CN102516387A - Affinity medium for human urinary trypsin inhibitor as well as synthesis and application for same - Google Patents
Affinity medium for human urinary trypsin inhibitor as well as synthesis and application for same Download PDFInfo
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Abstract
The invention relates to an affinity medium for human urinary trypsin inhibitor, formed by connecting an affinity ligand for human urinary trypsin inhibitor with a chromatography medium. Preferably, the affinity ligand for human urinary trypsin inhibitor is an analogue of the site S1 of serine protease, the chromatography medium is Sepharose, chitosan, silica gel and ceramic particles, and the affinity ligand for human urinary trypsin inhibitor is connected with the chromatography medium by using cyanuric chloride as a spacer arm. A synthesis method for the affinity medium for human urinary trypsin inhibitor, and a method for purifying human urinary trypsin inhibitor in a large scale by using the affinity medium for human urinary trypsin inhibitor are further provided. Human urinary trypsin inhibitor can be rapidly separated and purified in a large scale by using the affinity medium for human urinary trypsin inhibitor, and human urinary trypsin inhibitor pure product can be obtained in case of only one-step affinity chromatography; and the human urinary trypsin inhibitor pure product is short in the consumed time of whole purification step, high in the recovery rates of protein and activity, and suitable for large-scale popularization and application.
Description
Technical field
The present invention relates to technical field of bioengineering, the enzyme production technical field is used in particularly medical treatment, diagnosis, specifically is meant a kind of human urine trypsin inhibitor affinity media and synthetic and application thereof.
Background technology
Human urine trypsin inhibitor is a kind of broad-spectrum protease suppressor factor of Kunitz type, can effectively suppress the activity of trypsinase, Chymotrypsin, Sumylact L, lypase, Unidasa and other pancreas enzymes.In addition, it has stable lysosome membrane, suppresses the release of lysosomal enzyme, suppresses myocardial depressant factor (MDF) (MDF) and produces, and removes the effect that oxyradical and inflammation-inhibiting medium discharge.Human urine trypsin inhibitor also can improve operation stimulates the unusual and renal function of lower immune function, the Proteometabolism cause to reduce, prevent to perform the operation stimulate cause to internal organs with cells injury and improve recurrent state when suffering a shock etc.
It is widely used in clinical treatment; Suitable disease comprises the acute worsening period of acute pancreatitis (acute pancreatitis that comprises traumatic, postoperative and endoscope retrogradation property ERCP postoperative), chronic recurrent pancreatitis, acute circulatory failure (hemorrhagic shock, bacillary shock, traumatic shock, burn shock); Also be widely used in thoracic surgery, Digestive tract operation, tumor operation, organ transplantation, organ removal operation and CPB operation; Also be used to treat the renal tubal dysfunction that produces with the prophylaxis of tumours chemotherapy.
At present, and the Mochida Pharmaceutical of Japan (Tokyo, Japan) there is more sophisticated product in company, and molecular weight is～67kDa that domestic also have enterprise from the healthy male urine, to extract this albumen.Yet the purification procedures of human urine trypsin inhibitor has generally included the ammonium sulfate precipitation of multistep, and surfactant is handled, thermal treatment, ion exchange chromatography, sieve chromatography etc.And too much purification step has caused the purifying time longer, the result that albumen and activity recovery are lower.
For the scale operation human urine trypsin inhibitor, must reduce the step of separation and purification.Therefore, rapidly and efficiently human urine trypsin inhibitor separation method need be provided.
Summary of the invention
The objective of the invention is to have overcome above-mentioned shortcoming of the prior art; A kind of human urine trypsin inhibitor affinity media and synthetic and application thereof are provided, utilize this human urine trypsin inhibitor affinity media fast separating and purifying human urine trypsin inhibitor on a large scale, can only go on foot under the situation of affinity chromatography with one; Obtain the pure article of human urine trypsin inhibitor; Whole purification step elapsed time is short, and albumen and activity recovery are higher, are suitable for large-scale promotion application.
To achieve these goals; In first aspect of the present invention; A kind of human urine trypsin inhibitor affinity media is provided, has been characterized in, said human urine trypsin inhibitor affinity media is formed by connecting human urine trypsin inhibitor affinity ligand and chromatography media.
Preferably, said human urine trypsin inhibitor affinity ligand is a Tryase S1 site analog, and said chromatography media is Sepharose, chitosan, silica gel or ceramic particle.
More preferably, said Tryase S1 site analog be-ASP-GLY or-SER-GLY.
Preferably, said human urine trypsin inhibitor affinity media is connected as spacerarm through cyanuric chloride with chromatography media by the human urine trypsin inhibitor affinity ligand.
In second aspect of the present invention, a kind of compound method of above-mentioned human urine trypsin inhibitor affinity media is provided, be characterized in, said human urine trypsin inhibitor affinity ligand is connected with said chromatography media obtains.
Preferably, said human urine trypsin inhibitor affinity media is connected as spacerarm through cyanuric chloride with chromatography media by the human urine trypsin inhibitor affinity ligand.
In the third aspect of the invention; A kind of method of utilizing above-mentioned human urine trypsin inhibitor affinity media large scale purification human urine trypsin inhibitor is provided; Be characterized in; The solution stream that will contain human urine trypsin inhibitor is carried out affine absorption through said human urine trypsin inhibitor affinity media; Adopt scavenging solution to wash said human urine trypsin inhibitor affinity media then, at last with the human urine trypsin inhibitor that adsorbs on the said human urine trypsin inhibitor affinity media of elution buffer wash-out, thus the purifying human urine trypsin inhibitor.
Preferably, the described solution that contains human urine trypsin inhibitor is to adopt to obtain redissolving after human urine filtration and the freeze-drying processing.
Preferably, the condition of said affine absorption is a pH value 6.0～7.0, and specific conductivity is 5-10ms/cm.
Preferably, the cleaning condition that adopts scavenging solution to wash said human urine trypsin inhibitor affinity media is a pH value 6.0～7.0, specific conductivity 5～10ms/cm.
Preferably, use the elution requirement of the human urine trypsin inhibitor that adsorbs on the said human urine trypsin inhibitor affinity media of elution buffer wash-out to be pH value 2.0～3.0.
Beneficial effect of the present invention is specific as follows:
1, the present invention is through adopting Tryase S1 site analog as the human urine trypsin inhibitor affinity ligand; Be connected synthetic human urine trypsin inhibitor affinity media with chromatography media; Rapidly and efficiently the purifying human urine trypsin inhibitor is suitable for large-scale promotion application;
2, the present invention makes the purification efficiency of human urine trypsin inhibitor significantly increase through optimizing the protein adsorption condition, has big industrial application potentiality, embodies bigger economic benefit.
Description of drawings
Figure 1A is the structural representation of human urine trypsin inhibitor affinity ligand of first specific embodiment (being abbreviated as S-D-G) of human urine trypsin inhibitor affinity media of the present invention.
Figure 1B is the structural representation of human urine trypsin inhibitor affinity ligand of second specific embodiment (being abbreviated as S-S-G) of human urine trypsin inhibitor affinity media of the present invention.
Fig. 2 be first specific embodiment shown in Figure 1A and with second specific embodiment shown in Figure 1B when human urine trypsin inhibitor is adsorbed the pH value to the influence of human urine trypsin inhibitor adsorptive capacity.
Fig. 3 be under the pH6.0 first specific embodiment shown in Figure 1A and with second specific embodiment shown in Figure 1B to the adsorptive capacity of human urine trypsin inhibitor, wherein X-coordinate is represented in the Static Adsorption process, the free not human urine trypsin inhibitor of absorption; Ordinate zou is the human urine trypsin inhibitor of affinity media absorption.
Fig. 4 be with first specific embodiment shown in Figure 1A and with second specific embodiment shown in Figure 1B when being the human urine trypsin inhibitor affinity media elution buffer to the influence of the human urine trypsin inhibitor recovery.
Embodiment
In order more to be expressly understood technology contents of the present invention, the special following examples of lifting specify.
The preparation of people's trypsin inhibitor solution that following embodiment 1-2 and Comparative Examples 1 are used: the urine that will come from healthy male is carried out natural subsidence 1h; Get 200ml liquid; Use filter paper filtering to remove particle, it is Powdered behind-80 degrees centigrade of freezing 2h, using the vacuum freeze drier freeze-drying.(pH～6.0, redissolve by specific conductivity 5～10ms/cm) with adding 200ml damping fluid (20mM PBs pH 6.0) for powder.Get the centrifugal 15min of dissolving back solution 12000rpm, get supernatant and use.
The employing scavenging solution that relates to is a pH value 6.0～7.0 for the cleaning condition of the said people's trypsin inhibitor affinity media of flushing, specific conductivity 5～10ms/cm.For example adopt the scavenging solution of 20mM PBs pH 6.0.
Synthetic and the application of embodiment 1:Sepharose-ASP-GLY
Sepharose CL 4B (100g) drains into wet pie with the deionized water wash of 10 times of volumes; Be suspended in 50ml activation damping fluid (0.8M NaOH; 20% methyl-sulphoxide, 10% epoxy chloropropane, 0.5mg/ml Peng Qinghuana) 40 ℃ of shaking table concussion 2.5h; Pour into then in the glass frosted funnel; Under suction filtration,, to neutral, draining into wet pie up to washings pH through the distilled water wash of each 10 times of volumes.Activatory Sepharose CL 4B dielectric suspension adds the 20ml ethylenediamine solution in 500ml0.1M NaOH solution, and gel is stirring (200rpm) following 30 ℃ of constant temperature 12h.Use washed with de-ionized water.NH
2-Sepharose CL 4B is suspended in 350ml 50% (v/v) the ice bath acetone soln, subsequently 4g three nitrogen piperazines is dissolved in 80ml-20 ℃ of precooling acetone, adds dielectric suspensions fast, detects the pH value in real time, uses saturated NaHCO
3PH is maintained between the 6.5-7.0, and maintain continues to stir 2-4h at 0-4 ℃, and to the white disappearance, mixture is the transparence stopped reaction.With the acetone of 3 times of medium volumes, acetone: deionized water (1: 1), deionized water, the order washing reaction gets dichloro three nitrogen piperazine-amino-Sepharose CL 4B.Take by weighing 40g dichloro three nitrogen piperazine-amino-Sepharose CL 4B, take by weighing the 10g aspartic acid, be dissolved in the 80ml deionized water, 50-60 ℃ of vibration 24h, running audit pH in the reaction process remains between the 11-12 solution with 1M NaOH.Reaction finishes, and with 500ml deionized water thorough washing, drains.Take by weighing the 20g glycocoll, be dissolved in the 80ml deionized water, the medium of draining with the last step mixes, 85-95 ℃ of vibration 60h; Running audit pH in the reaction process remains between the 11-12 solution with 1M NaOH.Reaction finishes, and with 500ml deionized water thorough washing, drains.
(1) optimal adsorption pH value confirms
Get the human urine trypsin inhibitor standard substance, using deionized water to be diluted to concentration is 600 μ g/ml, is divided into 6 groups, and it is 4 that every group of solution is regulated the pH value; 5,6,7,8; 9, volume 1ml adds the 0.01g humid medium respectively, behind 4 ℃ of 2h that fully vibrate; Measure supernatant human urine trypsin inhibitor concentration, the adsorptive capacity of calculation medium, the pH value of selection optimal adsorption.
Initial human urine trypsin inhibitor～0.6mg/l is fully vibrating behind the 2h with medium different pH values under, and a large amount of human urine trypsin inhibitors are adsorbed by affinity media, the content reduction in the supernatant, and adsorptive capacity and pH value concern sees Fig. 2.Preferable adsorption conditions does, temperature is 4 ℃, and pH is 6.0-7.0, and specific conductivity is 5-10ms/cm.The optimal adsorption condition does, temperature is 4 ℃, and pH is 6, and specific conductivity is 5～10ms/cm.
(2) the medium maximal absorptive capacity confirms
On the basis that the pH value is optimized, confirm the maximal absorptive capacity of medium.Get the human urine trypsin inhibitor standard substance, using deionized water to be diluted to concentration is 100,200,300; 400,500,600,700; 800,900 μ g/ml, regulating the pH value is 7.8, adds the 0.01g humid medium respectively; Behind 4 ℃ of 2h that fully vibrate, measure supernatant human urine trypsin inhibitor concentration, the maximal absorptive capacity of calculation medium.Adsorptive capacity and supernatant protein concentration relation are seen Fig. 3, and Units of Account medium maximal absorptive capacity is the 31.7mg/g medium.
(3) optimum washing engaging condition confirms
On the basis of cultivating adsorption conditions and maximal absorptive capacity, investigate of the influence of elutriant pH value to the wash-out result, elutriant is 0.01M HCl, the result sees Fig. 4.Visible from figure, preferable elution requirement is a pH value 2.0～3.0; The righttest elution requirement is that the pH value is 2.0.
Synthetic and the application of embodiment 2:Sepharose-SER-GLY
Sepharose CL 4B (100g) drains into wet pie with the deionized water wash of 10 times of volumes; Be suspended in 50ml activation damping fluid (0.8M NaOH; 20% methyl-sulphoxide, 10% epoxy chloropropane, 0.5mg/ml Peng Qinghuana) 40 ℃ of shaking table concussion 2.5h; Pour into then in the glass frosted funnel; Under suction filtration,, to neutral, draining into wet pie up to washings pH through the distilled water wash of each 10 times of volumes.Activatory Sepharose CL 4B dielectric suspension adds the 20ml ethylenediamine solution in 500ml0.1M NaOH solution, and gel is stirring (200rpm) following 30 ℃ of constant temperature 12h.Use washed with de-ionized water.NH
2-Sepharose CL 4B is suspended in 350ml 50% (v/v) the ice bath acetone soln, subsequently 4g three nitrogen piperazines is dissolved in 80ml-20 ℃ of precooling acetone, adds dielectric suspensions fast, detects the pH value in real time, uses saturated NaHCO
3PH is maintained between the 6.5-7.0, and maintain continues to stir 2-4h at 0-4 ℃, and to the white disappearance, mixture is the transparence stopped reaction.With the acetone of 3 times of medium volumes, acetone: deionized water (1: 1), deionized water, the order washing reaction gets dichloro three nitrogen piperazine-amino-Sepharose CL 4B.Take by weighing 40g dichloro three nitrogen piperazine-amino-Sepharose CL 4B, take by weighing the 10g Serine, be dissolved in the 80ml deionized water, 50-60 ℃ of vibration 24h, running audit pH in the reaction process remains between the 11-12 solution with 1M NaOH.Reaction finishes, and with 500ml deionized water thorough washing, drains.Take by weighing the 20g glycocoll, be dissolved in the 80ml deionized water, the medium of draining with the last step mixes, 85-95 ℃ of vibration 60h; Running audit pH in the reaction process remains between the 11-12 solution with 1M NaOH.Reaction finishes, and with 500ml deionized water thorough washing, drains.
(1) optimal adsorption pH value confirms
Get the human urine trypsin inhibitor standard substance, using deionized water to be diluted to concentration is 600 μ g/ml, is divided into 6 groups, and it is 4 that every group of solution is regulated the pH value; 5,6,7,8; 9, volume 1ml adds the 0.01g humid medium respectively, behind 4 ℃ of 2h that fully vibrate; Measure supernatant human urine trypsin inhibitor concentration, the adsorptive capacity of calculation medium, the pH value of selection optimal adsorption.
Initial human urine trypsin inhibitor～0.6mg/l is fully vibrating behind the 2h with medium different pH values under, and a large amount of human urine trypsin inhibitors are adsorbed by affinity media, the content reduction in the supernatant, and adsorptive capacity and pH value concern sees Fig. 2.Preferable adsorption conditions does, temperature is 4 ℃, and pH is 6.0-7.0, and specific conductivity is 5-10ms/cm.The optimal adsorption condition does, temperature is 4 ℃, and pH is 6, and specific conductivity is 5～10ms/cm.
(2) the medium maximal absorptive capacity confirms
On the basis that the pH value is optimized, confirm the maximal absorptive capacity of medium.Get the human urine trypsin inhibitor standard substance, using deionized water to be diluted to concentration is 100,200,300; 400,500,600,700; 800,900 μ g/ml, regulating the pH value is 7.8, adds the 0.01g humid medium respectively; Behind 4 ℃ of 2h that fully vibrate, measure supernatant human urine trypsin inhibitor concentration, the maximal absorptive capacity of calculation medium.Adsorptive capacity and supernatant protein concentration relation are seen Fig. 3, and Units of Account medium maximal absorptive capacity is the 30.1mg/g medium.
(3) optimum washing engaging condition confirms
On the basis of cultivating adsorption conditions and maximal absorptive capacity, investigate of the influence of elutriant pH value to the wash-out result, elutriant is 0.01M HCl, the result sees Fig. 4.Visible from figure, preferable wash-out is adjusted to pH value 2.0～3.0; The righttest elution requirement is that the pH value is 2.0.
Synthetic and the application of Comparative Examples 1:Sepharose-GLY-GLY
Sepharose CL 4B (100g) drains into wet pie with the deionized water wash of 10 times of volumes; Be suspended in 50ml activation damping fluid (0.8M NaOH; 20% methyl-sulphoxide, 10% epoxy chloropropane, 0.5mg/ml Peng Qinghuana) 40 ℃ of shaking table concussion 2.5h; Pour into then in the glass frosted funnel; Under suction filtration,, to neutral, draining into wet pie up to washings pH through the distilled water wash of each 10 times of volumes.Activatory Sepharose CL 4B dielectric suspension adds the 20ml ethylenediamine solution in 500ml0.1M NaOH solution, and gel is stirring (200rpm) following 30 ℃ of constant temperature 12h.Use washed with de-ionized water.NH2-SepharoseCL 4B is suspended in 350ml 50% (v/v) the ice bath acetone soln; Subsequently 4g three nitrogen piperazines are dissolved in 80ml-20 ℃ of precooling acetone, add dielectric suspensions fast, detect the pH value in real time; With saturated NaHCO3 pH is maintained between the 6.5-7.0; Maintain continues to stir 2-4h at 0-4 ℃, and to the white disappearance, mixture is the transparence stopped reaction.With the acetone of 3 times of medium volumes, acetone: deionized water (1: 1), deionized water, the order washing reaction gets dichloro three nitrogen piperazine-amino-Sepharose CL4B.Take by weighing 40g dichloro three nitrogen piperazine-amino-Sepharose CL 4B, take by weighing the 10g glycocoll, be dissolved in the 80ml deionized water, 50-60 ℃ of vibration 24h, running audit pH in the reaction process remains between the 7.5-8.0 solution with 1M NaOH.Reaction finishes, and with 500ml deionized water thorough washing, drains.Take by weighing the 20g glycocoll, be dissolved in the 80ml deionized water, the medium of draining with the last step mixes, 85-95 ℃ of vibration 60h; Running audit pH in the reaction process remains between the 11-12 solution with 1M NaOH.Reaction finishes, and with 500ml deionized water thorough washing, drains.
Adopt Sepharose-G-G to carry out the confirming of optimal adsorption pH value, confirming of medium maximal absorptive capacity and confirming of optimum washing engaging condition, experimentation is with experimentation accordingly among the embodiment 1, and the result shows that Sepharose-G-G does not have adsorptive power basically.
Therefore; The invention provides a kind of method with separation and purification human urine trypsin inhibitor of industrial application potentiality; It uses Sepharose-ASP-GLY affinity chromatography one step separation and purification human urine trypsin inhibitor; Protein recovery is～1.4%, and activity recovery is～77.5%, and specific activity is～2840U/mg.It uses Sepharose-SER-GLY affinity chromatography one step separation and purification human urine trypsin inhibitor, and protein recovery is～11%, and activity recovery is～73.2%, and specific activity is～3210U/mg.
In this specification sheets, the present invention is described with reference to its certain embodiments.But, still can make various modifications and conversion obviously and not deviate from the spirit and scope of the present invention.Therefore, specification sheets and accompanying drawing are regarded in an illustrative, rather than a restrictive.
Claims (10)
1. a human urine trypsin inhibitor affinity media is characterized in that, said human urine trypsin inhibitor affinity media is formed by connecting human urine trypsin inhibitor affinity ligand and chromatography media.
2. human urine trypsin inhibitor affinity media according to claim 1; It is characterized in that; Said human urine trypsin inhibitor affinity ligand is a Tryase S1 site analog, and said chromatography media is Sepharose, chitosan, silica gel or ceramic particle.
3. human urine trypsin inhibitor affinity media according to claim 2 is characterized in that, said Tryase S1 site analog is-ASP-GLY or-SER-GLY.
4. human urine trypsin inhibitor affinity media according to claim 1 is characterized in that, said human urine trypsin inhibitor affinity media is connected as spacerarm through cyanuric chloride with chromatography media by the human urine trypsin inhibitor affinity ligand.
5. the compound method of a human urine trypsin inhibitor affinity media according to claim 1 is characterized in that, said human urine trypsin inhibitor affinity ligand is connected with said chromatography media obtain.
6. method of utilizing human urine trypsin inhibitor affinity media large scale purification human urine trypsin inhibitor according to claim 1; It is characterized in that; The solution stream that will contain human urine trypsin inhibitor is carried out affine absorption through said human urine trypsin inhibitor affinity media; Adopt scavenging solution to wash said human urine trypsin inhibitor affinity media then; At last with the human urine trypsin inhibitor that adsorbs on the said human urine trypsin inhibitor affinity media of elution buffer wash-out, thus the purifying human urine trypsin inhibitor.
7. the method for human urine trypsin inhibitor affinity media large scale purification human urine trypsin inhibitor according to claim 6; It is characterized in that the described solution that contains human urine trypsin inhibitor is to adopt to obtain redissolving after human urine filtration and the freeze-drying processing.
8. the method for human urine trypsin inhibitor affinity media large scale purification human urine trypsin inhibitor according to claim 6 is characterized in that the condition of said affine absorption is a pH value 6.0～7.0, and specific conductivity is 5-10ms/cm.
9. the method for human urine trypsin inhibitor affinity media large scale purification human urine trypsin inhibitor according to claim 6; It is characterized in that; The cleaning condition that adopts scavenging solution to wash said human urine trypsin inhibitor affinity media is a pH value 6.0～7.0, specific conductivity 5～10ms/cm.
10. the method for human urine trypsin inhibitor affinity media large scale purification human urine trypsin inhibitor according to claim 6; It is characterized in that, use the elution requirement of the human urine trypsin inhibitor that adsorbs on the said human urine trypsin inhibitor affinity media of elution buffer wash-out to be pH value 2.0～3.0.
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Cited By (4)
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| CN103880951A (en) * | 2014-03-31 | 2014-06-25 | 南昌市万华生化制品有限公司 | Method for preparing pure ulinastatin from ulinastatin affinity chromatography medium |
| CN104140473A (en) * | 2014-06-10 | 2014-11-12 | 盐城市印染有限公司 | Chitosan s-triazine sulfonate derivative as well as preparation method and application of silicon hybridization solution thereof |
| CN105219755A (en) * | 2015-10-09 | 2016-01-06 | 长沙仁泽生物技术有限公司 | A kind of extraction of chymotrypsinogen and purification process |
| CN111778231A (en) * | 2020-07-29 | 2020-10-16 | 珠海冀百康生物科技有限公司 | Purification method of lysyl endopeptidase |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103880951A (en) * | 2014-03-31 | 2014-06-25 | 南昌市万华生化制品有限公司 | Method for preparing pure ulinastatin from ulinastatin affinity chromatography medium |
| CN103880951B (en) * | 2014-03-31 | 2016-03-30 | 南昌市万华生化制品有限公司 | A kind of method being prepared sterling ulinastatin by ulinastatin affinity chromatography medium |
| CN104140473A (en) * | 2014-06-10 | 2014-11-12 | 盐城市印染有限公司 | Chitosan s-triazine sulfonate derivative as well as preparation method and application of silicon hybridization solution thereof |
| CN105219755A (en) * | 2015-10-09 | 2016-01-06 | 长沙仁泽生物技术有限公司 | A kind of extraction of chymotrypsinogen and purification process |
| CN111778231A (en) * | 2020-07-29 | 2020-10-16 | 珠海冀百康生物科技有限公司 | Purification method of lysyl endopeptidase |
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Effective date of registration: 20220119 Address after: 750021 No. 1, east of floor 3, north of No. 16 plant, strategic emerging material processing zone, Yinchuan Development Zone, Yinchuan City, Ningxia Hui Autonomous Region Patentee after: NINGXIA MIAOLANG BIOTECHNOLOGY CO.,LTD. Address before: 116006 No. 1, floor 10, No. 9, Renmin Road, Zhongshan District, Dalian City, Liaoning Province Patentee before: Dalian Xinxiang Technology Co.,Ltd. |