trypsin-immobilized monolithic microreactor for fast and efficient proteolysis | |
| Tengattini et al. | Enterokinase monolithic bioreactor as an efficient tool for biopharmaceuticals preparation: on-line cleavage of fusion proteins and analytical characterization of released products | |
| CN105518452B (en) | Mark the modulator approach of sugar chain sample | |
| CN1325516C (en) | Biomimetic affinity purification method of vitellus immune globulin | |
| Pastinen et al. | Cross-linked glucose isomerase crystals as a liquid chromatographic separation material | |
| CN114606221B (en) | Immobilized enzyme, preparation method and application thereof | |
| CN101185881A (en) | Chromatogram medium for immunoglobulin class protein separation purification and preparation method thereof | |
| US20080124781A1 (en) | Enzymes with modified amino acids | |
| CN101880694B (en) | Method for preparing chiral 3R, 5S-dihydroxyl compound by nonaqueous phase | |
| EP0167152A2 (en) | Protease adsorbent and process for purifying TPA utilizing the same | |
| Ishii et al. | [64] Affinity methods using argininal derivatives | |
| CN113262768B (en) | Molecularly imprinted monolithic column of myristoylation tetrapeptide, preparation method and application | |
| Yang et al. | Immobilized metal affinity composite membrane based on cellulose for separation of biopolymers | |
| CA2492752A1 (en) | Rapid process for purification and concentration of plasmin | |
| Sada | Engineering aspects of bioaffinity separation | |
| CN111957073B (en) | Affinity chromatography filler for purifying urokinase, preparation method and application | |
| Uzunoğlu et al. | Preparing and optimizing a newly affinity gel for purification of carbonic anhydrase isoenzymes | |
| del Valle et al. | Separation of asparaginase and trypsin by affinity chromatography combined with batchwise adsorption and columnwise desorption | |
| CN100378125C (en) | Bionic affinity purifying process of interleukin I natural agonist |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| ASS | Succession or assignment of patent right |
Owner name: SHANGHAI UNJA BIOTECHNOLOGY LTD. Free format text: FORMER OWNER: SHANGHAI JIAO TONG UNIVERSITY Effective date: 20110217 |
|
| C41 | Transfer of patent application or patent right or utility model | ||
| COR | Change of bibliographic data |
Free format text: CORRECT: ADDRESS; FROM: 200240 NO. 800, DONGCHUAN ROAD, MINHANG DISTRICT, SHANGHAI TO: 201109 NO. 328, WUHE ROAD, MINHANG DISTRICT, SHANGHAI |
|
| TR01 | Transfer of patent right |
Effective date of registration: 20110217 Address after: 201109 Shanghai city Minhang District Wu River Road No. 328 Patentee after: Shanghai Si Jia Biological Technology Co. Ltd. Address before: 200240 Dongchuan Road, Shanghai, No. 800, No. Patentee before: Shanghai Jiao Tong University |
|
| ASS | Succession or assignment of patent right |
Owner name: SHANGHAI RONGJUN BIO-PHARMACEUTICAL TECHNOLOGY CO. Free format text: FORMER OWNER: SHANGHAI SIJIA BIOTECHNOLOGY CO., LTD. Effective date: 20110808 |
|
| C41 | Transfer of patent application or patent right or utility model | ||
| COR | Change of bibliographic data |
Free format text: CORRECT: ADDRESS; FROM: 201109 MINHANG, SHANGHAI TO: 200240 MINHANG, SHANGHAI |
|
| TR01 | Transfer of patent right |
Effective date of registration: 20110808 Address after: 865 Lane 200240, Dongchuan Road, Shanghai, 44-102 Patentee after: CHANGZHOU RONGJUN BIOLOGICAL MEDICAL TECHNOLOGY CO., LTD. Address before: 201109 Shanghai city Minhang District Wu River Road No. 328 Patentee before: Shanghai Si Jia Biological Technology Co. Ltd. |
|
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20070905 Termination date: 20141020 |
|
| EXPY | Termination of patent right or utility model |