CN113913415A - Method for separating human urinary kallidinogenase and thrombin regulatory protein - Google Patents
Method for separating human urinary kallidinogenase and thrombin regulatory protein Download PDFInfo
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- 229960004072 thrombin Drugs 0.000 title claims abstract description 27
- 238000000034 method Methods 0.000 title claims abstract description 25
- 229960003709 kallidinogenase Drugs 0.000 title claims abstract description 20
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- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 claims abstract description 5
- 229920000642 polymer Polymers 0.000 claims abstract description 5
- HTIRHQRTDBPHNZ-UHFFFAOYSA-N Dibutyl sulfide Chemical compound CCCCSCCCC HTIRHQRTDBPHNZ-UHFFFAOYSA-N 0.000 claims abstract description 4
- 239000000243 solution Substances 0.000 claims description 87
- 238000000108 ultra-filtration Methods 0.000 claims description 54
- 239000003480 eluent Substances 0.000 claims description 51
- 238000005406 washing Methods 0.000 claims description 35
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- 238000011068 loading method Methods 0.000 claims description 27
- 210000002700 urine Anatomy 0.000 claims description 22
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- 102000002397 Kinins Human genes 0.000 description 1
- 108010093008 Kinins Proteins 0.000 description 1
- 102000012479 Serine Proteases Human genes 0.000 description 1
- 108010022999 Serine Proteases Proteins 0.000 description 1
- 102000003990 Urokinase-type plasminogen activator Human genes 0.000 description 1
- 108090000435 Urokinase-type plasminogen activator Proteins 0.000 description 1
- 230000001154 acute effect Effects 0.000 description 1
- 125000000539 amino acid group Chemical group 0.000 description 1
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- 206010008118 cerebral infarction Diseases 0.000 description 1
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
- C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
- C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
- C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
- C12N9/6424—Serine endopeptidases (3.4.21)
- C12N9/6456—Plasminogen activators
- C12N9/6462—Plasminogen activators u-Plasminogen activator (3.4.21.73), i.e. urokinase
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
- C12Y304/21—Serine endopeptidases (3.4.21)
- C12Y304/21073—Serine endopeptidases (3.4.21) u-Plasminogen activator (3.4.21.73), i.e. urokinase
Abstract
The invention discloses a method for separating human urinary kallidinogenase and thrombin regulatory protein in the field of biotechnology. The method comprises the steps of separating a human urinary kallidinogenase crude product and a thrombin regulating protein crude product for the first time by adopting a hydrophobic chromatography one step, wherein a hydrophobic ligand of a hydrophobic chromatography column is one of phenyl, butyl sulfide and octyl, and a filler main body framework of the hydrophobic chromatography column is one of agarose, cellulose or a polymer. The method has the advantages of simple operation, low cost, easy industrial amplification, high yield of two separated products and obvious impurity removal effect.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a method for separating human urinary kallidinogenase and thrombin regulatory protein.
Background
Human urine contains hundreds of proteins, and at present, urokinase, a human urinary trypsin inhibitor, human urinary kininogenase and the like are mainly developed and extracted, and the components have important clinical treatment values.
Among them, human urokininogenase (KN), a glycoprotein consisting of 238 amino acids isolated and extracted from human urine, has an isoelectric point of about 4.0 and a molecular weight of about 54,000D, and belongs to serine proteases. It can activate the conversion of human plasma kininogen to kinins. Currently, it has been developed as a drug for treating acute cerebral infarction.
The soluble thrombin regulatory protein (TM) exists mainly in human plasma and human urine, is a glycoprotein consisting of 557 amino acid residues, has a molecular weight of about 60,300D and an isoelectric point of about 3.8, and was originally found in experiments by n.l. esmon, et al, 1981.
Chinese urine resources are very rich, and depending on the favorable condition, the urine protein industry has been developed from the end of the seventies of the last century and has been more than thirty years to date. On the basis of the research on the urine protein for years by companies, on the premise of a technology for collecting the urine protein in a large scale, the invention carries out proteomics research on the collected various urine proteins so as to continuously develop various urine protein products with medicinal value and supply the urine protein products to the market. The research of preliminary separation and extraction is carried out aiming at different physicochemical properties of human urinary kallidinogenase and soluble thrombin regulating protein, and the discovery that the separation of KN in a TM crude product as early as possible can not only avoid the interference of KN on the detection of TM, prevent a series of enzymatic reactions caused by KN from leading to the degradation of TM, but also can greatly improve the specific activity of two products, improve the purity of the crude product and be beneficial to the refining of the two products.
Through research, one-step chromatographic separation research aiming at KN and TM has not been carried out in the literature, and because the isoelectric points and molecular weights of the two proteins are very close, the two proteins cannot be completely separated by conventional methods such as ion exchange and gel filtration. In addition, according to different affinity characteristics of KN and TM for different substrates/reactants, affinity chromatography can be theoretically used for separating the KN and the TM, but the method is high in cost, complex in types of initial raw material protein components, and poor in using effect, and the affinity chromatography cannot achieve the highest resolution.
Disclosure of Invention
The invention aims to provide a simple, efficient and easily-amplified method for separating human urinary kallidinogenase and thrombin regulatory protein, KN and TM are separated by one step through a hydrophobic chromatography, the yield of each product after separation is high, and the impurity removal effect is obvious.
In order to achieve the above object, the method for separating human urinary kallidinogenase and thrombin regulatory protein of the present invention adopts the following technical scheme:
a method for isolating human urinary kallidinogenase and thrombin regulating protein comprising the steps of:
(1) preparing a balance liquid: the equilibrium solution is one of acetate buffer solution, phosphate buffer solution and sodium bicarbonate buffer solution, the concentration of the equilibrium solution is 0.02-0.2mol/L, and the equilibrium solution contains 1.2-1.5mol/L (NH)4)2SO4The pH value is 4.0-10.0;
(2) pretreating a crude product of urine protein: adding 5-10 times of pure water into the crude product of the urine protein, stirring and dissolving for 10-30min, centrifuging, collecting supernatant, adding ammonium sulfate to adjust the conductivity and pH, wherein the pH is 4.0-10.0, and the conductivity is 2-5mS/cm higher than that of the equilibrium solution, and preparing a sample solution;
(3) loading and washing with a balance solution: the method comprises the following steps that a hydrophobic chromatographic column is balanced by balance liquid in advance, a hydrophobic ligand of the hydrophobic chromatographic column is one of phenyl, butyl sulfide and octyl, and a filler main body framework of the hydrophobic chromatographic column is one of agarose, cellulose or polymer; then loading the sample loading solution prepared in the step (2) into a hydrophobic chromatographic column, flushing the column by using a balance solution with the volume of 3-5 times of the column volume after the sample loading is finished, and then flushing the column by using a flushing solution A with the volume of 3-5 times of the column volume, wherein the flushing solution A contains 0.9-1.2mol/L (NH)4)2SO4The pH value is 4.0-10.0;
(4) eluting human urinary kallidinogenase: washing the column with 3-5 column volumes of eluent B containing 0.4-0.9mol/L (NH)4)2SO4The pH value is 4.0-10.0;
(5) elution of Thrombin regulatory protein: washing the column with 3-5 column volumes of eluent C containing 0-0.4mol/L (NH)4)2SO4The pH value is 4.0-10.0;
(6) and (3) ultrafiltration: carrying out ultrafiltration on the eluent B obtained in the step (4) to obtain an ultrafiltration concentrated solution B; carrying out ultrafiltration on the eluent C obtained in the step (5) to obtain an ultrafiltration concentrated solution C;
(7) ammonium sulfate precipitation to obtain a crude product: carrying out saturated ammonium sulfate precipitation on the ultrafiltration concentrated solution B in the step (6), stirring and dissolving for 10-30min, standing for 3-12h, and then centrifuging or suction filtering to obtain a powdery crude product of human urinary kallidinogenase; and (4) carrying out saturated ammonium sulfate precipitation on the ultrafiltration concentrated solution C in the step (6), stirring and dissolving for 10-30min, standing for 3-12h, and then centrifuging or carrying out suction filtration to obtain a powdery crude thrombin regulating protein powder.
Preferably, the washing liquid A is one of acetate, phosphate and sodium bicarbonate buffer solution, and the buffer concentration of the washing liquid A is 0.02-0.2 mol/L; the eluent B is one of acetate, phosphate and sodium bicarbonate buffer solution, and the buffer concentration of the eluent B is 0.02-0.2 mol/L; the eluent C is one of acetate, phosphate and sodium bicarbonate buffer solution, and the buffer concentration of the eluent C is 0.02-0.2 mol/L.
Preferably, the washing solution A is 0.02mol/L phosphate buffer solution containing 1.2mol/L (NH)4)2SO4The pH was 7.0.
Preferably, the eluent B is 0.02mol/L phosphate buffer solution, and the eluent B contains 0.7mol/L (NH)4)2SO4The pH was 7.0.
Preferably, the eluent C is 0.02mol/L phosphate buffer solution, and the eluent C contains 0.3mol/L (NH)4)2SO4The pH was 7.0.
Preferably, the equilibrium solution is 0.02mol/L phosphate bufferThe equilibrium liquid contains 1.5mol/L (NH)4)2SO4The pH was 7.0.
Preferably, the filler of the hydrophobic chromatography column is one of phenyl-sepharose, butyl sulfide-sepharose and octyl-sepharose.
Preferably, in the step (6), an ultrafiltration membrane with the molecular weight of 5000-10000Da is adopted for ultrafiltration of the eluent B, and the material of the ultrafiltration membrane is polysulfone; in the step (6), an ultrafiltration membrane with the molecular weight of 10000-30000Da is adopted for ultrafiltration of the eluent C, and the material of the ultrafiltration membrane is polysulfone.
Compared with the prior art, the invention has the beneficial effects that:
1. the method adopts a hydrophobic chromatographic column one-step method to separate KN and TM, simplifies the operation steps, reduces the cost, ensures that the yield is higher than 85 percent, and is easy for industrial production; the invention aims to separate KN and TM, the KN and TM have different amino acid compositions, structures and physicochemical properties, and the exposure conditions of surface hydrophobic groups of the two proteins in the same high-salt environment are greatly different, so that the apparent hydrophobicity of the two proteins is different, and the two proteins can be distinguished by a hydrophobic chromatography method. The hydrophobic ligand of the hydrophobic chromatographic column is one of phenyl, butyl, butylthio and octyl, and the main filler framework of the hydrophobic chromatographic column is one of agarose, cellulose or polymer, so that the effect of separating KN and TM in one step is achieved, and the operation steps are simplified;
2. according to the TM crude product subjected to hydrophobic chromatography, most of impurities with weak hydrophobicity, including KN, are removed, the purity of the TM crude product is improved, and convenience is provided for later TM purification and detection.
Detailed Description
The present invention is further illustrated by the following detailed description, which is to be construed as merely illustrative and not limitative of the remainder of the disclosure, and modifications and variations such as those ordinarily skilled in the art are intended to be included within the scope of the present invention as defined in the appended claims.
A method for isolating human urinary kallidinogenase and thrombin regulating protein comprising the steps of:
(1) preparing a balance liquid: the equilibrium solution is one of acetate buffer solution, phosphate buffer solution and sodium bicarbonate buffer solution, the concentration of the equilibrium solution is 0.02-0.2mol/L, and the equilibrium solution contains 1.2-1.5mol/L (NH)4)2SO4The pH value is 4.0-10.0;
(2) pretreating a crude product of urine protein: adding 5-10 times of pure water into the crude product of the urine protein, stirring and dissolving for 10-30min, centrifuging, collecting supernatant, adding ammonium sulfate to adjust the conductivity and pH, wherein the pH is 4.0-10.0, and the conductivity is 2-5mS/cm higher than that of the equilibrium solution, and preparing a sample solution;
(3) loading and washing with a balance solution: the method comprises the following steps that a hydrophobic chromatographic column is balanced by balance liquid in advance, a hydrophobic ligand of the hydrophobic chromatographic column is one of phenyl, butyl sulfide and octyl, and a filler main body framework of the hydrophobic chromatographic column is one of agarose, cellulose or polymer; then loading the sample loading solution prepared in the step (2) into a hydrophobic chromatographic column, flushing the column by using a balance solution with the volume of 3-5 times of the column volume after the sample loading is finished, and then flushing the column by using a flushing solution A with the volume of 3-5 times of the column volume, wherein the flushing solution A contains 0.9-1.2mol/L (NH)4)2SO4The pH value is 4.0-10.0;
(4) eluting human urinary kallidinogenase: washing the column with 3-5 column volumes of eluent B containing 0.4-0.9mol/L (NH)4)2SO4The pH value is 4.0-10.0;
(5) elution of Thrombin regulatory protein: washing the column with 3-5 column volumes of eluent C containing 0-0.4mol/L (NH)4)2SO4The pH value is 4.0-10.0;
(6) and (3) ultrafiltration: carrying out ultrafiltration on the eluent B obtained in the step (4) to obtain an ultrafiltration concentrated solution B; carrying out ultrafiltration on the eluent C obtained in the step (5) to obtain an ultrafiltration concentrated solution C;
(7) ammonium sulfate precipitation to obtain a crude product: carrying out saturated ammonium sulfate precipitation on the ultrafiltration concentrated solution B in the step (6), stirring and dissolving for 10-30min, standing for 3-12h, and then centrifuging or suction filtering to obtain a powdery crude product of human urinary kallidinogenase; and (4) carrying out saturated ammonium sulfate precipitation on the ultrafiltration concentrated solution C in the step (6), stirring and dissolving for 10-30min, standing for 3-12h, and then centrifuging or carrying out suction filtration to obtain a powdery crude thrombin regulating protein powder.
Example 1
(1) Preparing a balance liquid: the equilibrium solution is 0.02mol/L acetate buffer solution containing 1.5mol/L (NH)4)2SO4pH is 4.0, and conductivity is 180 ms/cm;
(2) pretreating a crude product of urine protein: taking 5.00g of crude urine protein (wherein the KN content is 7.0PNA// g, the TM content is 0.4ug/g), adding 8 times of pure water, stirring and dissolving for 10min, centrifuging at 4000rpm for 10min, keeping the supernatant, adding ammonium sulfate to adjust the conductance and the pH, wherein the pH is 4.0, and the conductance is adjusted to 183mS/cm to obtain a sample loading solution;
(3) loading and washing with a balance solution: pre-loading 50mL of phenyl-sepharose hydrophobic chromatography column, and balancing 5 column volumes with a balance solution of 0.02mol/L acetate buffer solution containing 1.5mol/L (NH)4)2SO4And (3) after the pH value is 4.0 and the conductivity is 180ms/cm, loading the sample loading solution prepared in the step (2) into a chromatographic column, washing the column by using a balance solution for 4 times of the column volume, and then washing the column by using a washing solution A with 5 times of the column volume, wherein the washing solution A is 0.02mol/L acetate buffer solution and contains 1.2mol/L (NH)4)2SO4pH is 4.0, and conductivity is 139 ms/cm;
(4) eluting human urinary kallidinogenase: washing the column with 5 column volumes of eluent B, which is 0.02mol/L acetate buffer solution containing 0.9mol/L (NH)4)2SO4Collecting eluent B, wherein the pH value is 4.0, the conductivity is 119 ms/cm;
(5) elution of Thrombin regulatory protein: washing the column with 5 column volumes of eluent C, 0.02mol/L acetate buffer solution containing 0.4mol/L (NH)4)2SO4Collecting eluent C, wherein the pH value is 4.0, the conductivity is 66 ms/cm;
(6) and (3) ultrafiltration: ultrafiltering and concentrating the eluent B obtained in the step (4) to about 50mL by using a 5000Da polysulfone ultrafiltration membrane to obtain an ultrafiltration concentrated solution B; ultrafiltering and concentrating the eluent C obtained in the step (5) to about 50mL by using a 10000Da polysulfone ultrafiltration membrane to obtain an ultrafiltration concentrated solution C;
(7) ammonium sulfate precipitation to obtain a crude product: adding 32.3g of ammonium sulfate into the ultrafiltration concentrated solution B in the step (6), adjusting the pH to 6.0, stirring for 30min, standing for 3h, and centrifuging at 4000rpm for 10min to obtain a KN crude product; and (4) adding 32.5g of ammonium sulfate into the ultrafiltration concentrated solution C in the step (6), adjusting the pH to 6.0, stirring for 30min, standing for 3h, and centrifuging at 4000rpm for 10min to obtain a TM crude product.
Example 2
(1) Preparing a balance liquid: the equilibrium solution is 0.02mol/L phosphate buffer solution containing 1.5mol/L (NH)4)2SO4pH of 7.0 and conductivity of 182 ms/cm;
(2) pretreating a crude product of urine protein: taking 5.00g of crude urine protein (wherein the KN content is 5.0PNA// g, the TM content is 0.3ug/g), adding 10 times of pure water, stirring and dissolving for 10min, centrifuging at 4000rpm for 10min, keeping supernatant, adding ammonium sulfate to adjust the conductance and pH, wherein the pH is 7.0, and the conductance is adjusted to 184mS/cm to obtain a sample loading solution;
(3) loading and washing with a balance solution: pre-loading 50mL of butyl-sepharose hydrophobic chromatography column, and balancing 5 times column volume with 0.02mol/L phosphate buffer solution containing 1.5mol/L (NH)4)2SO4And (3) after the pH is 7.0 and the conductivity is 182ms/cm, loading the loading solution prepared in the step (2) into a chromatographic column, washing the column by using a balance solution for 4 times of the column volume, and then washing the column by using a washing solution A with 5 times of the column volume, wherein the washing solution A is 0.02mol/L phosphate buffer solution and contains 1.2mol/L (NH)4)2SO4The pH value is 7.0, and the conductivity is 141 ms/cm;
(4) eluting human urinary kallidinogenase: washing the column with 5 column volumes of eluent B, which is 0.02mol/L phosphate buffer solution containing 0.7mol/L (NH)4)2SO4Collecting eluent B, wherein the pH value is 7.0, the conductivity is 102 ms/cm;
(5) elution of Thrombin regulatory protein: washing the column with 5 column volumes of eluent C, 0.02mol/L phosphate buffer solution containing 0.3mol/L (NH)4)2SO4Collecting eluent C, wherein the pH value is 7.0, the conductivity is 51 ms/cm;
(6) and (3) ultrafiltration: ultrafiltering and concentrating the eluent B obtained in the step (4) to about 50mL by using a 10000Da polysulfone ultrafiltration membrane to obtain an ultrafiltration concentrated solution B; ultrafiltering and concentrating the eluent C obtained in the step (5) to about 50mL by using a 10000Da polysulfone ultrafiltration membrane to obtain an ultrafiltration concentrated solution C;
(7) ammonium sulfate precipitation to obtain a crude product: adding 32.5g of ammonium sulfate into the ultrafiltration concentrated solution B in the step (6), adjusting the pH to 7.0, stirring for 30min, standing for 3h, and centrifuging at 4000rpm for 10min to obtain a KN crude product; and (4) adding 32.4g of ammonium sulfate into the ultrafiltration concentrated solution C in the step (6), adjusting the pH to 7.0, stirring for 30min, standing for 3h, and centrifuging at 4000rpm for 10min to obtain a TM crude product.
Example 3
(1) Preparing a balance liquid: the equilibrium solution is 0.2mol/L sodium bicarbonate buffer solution containing 1.3mol/L (NH)4)2SO4The pH value is 10.0, and the conductivity is 153 ms/cm;
(2) pretreating a crude product of urine protein: adding 5 times of pure water into 100.00g of crude urine protein (wherein the KN content is 5.0PNA// g, the TM content is 3.0ug/g), stirring and dissolving for 30min, centrifuging at 4000rpm for 10min, keeping the supernatant, adding ammonium sulfate to adjust the conductance and the pH, wherein the pH is 10.0, and the conductance is adjusted to 158mS/cm to obtain a sample loading solution;
(3) loading and washing with a balance solution: 1000mL of phenyl-sepharose hydrophobic chromatography column was packed in advance, and the column volume was 5 times of the column volume, which was equilibrated with an equilibration solution containing 0.2mol/L sodium bicarbonate buffer solution and 1.3mol/L (NH)4)2SO4And (3) loading the sample loading solution prepared in the step (2) into a chromatographic column, washing the column by using a balance solution with the volume 4 times that of the column after the sample loading is finished, and then washing the column by using a washing solution A with the volume 5 times that of the column, wherein the washing solution A is 0.2mol/L sodium bicarbonate buffer solution and contains 1.1mol/L (NH)4)2SO4pH is 10.0, and conductivity is 135 ms/cm;
(4) eluting human urinary kallidinogenase: washing the column with 5 column volumes of eluent B, which is 0.2mol/L sodium bicarbonate buffer containing 0.4mol/L (NH)4)2SO4Collecting eluent B, wherein the pH value is 10.0, the conductivity is 71 ms/cm;
(5) elution of Thrombin regulatory protein: washing the column with 5 column volumes of eluent C, eluent C is0.2mol/L sodium bicarbonate buffer containing 0.05mol/L (NH)4)2SO4Collecting eluent C, wherein the pH value is 10.0, the conductivity is 12 ms/cm;
(6) and (3) ultrafiltration: ultrafiltering and concentrating the eluent B obtained in the step (4) to about 100mL by using a 8000Da polysulfone ultrafiltration membrane to obtain an ultrafiltration concentrated solution B; ultrafiltering and concentrating the eluent C obtained in the step (5) to about 100mL by using a 30000Da polysulfone ultrafiltration membrane to obtain an ultrafiltration concentrated solution C;
(7) ammonium sulfate precipitation to obtain a crude product: adding 65.0g of ammonium sulfate into the ultrafiltration concentrated solution B in the step (6), adjusting the pH to 7.0, stirring for 30min, standing for 3h, and centrifuging at 4000rpm for 10min to obtain a KN crude product; and (4) adding 65.1g of ammonium sulfate into the ultrafiltration concentrated solution C in the step (6), adjusting the pH to 7.0, stirring for 30min, standing for 12h, and centrifuging at 4000rpm for 10min to obtain a TM crude product.
Firstly, content detection
After sampling the KN crude product and the TM crude product prepared in examples 1 to 3, performing detection, wherein detection data are shown in the following table, table 1 is a detection data table of the KN crude product and the TM crude product in example 1, table 2 is a detection data table of the KN crude product and the TM crude product in example 2, and table 3 is a detection data table of the KN crude product and the TM crude product in example 3;
TABLE 1 data sheet for the detection of crude KN and crude TM in example 1
As can be seen from the above table 1, the yield of the KN crude product reaches 85.12%, the yield of the TM crude product reaches 89.76%, the two separated products have high yield, the separation is successful, and the industrial production is easy.
TABLE 2 KN and TM crude detection data tables of example 2
As can be seen from the above table 2, the yield of the KN crude product reaches 87.38%, the yield of the TM crude product reaches 90.25%, the two separated products have high yield, the separation is successful, and the industrial production is easy.
TABLE 3 KN and TM crude detection data tables of example 3
As can be seen from the above table 2, the yield of the KN crude product reaches 90.25%, the yield of the TM crude product reaches 87.22%, the two separated products have high yield, the separation is successful, and the industrial production is easy.
Claims (8)
1. A method for isolating human urinary kallidinogenase and thrombin regulating protein comprising the steps of:
(1) preparing a balance liquid: the equilibrium solution is one of acetate buffer solution, phosphate buffer solution and sodium bicarbonate buffer solution, the concentration of the equilibrium solution is 0.02-0.2mol/L, and the equilibrium solution contains 1.2-1.5mol/L (NH)4)2SO4The pH value is 4.0-10.0;
(2) pretreating a crude product of urine protein: adding 5-10 times of pure water into the crude product of the urine protein, stirring and dissolving for 10-30min, centrifuging, collecting supernatant, adding ammonium sulfate to adjust the conductivity and pH, wherein the pH is 4.0-10.0, and the conductivity is 2-5mS/cm higher than that of the equilibrium solution, and preparing a sample solution;
(3) loading and washing with a balance solution: the method comprises the following steps that a hydrophobic chromatographic column is balanced by balance liquid in advance, a hydrophobic ligand of the hydrophobic chromatographic column is one of phenyl, butyl sulfide and octyl, and a filler main body framework of the hydrophobic chromatographic column is one of agarose, cellulose or polymer; then loading the sample loading solution prepared in the step (2) into a hydrophobic chromatographic column, flushing the column by using a balance solution with the volume of 3-5 times of the column volume after the sample loading is finished, and then flushing the column by using a flushing solution A with the volume of 3-5 times of the column volume, wherein the flushing solution A contains 0.9-1.2mol/L (NH)4)2SO4The pH value is 4.0-10.0;
(4) eluting human urinary kallidinogenase: washing the column with 3-5 column volumes of eluent B containing 0.4-0.9mol/L (NH)4)2SO4The pH value is 4.0-10.0;
(5) washing machineThrombin-free regulatory protein: washing the column with 3-5 column volumes of eluent C containing 0-0.4mol/L (NH)4)2SO4The pH value is 4.0-10.0;
(6) and (3) ultrafiltration: carrying out ultrafiltration on the eluent B obtained in the step (4) to obtain an ultrafiltration concentrated solution B; carrying out ultrafiltration on the eluent C obtained in the step (5) to obtain an ultrafiltration concentrated solution C;
(7) ammonium sulfate precipitation to obtain a crude product: carrying out saturated ammonium sulfate precipitation on the ultrafiltration concentrated solution B in the step (6), stirring and dissolving for 10-30min, standing for 3-12h, and then centrifuging or suction filtering to obtain a powdery crude product of human urinary kallidinogenase; and (4) carrying out saturated ammonium sulfate precipitation on the ultrafiltration concentrated solution C in the step (6), stirring and dissolving for 10-30min, standing for 3-12h, and then centrifuging or carrying out suction filtration to obtain a powdery crude thrombin regulating protein powder.
2. The method of isolating human urinary kininogenase and thrombin regulating protein according to claim 1, wherein: the flushing fluid A is one of acetate, phosphate and sodium bicarbonate buffer solution, and the buffer concentration of the flushing fluid A is 0.02-0.2 mol/L; the eluent B is one of acetate, phosphate and sodium bicarbonate buffer solution, and the buffer concentration of the eluent B is 0.02-0.2 mol/L; the eluent C is one of acetate, phosphate and sodium bicarbonate buffer solution, and the buffer concentration of the eluent C is 0.02-0.2 mol/L.
3. The method of isolating human urinary kininogenase and thrombin regulating protein according to claim 2, wherein: the washing solution A is 0.02mol/L phosphate buffer solution, and the buffer solution contains 1.2mol/L (NH)4)2SO4The pH was 7.0.
4. The method of isolating human urinary kininogenase and thrombin regulating protein according to claim 2, wherein: the eluent B is 0.02mol/L phosphate buffer solution, and the eluent B contains 0.7mol/L (NH)4)2SO4The pH was 7.0.
5. The method of isolating human urinary kininogenase and thrombin regulating protein according to claim 2, wherein: the eluent C is 0.02mol/L phosphate buffer solution, and the eluent C contains 0.3mol/L (NH)4)2SO4The pH was 7.0.
6. The method of isolating human urinary kininogenase and thrombin regulating protein according to claim 1, wherein: the equilibrium solution is 0.02mol/L phosphate buffer solution, and the equilibrium solution contains 1.5mol/L (NH)4)2SO4The pH was 7.0.
7. The method of isolating human urinary kininogenase and thrombin regulating protein according to claim 1, wherein: the filler of the hydrophobic chromatographic column is one of phenyl-sepharose, butyl sulfide-sepharose and octyl-sepharose.
8. The method of isolating human urinary kininogenase and thrombin regulating protein according to claim 1, wherein: performing ultrafiltration on the eluent B in the step (6) by adopting an ultrafiltration membrane with the molecular weight of 5000-10000Da, wherein the ultrafiltration membrane is made of polysulfone; in the step (6), an ultrafiltration membrane with the molecular weight of 10000-30000Da is adopted for ultrafiltration of the eluent C, and the material of the ultrafiltration membrane is polysulfone.
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| CN101134952A (en) * | 2007-07-02 | 2008-03-05 | 广东天普生化医药股份有限公司 | Human urine kininogenase and method for making same |
| CN105753975A (en) * | 2016-04-28 | 2016-07-13 | 广东天普生化医药股份有限公司 | Ulinastatin purification method based on hydrophobic interaction column |
| CN109354621A (en) * | 2018-12-11 | 2019-02-19 | 江苏艾迪药业有限公司 | A kind of purification process of natural thrombomodulin |
| CN112266415A (en) * | 2020-10-30 | 2021-01-26 | 江苏艾迪药业股份有限公司 | Method for large-scale production of thrombin regulatory protein |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101134952A (en) * | 2007-07-02 | 2008-03-05 | 广东天普生化医药股份有限公司 | Human urine kininogenase and method for making same |
| CN105753975A (en) * | 2016-04-28 | 2016-07-13 | 广东天普生化医药股份有限公司 | Ulinastatin purification method based on hydrophobic interaction column |
| CN109354621A (en) * | 2018-12-11 | 2019-02-19 | 江苏艾迪药业有限公司 | A kind of purification process of natural thrombomodulin |
| CN112266415A (en) * | 2020-10-30 | 2021-01-26 | 江苏艾迪药业股份有限公司 | Method for large-scale production of thrombin regulatory protein |
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