CN1294256C - Gene-expressed human tissue kallikrein protein separating and purifying method - Google Patents
Gene-expressed human tissue kallikrein protein separating and purifying method Download PDFInfo
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- CN1294256C CN1294256C CNB021331642A CN02133164A CN1294256C CN 1294256 C CN1294256 C CN 1294256C CN B021331642 A CNB021331642 A CN B021331642A CN 02133164 A CN02133164 A CN 02133164A CN 1294256 C CN1294256 C CN 1294256C
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Abstract
The present invention relates to extraction of human tissue kallikrein, particularly to a method for separating and purifying genes expressing human tissue kallikrein. The present invention has the following processes of supernatant liquid separation, chromatography which is step gradient elution, and affinity chromatography separation and purification, wherein columns are balanced by 20 to 30mM of buffer solution with the pH value of 7 to 8; the supernatant liquid is pumped into the columns; the buffer solution is eluted by a buffer solution containing 0.1 to 0.2M of NaCl and is then changed eluted by a buffer solution containing 0.3 to 0.5M of NaCl, fractioning is collected for standby; columns are firstly balanced by 10 to 20mMof NaAc with the pH value of 5.0 to 6.0, and then, fractioning is collected through step gradient elution; the solution is eluted by linear gradient, the eluant contains 0.1 to 0.5 of NaCl, and fractioning is collected for preservation. The present invention has the advantages of low operating cost, application to large-scale production, and good separating effect.
Description
Technical field
The present invention relates to the extraction of human tissue kallikrein, specifically a kind of separation purification method of gene-expressed human tissue kallikrein protein.
Background technology
Nineteen thirty has the people to find a kind of protein with specific function first in urine and pancreas, and is referred to as tissue kallikrein (kallikrein).In blood plasma and many tissues, also find to exist the kassinin kinin release system later on.This system also has important endogenous hypotensive effect except that participating in inflammatory reaction, causing the effect such as pain, also is related with the morbidity of diseases such as hypertension, ephrosis.The last century the eighties abroad just has been noted that the different physiological roles of kallikrein, and it has been carried out systematic research.About existing many reports such as the research of its gene locus, tissue expression and physiological function etc.
Kallikrein is a kind of serine protease, and belonging to the serine residue is a subclass under the silk-protein enzyme family in active centre.Kallikrein comprises blood plasma kallikrein and organizes two kinds of kallikrein; Under the normal circumstances, the overwhelming majority is that the precursor forms with non-activity exists.Organize kallikrein extensively to be present in many tissues such as kidney, pancreas, gastrointestinal tract mucous and central nervous system, it is synthetic in tissue with the form of prekallikrein, activate formative tissue kallikrein through trypsinase again, and can be released into blood circulation.Organize the former I of the kallikrein specific cracking plasmakinin of energy in vivo, generate the active result pancreokinin, and can enter blood plasma, generate bradykinin through the aminopeptidase hydrolysis.Kassinin kinin and its receptors bind can produce exciting and multiple biological effect such as suppressing of vasodilation, ion transportation, smooth muscle loosening or contraction, cell growth; Therefore kallikrein can play and keep localised blood pressure, stablizes unobstructed blood vessel, participate in physiological regulatory actions such as blood and electrolyte balance, and the activation of proenzyme, the activation of somatomedin, the processing of hormone precursor are also played an important role.Experimentation on animals result in recent years shows that kallikrein can alleviate the cardiac hypertrophy of lab mice greatly, infarct size and heart cell death and fibrosis.For the lab mice that suffers from the arteriosclerosis disease, kallikrein can reduce the endothelial injury endocardium damage afterwards that air bag brings out, and its provide protection is by β
1And β
2Acceptor is finished.The kidney injury that Kallikrein can also reduce and avoid medicine or salt to bring out comprises tubulorrhexis, glomerulosclerosis, the accumulation of protein pipe and inflammation.Kallikrein can also reduce the bump mortality ratio of the responsive hypertension mouse of salt, can accelerate its blood flow rate for the ischemic lab mice of hind leg, this shows that kallikrein all has the potential curative effect for hypertension, heart disease, kidney disease etc.
The separation and purification difficulty of kallikrein of directly carrying out human body is bigger, and the people such as Wang Zhengrong of West China Center of Medical Sciences of Sichuan University produce the animal kallikrein and application facet was done some useful work at genetic engineering technique.The people that the Chao of medical college of American South card university adopts bionic method to obtain from the fetal kidney cell at first organizes the kallikrein gene to clone, and constructs the recombinant virus that is loaded with the kallikrein gene.Shenzhen people's hospital Lee body in 2000 far waits the people to adopt the success that takes the lead at home of similar method to clone people kallikrein gene, through Shanghai Sangon bio-engineering corporation sequencing analysis, the Chinese visible human of this new clone organizes the kallikrein gene in full accord with the gene order of international report.Recently, the recombinant virus that the investigator of Wuhan University will be loaded with people kallikrein gene is expelled in the vein of Hypertensive Rats, can make the blood pressure of rat be reduced to normal level and keep steadily long-time.In order to overcome the drawback that virus is expressed in vivo, employing insect cell lines such as Chao are expressed.The people kallikrein that this method obtains is comparatively safe, reliable, is to realize that the genetically engineered people organizes the feasible direction of kallikrein large-scale production.
The kallikrein that experiment is at present used is general, and the method for extracting from human urine that adopts obtains, and almost all derives from from animal pancreas as the kallikrein product of medicinal application and extracts.Extracting kallikrein from people's urine must handle through enrichment earlier, and further carries out separation and purification by methods such as membrane chromatographies.Because kallikrein content in biological tissue and body fluid is few, in 1000L urine, can only extract at most and obtain 17mgkallikrein, preparation cost is high.
Extract kallikrein from animal tissues, at first adopt historrhexis, methods such as further employing is saltoutd, chromatographic separation are carried out purifying.Since animal kallikrein and people's kallikrein difference structurally, degree of glycosylation difference, so the sample of different sources, and because the difference that its background is formed, separation purification method also is not quite similar.Its weak point is to saltout, chromatographic separation is to finish under the rapid operation of multistep, very numerous Xu, and the multistep operational losses is bigger, is unsuitable for scale operation.
Summary of the invention
The object of the present invention is to provide a kind of separation purification method of simple to operate, the gene-expressed human tissue kallikrein protein that is suitable for scale operation.
For achieving the above object, the technical solution adopted in the present invention is:
Steps such as that separation purification method of the present invention comprises is centrifugal, ion exchange chromatography, affinity chromatography, detailed process is:
(1) separation of cell culture supernatant: the medium centrifugal that will contain human tissue kallikrein separates, and it is standby to get supernatant liquor;
(2) ion exchange chromatography separates:
1. at first adopt the step gradient elution, to reclaim the human tissue kallikrein in the substratum as much as possible; Be specially:
Damping fluid balance pillar with 20～30mM, pH7～8 pumps into supernatant liquor in the post then; Include the buffer solution elution of 0.1～0.2M NaCl with 20～30mM pH7～8, measured value to be checked changes the damping fluid that 20～30mM pH7～8 include 0.3～0.5M NaCl after being lower than below 0.05, human tissue kallikrein desorption this moment, in post, flow out, collect the cut of this moment, 4 ℃ of storages are standby; Pillar regeneration is standby;
Wherein damping fluid can be Tris-HCl, phosphate buffered saline buffer, HEPES damping fluid etc.;
NaCl also can substitute with KCl;
2. the method that adopts the step gradient to combine with linear gradient is separated human tissue kallikrein and other foreign protein as far as possible;
Sample pretreatment: the described step 1. cut ultrafiltration of intermediate ion exchange column collection (adopting Centriprep10P) molecular weight cut-off is the above filter concentrating sample of 10Kda, will concentrate the back sample then and be dissolved in again in the damping fluid of 10～20mM NaAc pH5.0～6.0;
Sample separation: earlier with 10～20mM NaAc pH5.0～6.0 balance pillars, then the sample of handling well is written in the post, continues towards post, up to A with level pad
280Absorption value is reduced to below 0.05, includes the buffer solution elution of 0.05～0.1M NaCl with 10～20mM NaRc pH5.0～6.0 again, collects cut a; Include the buffer solution elution of 0.1～0.15M NaCl with 10～20mM NaRc pH5.0～6.0 then, collect cut b, then use linear gradient elution, the salt component scope is 0.1M NaCl to 0.5M NaCl in the elutriant, a large amount of human tissue kallikreins are eluted, collect cut, 4 ℃ of preservations; In described cut a and b, also there is a small amount of human tissue kallikrein to elute, can collects and cross post again;
NaCl also can substitute with KCL;
Employed ion-exchanger can be polyose ion-exchangers such as DEAE-cellulose, DEAE-sephadex or DEAE-sepharose in the ion exchange chromatography sepn process, also can adopt crosslinked agar carbohydrate ion exchange resin such as DEAESepharose CL-6B.In general, weak anion exchange resin all can be used for this step separation;
(3) affinity chromatography separation and purification: operation according to a conventional method gets product.
Human tissue kallikrein belongs to serine protease, and available specific immunity is affine, phenylformic acid amine (PAB), Trypsin inhibitor,Trasylol multiple affinity chromatography separating mediums such as (Aprotinin) carry out purifying.
Aprotinin-Agrose column purification method a:
After active fractions suitably concentrated (ultrafiltration), be transferred on the affinity column, use 15-25mMTris-HCl, pH6.5～7.5 damping fluid thorough washing; Remove the not protein of absorption, to A
280Absorption value is reduced to baseline; Use 0.05～0.15mol/L GIy-HCI then, pH2.0～3.0 buffer solution elution; The elution peak that occurs is target product; Neutralize with 0.3～0.5mol/L Tris immediately; The back solution that will neutralize is dialysed to 20～25mM phosphoric acid buffer; Lyophilize.Aprotinin-Agrose column purification method b:
Include the damping fluid balance pillar of 200～250mM NaCl earlier with 15～25mM Tris-HCl, pH7.5～8.5, sample is written in the post, take off with 0.1～0.15M glycine/HCl, pH2.0～3.0 suctions, collect elution peak, with 0.3～0.5mol/L Tris neutralization, the back solution that will neutralize is dialysed to 20～25mM phosphoric acid buffer immediately; Lyophilize.
The present invention has following advantage:
1. the present invention has set up a kind of centrifugal, ion exchange chromatography, affine three step separation purification method; Reduce running cost, be suitable for large-scale production and application.
2. the present invention utilizes weak anion exchange resin in the ion exchange chromatography step, adopts PH and ionic strength gradient bonded method to remove most of foreign protein, and remaining foreign protein content is extremely low, almost only remains two kinds of associated protein after the separation; Its selected condition does not influence its lytic activity.
3. the present invention adopts Trypsin inhibitor,Trasylol as separating medium in affine separating step, and effect is better, and can repeatedly use repeatedly.
4. the present invention adopts Tris-HCl damping fluid and glycine/HCl to wash in the affinity chromatography step, and its condition relaxes, and target product and affinity media are all had no adverse effects.
5. after occurring, target product of the present invention peak neutralizes with Tris immediately, to keep its stability.
6. the present invention dialyses the affinity chromatography cut to phosphoric acid buffer, carries out the solvent displacement, is beneficial to preservation.
Description of drawings
Fig. 1 is that the linear gradient that contains expressing gene engineering human tissue kallikrein cell culture supernatant is separated spectrogram.
Fig. 2 is the first separation spectrogram under the step gradient condition.
Separation spectrogram when Fig. 3 is a large amount of sample introduction.
Fig. 4 is the electrophoresis detection spectrogram of ion-exchange chromatography separating resulting.
Fig. 5 separates spectrogram for adopting the step gradient with the ion-exchange chromatography of linear gradient combination.
Fig. 6 is a gel chromatography purification result spectrogram.
Fig. 7 is an affinity chromatography separating resulting spectrogram.
Fig. 8 is the SDS-PAGE spectrogram of the finished product.
Fig. 9 separates spectrogram for adopting the step gradient with the ion-exchange chromatography of linear gradient combination.
Figure 10 is the SDS-PAGE spectrogram of product.
Embodiment
Embodiment 1
Steps such as that separation purification method of the present invention comprises is centrifugal, ion exchange chromatography, affinity chromatography; Detailed process comprises:
1. the separation of cell culture supernatant:
Cell culture fluid is placed the polycarbonate centrifuge tube, use the pasteur pipet balance; In supercentrifuge, 4 ℃, the centrifugal 20～30min of 2000～3000r/min.
Supernatant liquor poured in the polypropylene funnel that is equipped with filter cloth into filter and put in the polypropylene container, stored refrigerated is used for further ion exchange chromatography.
2. ion exchange chromatography separates:
DEAE-Sepharose ion exchange layer analysis method: chromatographic column: 16mm * 20cm glass packed column; Filler: DEAE-Sepharose ion-exchange packing; Peristaltic pump: miniplus3, Gilson company; Detect wavelength: 280nm; Flow velocity: 5ml/min
1. earlier with the damping fluid balance pillar of 20mM Tris-HCl pH7.6, then the supernatant liquor dilute with water is pumped in the post for one times.Include the buffer solution elution of 0.1M NaCl with 20mM Tris-HCl pH7.6, change the damping fluid that 20mM Tris-HCl pH7.6 includes 0.3M NaCl after measured value to be checked is lower than below 0.05, human tissue kallikrein desorption this moment flows out in post, collect the cut of this moment, 4 ℃ of storages are standby.Pillar is used the foreign protein of other absorption under the 3M NaCl wash-out again, and is standby with the damping fluid balance.
Its result as shown in Figure 2.The SDS-PAGE detected result is as shown in Figure 4: wherein swimming lane 1 is: the low molecular protein standard; Swimming lane 2 is: the pure product of genetically engineered human tissue kallikrein (rHUK) that Lee Chao gives in the laboratory; Swimming lane 3 is: the rHUK of Fractional Collections; Swimming lane 4 is: the fraction of collecting when 20mM Tris-Hcl pH7.6with 0.3M NaCl.Separating resulting when Fig. 3 strengthens 100 times for sample size, other experiment condition is identical with Fig. 2.
2. sample preparation: the cut that the back ion exchange column is collected is the filter concentrating sample of 10Kda with Centriprep 10P molecular weight cut-off, and will concentrating afterwards then, sample is dissolved in the damping fluid of 10mM NaAc pH5.0 again.
Pillar with 10mM NaAc pH5.0 balance, is written into the sample of handling well in the post earlier then, continues towards post, up to A with level pad
280Absorption value is reduced to below 0.05, includes the buffer solution elution of 0.1M NaCl again with 10mM NaAc pH5.0, collects cut a.Include the buffer solution elution of 0.15MNaCl then with 10mM NaAc pH5.0, collect cut b.Then use linear gradient elution, the salt component scope is 0.15M NaCl to 0.5M NaCl in the elutriant, and this moment, a large amount of human tissue kallikreins were eluted, and collected cut, 4 ℃ of preservations.In cut a that collects and b, also there is a small amount of human tissue kallikrein to elute, can collects and cross post again.
Its result is as shown in Figure 5: chromatographic column wherein: DEAE-Sepharose 16 * 200mm; Damping fluid: A:10mM NaAc pH5.0; B:10mM NaAc with0.5M NaCl; Flow velocity: 5ml/min detects wavelength: 280nm; Gradient condition; With 10mM NaAc pH5.0 flushing 30min, with 10mM NaAcwith0.10M NaCl flushing 30min, use linear gradient elution at last with 10mM NaAc with0.15M NaCl flushing 30min more earlier, the wash-out scope is 0.15M NaCl to 0.5M NaCl; RHUK is at linear gradient section wash-out.
3. affinity chromatography separation and purification
Human tissue kallikrein belongs to serine protease, therefore affine, the phenylformic acid amine (PAB) of available specific immunity, press down enzyme peptide multiple affinity chromatography separating mediums such as (Aprotinin) and carry out purifying.It is better that use presses down the latent effect of the affine look of enzyme peptide, and can repeatedly use repeatedly.
Concrete operations can be adopted following two kinds of modes:
Aprotinin-Agrose column purification method 1:
After active fractions suitably concentrated, be transferred on the affinity column, with 15-25mMTris～HCl, pH7.0 damping fluid thorough washing; Remove not the protein of absorption, reduce to baseline to absorbancy.Use 0.1mol/LGiy～HCI then, pH2.8～3.0 buffer solution elution; The peak that occurs is target product; Immediately with 0.5mol/L Tris neutralization; This solution is dialysed to the 20mM phosphoric acid buffer; Lyophilize.
Aprotinin-Agrose column purification method 2:
Pillar includes the damping fluid balance of 200mM NaCl earlier with 20mM Tris-HCl pH8.0, sample is written in the post, inhales with 0.1M glycine/HCl pH2.0 and takes off, and collects elution peak.With 0.5mol/L Tris neutralization, this solution is dialysed to the 20mM phosphoric acid buffer immediately; Lyophilize.
Its result is as shown in Figure 8: wherein swimming lane 1 is: the mark product; Swimming lane 2 is: human tissue kallikrein; Can prepare the pure rHUK of electrophoresis with this method as can be seen.
Its yield is 80%; The result is as shown in Figure 7: chromatographic column wherein: Shodex AFpak AAP-8948 * 50mm; Damping fluid: A:0.02Mtris-HCl with 0.2M NaCl pH8.0; B:0.1M glycine pH2.0; Flow velocity: 1ml/min detects wavelength: 280nm.
Comparative example 1:
1. the separation of cell culture supernatant: with embodiment 1;
2. ion exchange chromatography separates:
DEAE-Sepharose ion exchange layer analysis method: chromatographic column: 16mm * 20cm glass packed column; Filler: DEAE-Sepharose ion-exchange packing; Peristaltic pump: miniplus3, Gilson company; Detect wavelength: 280nm; Flow velocity: 5ml/min
Use the linear gradient elution supernatant liquor, its result as shown in Figure 1, wherein: chromatographic column: ion-exchange packing DEAE-Sepharose, 16 * 200mm glass packed column; Linear gradient elution; Buffer A: 20mMTris-HCl pH8.0, buffer B: 20mMtris-HCl, pH8.0with 0.5M NaCl; Flow velocity: 5ml/min; Detect wavelength: 280nm.Human tissue kallikrein flows out in by post when salt concn is 0.3M, and as can be seen from the figure kallikrein is mixed in other albumen, is difficult to determine the terminal of collecting when collecting sample, and the uncomfortable cooperation the first step is separated.
Comparative example 2:
1. the separation of cell culture supernatant: with embodiment 1;
2. ion exchange chromatography separates:
DEAE-Sepharose ion exchange layer analysis method: chromatographic column: 16mm * 20cm glass packed column; Filler: DEAE-Sepharose ion-exchange packing; Peristaltic pump: miniplus3, Gilson company; Detect wavelength: 280nm; Flow velocity: 5ml/min
1. with embodiment 1; Adopt gel chromatography to filter afterwards;
Chromatographic column: 16 * 400mm glass packed column; Filler: Sephcryal S-200; Buffer:0.005M sodium phosphate+0.09M NaCl; Flow velocity: 1ml/min detects wavelength: 280nm.
Its result as shown in Figure 6.From separating resulting, Sephcryal S-200 also is unrealized human tissue kallikrein is effectively separated this purpose with foreign protein, but also has enlarged volume, brings more troubles to subsequent disposal.
Comparative example 3:
Because of human tissue kallikrein contains a hydrophobic grouping, therefore consider to use hydrophobic chromatography as 1. going on foot purification step, as far as possible human tissue kallikrein and other foreign protein are separated.
1. the separation of cell culture supernatant: with embodiment 1;
2. hydrophobic chromatography is separated:
Chromatographic column: 16 * 200mm glass packed column; Filler: phenyl-sepharose 6B; Flow velocity: 3ml/min; Detect wavelength: 280nm
With 3M NaCl solution equilibria, the cell culture supernatant that will contain human tissue kallikrein then is made into 3M NaCl solution and pumps in the post chromatographic column earlier.Continue eluant solution, after measured value to 0.05 to be checked is following, collect sample with 3M NaCl.Detect finder tissue kallikrein through SDS-PAGE and all be not adsorbed on the post, the rate of recovery is low, and with the separating effect of other foreign protein not as ion-exchange chromatography.
Embodiment 2
1. the separation of cell culture supernatant: with embodiment 1;
2. ion exchange chromatography separates:
DEAE-Sepharose ion exchange layer analysis method: chromatographic column: 16mm * 20cm glass packed column; Filler: DEAE-Sepharose ion-exchange packing; Peristaltic pump: miniplus3, Gilson company; Detect wavelength: 280nm; Flow velocity: 5ml/min
1. earlier with the damping fluid balance pillar of 20mM Tris-HCl pH6, then the supernatant liquor dilute with water is pumped in the post for one times.Include the buffer solution elution of 0.1M NaCl with 20mM Tris-HCl pH6, collect cut a; Change 20mM Tris-HCl pH6 after measured value to be checked is lower than below 0.05 and include the buffer solution elution of 0.3M NaCl, collect cut b.SDS-PAGE detects the back and finds all to contain human tissue kallikrein in passing peak, cut a and cut b.Its result as shown in figure 10, among the figure left side several the 6th swimming lanes be cut b.
Following steps are with embodiment 1
The first step can't realize to greatest extent human tissue kallikrein being separated with other foreign protein that high yield this purpose is arranged simultaneously under this condition.Yield obviously reduces, and has only about 50%.
Embodiment 3
1. the separation of cell culture supernatant: with embodiment 1;
2. ion exchange chromatography separates:
DEAE-Sepharose ion exchange layer analysis method: chromatographic column: 16mm * 20cm glass packed column; Filler: DEAE-Sepharose ion-exchange packing; Peristaltic pump: miniplus3, Gilson company; Detect wavelength: 280nm; Flow velocity: 5ml/min
1. earlier with the damping fluid balance pillar of 20mM Tris-HCl pH8.5, then the supernatant liquor dilute with water is pumped in the post.Include the buffer solution elution of 0.1M NaCl with 20mM Tris-HCl pH8.5, collect cut a; Change 20mM Tris-HCl pH8.5 after measured value to be checked is lower than below 0.05 and include the buffer solution elution of 0.3M NaCl, collect cut b.
Following steps are with embodiment 1.
Yield 82%.But, bring a lot of troubles to follow-up work through the content showed increased that SDS-PAGE detects foreign protein among the first step component b.
Embodiment 4
1. the separation of cell culture supernatant: with embodiment 1;
2. ion exchange chromatography separates:
DEAE-Sepharose ion exchange layer analysis method: chromatographic column: 16mm * 20cm glass packed column; Filler: DEAE-Sepharose ion-exchange packing; Peristaltic pump: miniplus3, Gilson company; Detect wavelength: 280nm; Flow velocity: 5ml/min
1. with embodiment 1;
2. sample preparation: the cut that the back ion exchange column is collected is the above filter concentrating sample of 10Kda with Centriprep 10P molecular weight cut-off, will concentrate the direct upper prop of sample afterwards then.
Pillar with 10mM NaAc pH5.0 balance, is written into the sample of handling well in the post earlier then, includes the buffer solution elution of 0.1M NaCl with 10mM NaAc pH5.0.Include the buffer solution elution of 0.15M NaCl then with 10mM NaAc pH5.0.Then use linear gradient elution, the salt component scope is 0.15M NaCl to 0.5M NaCl in the elutriant, and this moment, a large amount of human tissue kallikreins were eluted, and collected cut.
As can be seen from Figure 9 this condition fails sample is separated, and all components all elutes in same elution peak, does not reach further isolating effect.
Embodiment 5
The first step DEAE-Sepharose ion exchange layer analysis method: chromatographic column: 16mm * 20cm glass packed column; Filler: DEAE-Sepharose ion-exchange packing; Peristaltic pump: miniplus3, Gilson company; Detect wavelength: 280nm; Flow velocity: 5ml/min
Experiment condition: go up behind the sample with 20mM Tris-HCl pH7.6 with 0.15M NaCl, change 20mM Tris-HCl with 0.25M NaCl into after being eluted to baseline, be eluted to and use 20mM Tris-HClwith 0.5M NaCl again instead behind the baseline and be eluted to baseline.Yield 76%.
Claims (7)
1. the separation purification method of a gene-expressed human tissue kallikrein protein is characterized in that the detailed process operation is as follows:
(1) separation of cell culture supernatant: the medium centrifugal that will contain human tissue kallikrein separates, and it is standby to get supernatant liquor;
(2) ion exchange chromatography separates:
1. adopt the step gradient elution: with the damping fluid balance pillar of 20～30mM, pH7～8,, pump in the post earlier then with the supernatant liquor dilute with water; Include the buffer solution elution of 0.1～0.2M NaCl again with 20～30mM, pH7～8, measured value to be checked is lower than below 0.05, changes the damping fluid that 20～30mM pH7～8 include 0.2～0.4MNaCl then, collects the cut of this moment, and is standby;
2. earlier with 10～20mM NaAc, pH5.0～6.0 balance pillars, then sample is written in the post, continues towards post, up to A with level pad
280Absorption value is reduced to below 0.05, includes the buffer solution elution of 0.05～0.1M NaCl again with 10～20mM NaAc, pH5.0～6.0, collects cut a; Include the buffer solution elution of 0.1～0.15M NaCl then with 10～20mMNaAc, pH5.0～6.0, collect cut b; Then use linear gradient elution, the salt component scope is 0.1M NaCl to 0.5M NaCl in the elutriant, collects cut and preserves;
(3) affinity chromatography separation and purification: operation according to a conventional method gets product;
Wherein: buffered soln is Tris-HCl; 2. before carry out sample pretreatment in described step: with described step 1. the ion exchange column cut ultrafiltration of collecting hold back, molecular weight cut-off is the filter concentrating sample of 10Kda, will concentrate the back sample then and be dissolved in again in the damping fluid of 10～20mM NaAc pH5.0～6.0; Concrete operations in step (3) affinity chromatography separation and purification are:
Aprotinin-Agrose column purification: active fractions is concentrated, be transferred to then on the affinity column, with 15～25mMTris-HCl, pH6.5～7.5 damping fluid thorough washing; Use 0.05～0.15mol/L GIy-HCI, pH2.0～3.0 buffer solution elution again; Collect target product;
Described operation or be: include the damping fluid balance pillar of 200～250mM NaCl earlier with 15～25mM Tris-HCl, pH7.5～8.5, sample is written in the post, take off the collection elution peak with 0.1～0.15M glycine/HCl, pH2.0～3.0 suctions.
2. according to the described separation purification method of claim 1, it is characterized in that: employed ion-exchanger is a weak anion exchange resin in the described ion exchange chromatography sepn process.
3. according to the described separation purification method of claim 2, it is characterized in that: described exchange resin is polyose ion-exchanger DEAE-cellulose, DEAE-sephadex, DEAE-sepharose or crosslinked agar carbohydrate ion exchange resin DEAE Sepharose CL-6B.
4. according to the described separation purification method of claim 1, it is characterized in that: employed NaCl substitutes with KCL in the sepn process of described claim 1 ion exchange chromatography.
5. according to the described separation purification method of claim 1, it is characterized in that: in step (3), adopt Trypsin inhibitor,Trasylol affinity chromatography separating medium to carry out purifying.
6. according to the described separation purification method of claim 1, it is characterized in that: neutralize with 0.3～0.5mol/L Tris immediately at step (3) affinity chromatography separation and purification after product.
7. according to the described separation purification method of claim 6, it is characterized in that: the back solution that will neutralize is dialysed to 20～25mM phosphoric acid buffer; Lyophilize.
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| CN103122028B (en) * | 2011-11-18 | 2016-03-30 | 复旦大学 | Trypsinase affinitive layer purification people recombinates the method for SPINK6 albumen |
| CN103865909B (en) * | 2012-12-28 | 2016-04-13 | 青岛九龙生物医药有限公司 | A kind of preparation method that can remove the urokinase of pyrogen and virus |
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| US3830791A (en) * | 1971-04-24 | 1974-08-20 | Bayer Ag | Purification of enzyme inhibitors by amphoteric ion exchange resins |
| US3905870A (en) * | 1972-11-03 | 1975-09-16 | Bayer Ag | Purification of kallikrein |
| US4393140A (en) * | 1981-01-31 | 1983-07-12 | Bayer Aktiengesellschaft | Process for the preparation of the highly pure enzyme kallikrein from swine pancreas extracts |
| JPH0948817A (en) * | 1995-08-07 | 1997-02-18 | Mitsubishi Chem Corp | Affinity resin for purification of plasma kallikrein |
-
2002
- 2002-10-11 CN CNB021331642A patent/CN1294256C/en not_active Expired - Fee Related
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US3830791A (en) * | 1971-04-24 | 1974-08-20 | Bayer Ag | Purification of enzyme inhibitors by amphoteric ion exchange resins |
| US3905870A (en) * | 1972-11-03 | 1975-09-16 | Bayer Ag | Purification of kallikrein |
| US4393140A (en) * | 1981-01-31 | 1983-07-12 | Bayer Aktiengesellschaft | Process for the preparation of the highly pure enzyme kallikrein from swine pancreas extracts |
| JPH0948817A (en) * | 1995-08-07 | 1997-02-18 | Mitsubishi Chem Corp | Affinity resin for purification of plasma kallikrein |
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| Publication number | Publication date |
|---|---|
| CN1488753A (en) | 2004-04-14 |
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