CN113913415B - Method for separating human urinary kallidinogenase and thrombin regulating protein - Google Patents
Method for separating human urinary kallidinogenase and thrombin regulating protein Download PDFInfo
- Publication number
- CN113913415B CN113913415B CN202111233287.9A CN202111233287A CN113913415B CN 113913415 B CN113913415 B CN 113913415B CN 202111233287 A CN202111233287 A CN 202111233287A CN 113913415 B CN113913415 B CN 113913415B
- Authority
- CN
- China
- Prior art keywords
- solution
- column
- thrombin
- eluent
- liquid
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 102000004169 proteins and genes Human genes 0.000 title claims abstract description 47
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 47
- 108060005987 Kallikrein Proteins 0.000 title claims abstract description 26
- 102000001399 Kallikrein Human genes 0.000 title claims abstract description 26
- 108090000190 Thrombin Proteins 0.000 title claims abstract description 26
- 229960003709 kallidinogenase Drugs 0.000 title claims abstract description 26
- 229960004072 thrombin Drugs 0.000 title claims abstract description 26
- 230000002485 urinary effect Effects 0.000 title claims abstract description 26
- 238000000034 method Methods 0.000 title claims abstract description 23
- 230000001105 regulatory effect Effects 0.000 title claims abstract description 23
- 230000002209 hydrophobic effect Effects 0.000 claims abstract description 31
- 239000012043 crude product Substances 0.000 claims abstract description 28
- 238000004587 chromatography analysis Methods 0.000 claims abstract description 14
- 239000000945 filler Substances 0.000 claims abstract description 7
- 125000000484 butyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 claims abstract description 6
- 229920000936 Agarose Polymers 0.000 claims abstract description 5
- -1 butylthio Chemical group 0.000 claims abstract description 5
- 229920002678 cellulose Polymers 0.000 claims abstract description 5
- 239000001913 cellulose Substances 0.000 claims abstract description 5
- 239000003446 ligand Substances 0.000 claims abstract description 5
- 125000002347 octyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 claims abstract description 5
- 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
- 239000000243 solution Substances 0.000 claims description 78
- 239000007788 liquid Substances 0.000 claims description 46
- 238000000108 ultra-filtration Methods 0.000 claims description 37
- 239000003480 eluent Substances 0.000 claims description 32
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical compound [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 claims description 28
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 claims description 24
- 238000011010 flushing procedure Methods 0.000 claims description 24
- 210000002700 urine Anatomy 0.000 claims description 22
- 238000005406 washing Methods 0.000 claims description 19
- 229910052921 ammonium sulfate Inorganic materials 0.000 claims description 18
- 235000011130 ammonium sulphate Nutrition 0.000 claims description 18
- 238000003756 stirring Methods 0.000 claims description 18
- 239000012528 membrane Substances 0.000 claims description 14
- 229910000030 sodium bicarbonate Inorganic materials 0.000 claims description 14
- 235000017557 sodium bicarbonate Nutrition 0.000 claims description 14
- 239000007853 buffer solution Substances 0.000 claims description 12
- 238000011068 loading method Methods 0.000 claims description 12
- 229920002492 poly(sulfone) Polymers 0.000 claims description 10
- 239000000872 buffer Substances 0.000 claims description 9
- 239000008055 phosphate buffer solution Substances 0.000 claims description 9
- 230000001376 precipitating effect Effects 0.000 claims description 9
- 239000008351 acetate buffer Substances 0.000 claims description 8
- 239000008363 phosphate buffer Substances 0.000 claims description 7
- QTBSBXVTEAMEQO-UHFFFAOYSA-M Acetate Chemical compound CC([O-])=O QTBSBXVTEAMEQO-UHFFFAOYSA-M 0.000 claims description 6
- 229910019142 PO4 Inorganic materials 0.000 claims description 6
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 claims description 6
- 239000010452 phosphate Substances 0.000 claims description 6
- 239000006228 supernatant Substances 0.000 claims description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 6
- 238000012870 ammonium sulfate precipitation Methods 0.000 claims description 3
- 238000001914 filtration Methods 0.000 claims description 3
- 238000000967 suction filtration Methods 0.000 claims description 3
- 239000011543 agarose gel Substances 0.000 claims 3
- 239000000047 product Substances 0.000 abstract description 16
- 230000000694 effects Effects 0.000 abstract description 5
- 239000012535 impurity Substances 0.000 abstract description 3
- 230000003321 amplification Effects 0.000 abstract 1
- 238000003199 nucleic acid amplification method Methods 0.000 abstract 1
- 235000018102 proteins Nutrition 0.000 description 34
- 239000000523 sample Substances 0.000 description 10
- 238000001514 detection method Methods 0.000 description 6
- 238000000926 separation method Methods 0.000 description 5
- 229920002684 Sepharose Polymers 0.000 description 4
- 238000011067 equilibration Methods 0.000 description 4
- 238000009776 industrial production Methods 0.000 description 4
- 238000011160 research Methods 0.000 description 4
- 238000012360 testing method Methods 0.000 description 4
- 239000012141 concentrate Substances 0.000 description 3
- 102000034356 gene-regulatory proteins Human genes 0.000 description 3
- 108091006104 gene-regulatory proteins Proteins 0.000 description 3
- 239000012488 sample solution Substances 0.000 description 3
- 239000012619 Butyl Sepharose® Substances 0.000 description 2
- 108090000288 Glycoproteins Proteins 0.000 description 2
- 102000003886 Glycoproteins Human genes 0.000 description 2
- 238000001042 affinity chromatography Methods 0.000 description 2
- 150000001413 amino acids Chemical class 0.000 description 2
- 210000002381 plasma Anatomy 0.000 description 2
- GUPXYSSGJWIURR-UHFFFAOYSA-N 3-octoxypropane-1,2-diol Chemical compound CCCCCCCCOCC(O)CO GUPXYSSGJWIURR-UHFFFAOYSA-N 0.000 description 1
- 108010077861 Kininogens Proteins 0.000 description 1
- 102000010631 Kininogens Human genes 0.000 description 1
- 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
- 230000003213 activating effect Effects 0.000 description 1
- 230000001154 acute effect Effects 0.000 description 1
- 125000000539 amino acid group Chemical group 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 206010008118 cerebral infarction Diseases 0.000 description 1
- 208000026106 cerebrovascular disease Diseases 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 238000013375 chromatographic separation Methods 0.000 description 1
- 238000005345 coagulation Methods 0.000 description 1
- 230000015271 coagulation Effects 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 208000035475 disorder Diseases 0.000 description 1
- 208000009190 disseminated intravascular coagulation Diseases 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000006911 enzymatic reaction Methods 0.000 description 1
- 239000006167 equilibration buffer Substances 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 230000002349 favourable effect Effects 0.000 description 1
- 238000002523 gelfiltration Methods 0.000 description 1
- 125000001165 hydrophobic group Chemical group 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 238000005342 ion exchange Methods 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 235000004252 protein component Nutrition 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 239000000376 reactant Substances 0.000 description 1
- 238000007670 refining Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 108010088854 urinastatin Proteins 0.000 description 1
- 229960005356 urokinase Drugs 0.000 description 1
Classifications
-
- 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
Landscapes
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Genetics & Genomics (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- General Health & Medical Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biochemistry (AREA)
- Medicinal Chemistry (AREA)
- General Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Toxicology (AREA)
- Gastroenterology & Hepatology (AREA)
- Biophysics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Enzymes And Modification Thereof (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention discloses a method for separating human urinary kallidinogenase and thrombin regulating protein in the field of biotechnology. The method adopts a hydrophobic chromatography to separate a crude product of human urinary kallidinogenase and a crude product of thrombin regulating protein for the first time, wherein the hydrophobic ligand of the hydrophobic chromatography column is one of phenyl, butyl, butylthio and octyl, and the filler main body framework of the hydrophobic chromatography column is one of agarose, cellulose or polymer. The method has the advantages of simple operation, low cost, easy industrialized amplification, high yield of the two separated products and obvious impurity removal effect.
Description
Technical Field
The invention relates to the field of biotechnology, in particular to a method for separating human urinary kallidinogenase and thrombin regulating protein.
Background
Human urine contains hundreds of proteins, and the main components which are developed and extracted at present are urokinase, a human urinary trypsin inhibitor, human urokininogenase and the like, and the components have important clinical treatment values.
Wherein human urokininogenase (Urinary Kallidinogenase, hereinafter abbreviated as KN) is a glycoprotein composed of 238 amino acids and isolated from human urine, has an isoelectric point of about 4.0 and a molecular weight of about 54,000D, and is a serine protease. It is capable of activating the conversion of human plasma kininogen to kinin. Currently, drugs for treating acute cerebral infarction have been developed.
The soluble thrombin regulatory protein (TM) is mainly present in human blood plasma and human urine, is a glycoprotein composed 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 in which N.L. Esmon is equal to 1981, and is clinically used mainly for the treatment of Disseminated Intravascular Coagulation (DIC) caused by coagulation system disorder.
The urine resource of Chinese is very abundant, and depending on the favorable condition, the urine protein industry has been developed from the last seventies of the last century for more than thirty years. On the basis of urine protein research for many years, the invention takes the large-scale urine protein collection technology as the premise, carries out proteomics research on a plurality of collected urine proteins, further continues to develop urine protein products with a plurality of medicinal values, and supplies the urine protein products to the market. Preliminary separation and extraction researches are carried out on different physicochemical properties of human urinary kallidinogenase and soluble thrombin regulatory protein, and the fact that KN in a crude product of the TM is separated as soon as possible is found, so that interference of KN on detection of the TM can be avoided, degradation of the TM caused by a series of enzyme reactions brought by the KN is prevented, meanwhile, the specific activity of two products can be greatly improved, the purity of the crude product is improved, and refining of the two products is facilitated.
Through investigation, one-step chromatographic separation research aiming at KN and TM has not been carried out in the literature, and the isoelectric points and molecular weights of two proteins are very close, so that the two proteins cannot be completely separated by using conventional ion exchange, gel filtration and other methods. In addition, according to the different affinity characteristics of KN and TM for different substrates/reactants, the KN and TM can be separated by adopting an affinity chromatography method theoretically, but the method has high cost, the types of initial raw material protein components are complex, the affinity chromatography can not reach the highest resolution, and the use effect is poor.
Disclosure of Invention
The invention aims to provide a simple, efficient and easily-amplified method for separating human urinary kallidinogenase and thrombin regulatory protein, which adopts a hydrophobic chromatography to separate KN and TM in one step, and each product has high yield and obvious impurity removal effect after separation.
In order to achieve the above object, the method for separating human urinary kallidinogenase and thrombin regulating 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 balancing solution: the balancing solution is one of acetate buffer solution, phosphate buffer solution and sodium bicarbonate buffer solution, the concentration of the balancing solution is 0.02-0.2mol/L, and the balancing solution contains 1.2-1.5mol/L (NH) 4 ) 2 SO 4 The pH is 4.0-10.0;
(2) Pretreatment of crude urine protein: adding pure water 5-10 times of the crude urine protein, stirring and dissolving for 10-30min, centrifuging, collecting supernatant, adding ammonium sulfate to adjust conductivity and pH to 4.0-10.0, and making conductivity higher than balance liquid by 2-5mS/cm to obtain sample liquid;
(3) Loading and flushing with a balancing solution: the hydrophobic chromatographic column is balanced by a balancing liquid in advance, wherein the hydrophobic ligand of the hydrophobic chromatographic column is one of phenyl, butyl, butylthio and octyl, and the filler main framework of the hydrophobic chromatographic column is one of agarose, cellulose or polymer; then loading the sample liquid prepared in the step (2) on a hydrophobic chromatography column, after loading, flushing the column with a balancing liquid for 3-5 times of column volume, and flushing the column with a flushing liquid A of 3-5 times of column volume, wherein the flushing liquid A contains 0.9-1.2mol/L (NH) 4 ) 2 SO 4 The pH 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 ) 2 SO 4 The pH is 4.0-10.0;
(5) Eluting thrombin modulating protein: washing the column with 3-5 column volumes of eluent C containing 0-0.4mol/L (NH) 4 ) 2 SO 4 The pH is 4.0-10.0;
(6) Ultrafiltration: ultrafiltering the eluted solution B obtained in the step (4) to obtain ultrafiltered concentrated solution B; ultrafiltering the eluted solution C obtained in the step (5) to obtain ultrafiltered concentrated solution C;
(7) Precipitating ammonium sulfate to obtain a crude product: precipitating the ultrafiltration concentrated solution B in the step (6) by saturated ammonium sulfate, stirring and dissolving for 10-30min, standing for 3-12h, and centrifuging or suction filtering to obtain powdery crude product of human urinary kallidinogenase; and (3) 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 thrombin regulating protein powdery crude product.
Preferably, the flushing liquid A is one of acetate, phosphate and sodium bicarbonate buffer solution, and the buffer concentration of the flushing liquid A is 0.02-0.2mol/L; the eluent B is one of acetate, phosphate and sodium bicarbonate buffer solution, and the buffer concentration of the eluent B is 0.02-0.2mol/L; the eluent C is one of acetate, phosphate and sodium bicarbonate buffer solution, and the buffer concentration of the eluent C is 0.02-0.2mol/L.
Preferably, the flushing liquid A is a phosphate buffer solution of 0.02mol/L, and the buffer solution contains 1.2mol/L (NH 4 ) 2 SO 4 The pH was 7.0.
Preferably, the eluent B is 0.02mol/L phosphate buffer, and the eluent B contains 0.7mol/L (NH 4 ) 2 SO 4 The pH was 7.0.
Preferably, the eluent C is 0.02mol/L phosphate buffer, and the eluent C contains 0.3mol/L (NH 4 ) 2 SO 4 The pH was 7.0.
Preferably, the equilibration liquid is 0.02mol/L phosphate buffer solution, the equilibration liquid contains 1.5mol/L (NH) 4 ) 2 SO 4 The pH was 7.0.
Preferably, the filler of the hydrophobic chromatography column is one of phenyl-sepharose, butyl-sepharose, butylthio-sepharose and octyl-sepharose.
Preferably, ultrafiltration is carried out on the eluted solution 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; and (3) ultrafiltering the eluted solution C in the step (6) by adopting an ultrafiltration membrane with the molecular weight of 10000-30000Da, wherein the ultrafiltration membrane is made of polysulfone.
Compared with the prior art, the invention has the beneficial effects that:
1. the method adopts a hydrophobic chromatography column one-step method to separate KN and TM, simplifies operation steps, reduces cost, ensures that the yield is higher than 85%, and is easy for industrial production; the invention aims to separate KN and TM, and the structure and physical and chemical properties are completely different due to different compositions of KN and TM amino acids, and the exposure condition of hydrophobic groups on the surfaces of two proteins in the same high-salt environment is 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 adopted hydrophobic chromatographic column is one of phenyl, butyl, butylthio and octyl, and the filler main body 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 method, the crude TM product subjected to hydrophobic chromatography is subjected to removal of most impurities with weaker hydrophobicity containing KN, so that the purity of the crude TM product is improved, and convenience is provided for purification and detection of the final TM product.
Detailed Description
The present invention is further illustrated below in conjunction with the specific embodiments, it being understood that these embodiments are meant to be illustrative of the invention only and not limiting the scope of the invention, and that modifications of the invention, which are equivalent to those skilled in the art to which the invention pertains, will fall within the scope of the invention as defined in the claims appended hereto.
A method for isolating human urinary kallidinogenase and thrombin regulating protein, comprising the steps of:
(1) Preparing a balancing solution: the balancing solution is one of acetate buffer solution, phosphate buffer solution and sodium bicarbonate buffer solution, the concentration of the balancing solution is 0.02-0.2mol/L, and the balancing solution contains 1.2-1.5mol/L (NH) 4 ) 2 SO 4 The pH is 4.0-10.0;
(2) Pretreatment of crude urine protein: adding pure water 5-10 times of the crude urine protein, stirring and dissolving for 10-30min, centrifuging, collecting supernatant, adding ammonium sulfate to adjust conductivity and pH to 4.0-10.0, and making conductivity higher than balance liquid by 2-5mS/cm to obtain sample liquid;
(3) Loading and flushing with a balancing solution: the hydrophobic chromatographic column is balanced by a balancing liquid in advance, the hydrophobic ligand of the hydrophobic chromatographic column is one of phenyl, butyl, butylthio and octyl, and the filler main body framework of the hydrophobic chromatographic columnIs one of agarose, cellulose or polymer; then loading the sample liquid prepared in the step (2) on a hydrophobic chromatography column, after loading, flushing the column with a balancing liquid for 3-5 times of column volume, and flushing the column with a flushing liquid A of 3-5 times of column volume, wherein the flushing liquid A contains 0.9-1.2mol/L (NH) 4 ) 2 SO 4 The pH 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 ) 2 SO 4 The pH is 4.0-10.0;
(5) Eluting thrombin modulating protein: washing the column with 3-5 column volumes of eluent C containing 0-0.4mol/L (NH) 4 ) 2 SO 4 The pH is 4.0-10.0;
(6) Ultrafiltration: ultrafiltering the eluted solution B obtained in the step (4) to obtain ultrafiltered concentrated solution B; ultrafiltering the eluted solution C obtained in the step (5) to obtain ultrafiltered concentrated solution C;
(7) Precipitating ammonium sulfate to obtain a crude product: precipitating the ultrafiltration concentrated solution B in the step (6) by saturated ammonium sulfate, stirring and dissolving for 10-30min, standing for 3-12h, and centrifuging or suction filtering to obtain powdery crude product of human urinary kallidinogenase; and (3) 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 thrombin regulating protein powdery crude product.
Example 1
(1) Preparing a balancing solution: the equilibrium solution is 0.02mol/L acetate buffer solution containing 1.5mol/L (NH) 4 ) 2 SO 4 pH 4.0, conductivity 180ms/cm;
(2) Pretreatment of crude urine protein: taking 5.00g of crude urine protein (wherein the KN content is 7.0PNA// g, and the TM content is 0.4 ug/g), adding 8 times of pure water, stirring and dissolving for 10min, centrifuging at 4000rpm for 10min, leaving supernatant, adding ammonium sulfate to adjust the conductivity and pH to 4.0, and adjusting the conductivity to 183mS/cm to obtain a sample solution;
(3) Loading and flushing with a balancing solution: 50mL of phenyl-sepharose hydrophobic chromatography column is pre-packed, and 5 times of column volume is balanced by using a balancing solutionThe equilibration buffer is 0.02mol/L acetate buffer containing 1.5mol/L (NH) 4 ) 2 SO 4 The pH value is 4.0, the conductivity is 180ms/cm, the sample liquid prepared in the step (2) is applied to the chromatographic column, after the sample application, the column volume is washed by the balance liquid for 4 times, the column is washed by the washing liquid A with 5 times of the column volume, the washing liquid A is 0.02mol/L acetate buffer solution, and the concentration of the washing liquid A is 1.2mol/L (NH) 4 ) 2 SO 4 pH 4.0, conductivity 139ms/cm;
(4) Eluting human urinary kallidinogenase: washing the column with 5 column volumes of eluent B to give an eluted solution B, the eluent B being 0.02mol/L acetate buffer containing 0.9mol/L (NH) 4 ) 2 SO 4 The pH value is 4.0, the conductivity is 119ms/cm, and the eluted solution B is collected;
(5) Eluting thrombin modulating protein: washing the column with 5 column volumes of eluent C to give an eluted solution C, the eluent C being 0.02mol/L acetate buffer containing 0.4mol/L (NH) 4 ) 2 SO 4 The pH value is 4.0, the conductivity is 66ms/cm, and the eluted solution C is collected;
(6) Ultrafiltration: ultrafiltering and concentrating the eluted solution B obtained in the step (4) to about 50mL by using a 5000Da polysulfone ultrafiltration membrane to obtain ultrafiltration concentrate B; ultrafiltering and concentrating the eluted solution C obtained in the step (5) to about 50mL by using a 10000Da polysulfone ultrafiltration membrane to obtain ultrafiltered concentrated solution C;
(7) Precipitating ammonium sulfate to obtain a crude product: adding 32.3g of ammonium sulfate into the ultrafiltration concentrated solution B in the step (6), regulating the pH to 6.0, stirring for 30min, standing for 3h, and centrifuging at 4000rpm for 10min to obtain a KN crude product; adding 32.5g of ammonium sulfate into the ultrafiltration concentrated solution C in the step (6), regulating the pH to 6.0, stirring for 30min, standing for 3h, and centrifuging at 4000rpm for 10min to obtain a crude product of the TM.
Example 2
(1) Preparing a balancing solution: the balance liquid is 0.02mol/L phosphate buffer solution containing 1.5mol/L (NH) 4 ) 2 SO 4 pH 7.0, conductivity 182ms/cm;
(2) Pretreatment of crude urine protein: taking 5.00g of crude urine protein (wherein the KN content is 5.0PNA// g, and the TM content is 0.3 ug/g), adding 10 times of pure water, stirring and dissolving for 10min, centrifuging at 4000rpm for 10min, leaving supernatant, adding ammonium sulfate to adjust the conductivity and pH to 7.0, and adjusting the conductivity to 184mS/cm to obtain a sample solution;
(3) Loading and flushing with a balancing solution: 50mL of a pre-packed butyl-sepharose hydrophobic chromatography column was equilibrated with a equilibration solution at 5 column volumes, 0.02mol/L phosphate buffer containing 1.5mol/L (NH) 4 ) 2 SO 4 The pH value is 7.0, the conductivity is 182ms/cm, the sample liquid prepared in the step (2) is applied to the chromatographic column, after the sample application, the column volume is washed by the balance liquid for 4 times, the column is washed by the washing liquid A with 5 times of the column volume, the washing liquid A is phosphate buffer solution with 0.02mol/L, and the pH value is 1.2mol/L (NH) 4 ) 2 SO 4 pH 7.0, conductivity 141ms/cm;
(4) Eluting human urinary kallidinogenase: washing the column with 5 column volumes of eluent B to give an eluted solution B of 0.02mol/L phosphate buffer containing 0.7mol/L (NH) 4 ) 2 SO 4 The pH value is 7.0, the conductivity is 102ms/cm, and the eluted solution B is collected;
(5) Eluting thrombin modulating protein: washing the column with 5 column volumes of eluent C to give an eluted solution C of 0.02mol/L phosphate buffer containing 0.3mol/L (NH) 4 ) 2 SO 4 The pH value is 7.0, the conductivity is 51ms/cm, and the eluted solution C is collected;
(6) Ultrafiltration: ultrafiltering and concentrating the eluted solution B obtained in the step (4) to about 50mL by using a 10000Da polysulfone ultrafiltration membrane to obtain ultrafiltration concentrate B; ultrafiltering and concentrating the eluted solution C obtained in the step (5) to about 50mL by using a 10000Da polysulfone ultrafiltration membrane to obtain ultrafiltered concentrated solution C;
(7) Precipitating ammonium sulfate to obtain a crude product: adding 32.5g of ammonium sulfate into the ultrafiltration concentrated solution B in the step (6), regulating the pH to 7.0, stirring for 30min, standing for 3h, and centrifuging at 4000rpm for 10min to obtain a KN crude product; adding 32.4g of ammonium sulfate into the ultrafiltration concentrated solution C in the step (6), regulating the pH to 7.0, stirring for 30min, standing for 3h, and centrifuging at 4000rpm for 10min to obtain a crude product of the TM.
Example 3
(1) Preparing a balancing solution: the balance liquid is 0.2mol/L sodium bicarbonate bufferLiquid containing 1.3mol/L (NH) 4 ) 2 SO 4 pH 10.0, conductivity 153ms/cm;
(2) Pretreatment of crude urine protein: taking 100.00g of crude urine protein (wherein KN content is 5.0PNA// g, TM content is 3.0 ug/g), adding 5 times of pure water, stirring and dissolving for 30min, centrifuging at 4000rpm for 10min, leaving supernatant, adding ammonium sulfate to adjust conductivity and pH to 10.0, and adjusting conductivity to 158mS/cm to obtain a sample solution;
(3) Loading and flushing with a balancing solution: 1000mL of a phenyl-sepharose hydrophobic chromatography column was packed in advance, and the column volume was 5 times equilibrated with a equilibration solution containing 0.2mol/L sodium bicarbonate buffer containing 1.3mol/L (NH) 4 ) 2 SO 4 The pH value is 10.0, the conductivity is 153ms/cm, the sample liquid obtained in the step (2) is applied to the chromatographic column, after the sample application, the column volume is washed by the balance liquid for 4 times, the column is washed by the washing liquid A with 5 times of the column volume, the washing liquid A is 0.2mol/L sodium bicarbonate buffer solution, and the pH value is 1.1mol/L (NH) 4 ) 2 SO 4 pH 10.0, conductivity 135ms/cm;
(4) Eluting human urinary kallidinogenase: washing the column with 5 column volumes of eluent B to give an eluted solution B, wherein the eluent B is 0.2mol/L sodium bicarbonate buffer containing 0.4mol/L (NH) 4 ) 2 SO 4 The pH value is 10.0, the conductivity is 71ms/cm, and the eluted solution B is collected;
(5) Eluting thrombin modulating protein: washing the column with 5 column volumes of eluent C to give an eluted solution C, wherein the eluent C is 0.2mol/L sodium bicarbonate buffer, containing 0.05mol/L (NH) 4 ) 2 SO 4 The pH value is 10.0, the conductivity is 12ms/cm, and the eluted solution C is collected;
(6) Ultrafiltration: ultrafiltering and concentrating the eluted solution B obtained in the step (4) to about 100mL by using an 8000Da polysulfone ultrafiltration membrane to obtain ultrafiltration concentrate B; ultrafiltering and concentrating the eluted solution C obtained in the step (5) to about 100mL by using a 30000Da polysulfone ultrafiltration membrane to obtain ultrafiltration concentrated solution C;
(7) Precipitating ammonium sulfate to obtain a crude product: adding 65.0g of ammonium sulfate into the ultrafiltration concentrated solution B in the step (6), regulating the pH to 7.0, stirring for 30min, standing for 3h, and centrifuging at 4000rpm for 10min to obtain a KN crude product; and (3) adding 65.1g of ammonium sulfate into the ultrafiltration concentrated solution C in the step (6), regulating the pH to 7.0, stirring for 30min, standing for 12h, and centrifuging at 4000rpm for 10min to obtain a crude product of the TM.
1. Content detection
The crude KN and crude TM products prepared in examples 1-3 were sampled and tested, the test data are shown in the following table, table 1 is the crude KN and crude TM products test data table in example 1, table 2 is the crude KN and crude TM products test data table in example 2, and Table 3 is the crude KN and crude TM products test data table in example 3;
TABLE 1 KN crude and TM crude detection data sheet of 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%, and the yields of the two separated products are high, so that the separation is successful, and the industrial production is easy.
TABLE 2 KN crude and TM crude detection data sheet 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%, and the yields of the two separated products are high, so that the separation is successful, and the industrial production is easy.
TABLE 3 KN crude and TM crude detection data sheet 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%, and the yields of the two separated products are high, so that the separation is successful, and the industrial production is easy.
Claims (6)
1. A method for isolating human urinary kallidinogenase and thrombin regulating protein, comprising the steps of:
(1) Preparing a balancing solution: the balancing solution is one of acetate buffer solution, phosphate buffer solution and sodium bicarbonate buffer solution, the concentration of the balancing solution is 0.02-0.2mol/L, and the balancing solution contains 1.2-1.5mol/L (NH) 4 ) 2 SO 4 The pH is 4.0-10.0;
(2) Pretreatment of crude urine protein: adding pure water 5-10 times of the crude urine protein, stirring and dissolving for 10-30min, centrifuging, collecting supernatant, adding ammonium sulfate to adjust conductivity and pH to 4.0-10.0, and making conductivity higher than balance liquid by 2-5mS/cm to obtain sample liquid;
(3) Loading and flushing with a balancing solution: the hydrophobic chromatographic column is balanced by a balancing liquid in advance, wherein the hydrophobic ligand of the hydrophobic chromatographic column is one of phenyl, butyl, butylthio and octyl, and the filler main framework of the hydrophobic chromatographic column is one of agarose, cellulose or polymer; then loading the sample liquid prepared in the step (2) on a hydrophobic chromatography column, after loading, flushing the column with a balancing liquid for 3-5 times of column volume, and flushing the column with a flushing liquid A of 3-5 times of column volume, wherein the flushing liquid A contains 0.9-1.2mol/L (NH) 4 ) 2 SO 4 The pH value is 4.0-10.0, and the filler of the hydrophobic chromatographic column is one of phenyl-agarose gel, butyl sulfur-agarose gel and octyl-agarose gel; the flushing liquid A is one of acetate, phosphate and sodium bicarbonate buffer solution, and the buffer concentration of the flushing liquid A is 0.02-0.2mol/L;
(4) Eluting human urinary kallidinogenase: washing the column with 3-5 column volumes of eluent B containing 0.4-0.9mol/L (NH) 4 ) 2 SO 4 The pH value is 4.0-10.0, the eluent B is one of acetate, phosphate and sodium bicarbonate buffer solution, and the buffer concentration of the eluent B is 0.02-0.2mol/L;
(5) Eluting thrombin modulating protein: washing the column with 3-5 column volumes of eluent C containing 0-0.4mol/L (NH) 4 ) 2 SO 4 The pH is 4.0-10.0; the eluent C isOne of acetate, phosphate and sodium bicarbonate buffer solution, wherein the buffer concentration of the eluent C is 0.02-0.2mol/L;
(6) Ultrafiltration: ultrafiltering the eluted solution B obtained in the step (4) to obtain ultrafiltered concentrated solution B; ultrafiltering the eluted solution C obtained in the step (5) to obtain ultrafiltered concentrated solution C;
(7) Precipitating ammonium sulfate to obtain a crude product: precipitating the ultrafiltration concentrated solution B in the step (6) by saturated ammonium sulfate, stirring and dissolving for 10-30min, standing for 3-12h, and centrifuging or suction filtering to obtain powdery crude product of human urinary kallidinogenase; and (3) 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 thrombin regulating protein powdery crude product.
2. The method of isolating human urinary kallidinogenase and thrombin regulating protein according to claim 1, wherein: the flushing liquid A is 0.02mol/L phosphate buffer solution, and the buffer solution contains 1.2mol/L (NH) 4 ) 2 SO 4 The pH was 7.0.
3. The method of isolating human urinary kallidinogenase and thrombin regulating protein according to claim 1, wherein: the eluent B is 0.02mol/L phosphate buffer, and the eluent B contains 0.7mol/L (NH) 4 ) 2 SO 4 The pH was 7.0.
4. The method of isolating human urinary kallidinogenase and thrombin regulating protein according to claim 1, wherein: the eluent C was 0.02mol/L phosphate buffer, and the eluent C contained 0.3mol/L (NH) 4 ) 2 SO 4 The pH was 7.0.
5. The method of isolating human urinary kallidinogenase and thrombin regulating protein according to claim 1, wherein: the equilibrium liquid is 0.02mol/L phosphate buffer solution, and the equilibrium liquid contains 1.5mol/L (NH) 4 ) 2 SO 4 The pH was 7.0.
6. The method of isolating human urinary kallidinogenase and thrombin regulating protein according to claim 1, wherein: in the step (6), the eluted solution B is ultrafiltered by adopting an ultrafiltration membrane with the molecular weight of 5000-10000Da, and the ultrafiltration membrane is made of polysulfone; and (3) ultrafiltering the eluted solution C in the step (6) by adopting an ultrafiltration membrane with the molecular weight of 10000-30000Da, wherein the ultrafiltration membrane is made of polysulfone.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111233287.9A CN113913415B (en) | 2021-10-22 | 2021-10-22 | Method for separating human urinary kallidinogenase and thrombin regulating protein |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111233287.9A CN113913415B (en) | 2021-10-22 | 2021-10-22 | Method for separating human urinary kallidinogenase and thrombin regulating protein |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN113913415A CN113913415A (en) | 2022-01-11 |
| CN113913415B true CN113913415B (en) | 2024-03-19 |
Family
ID=79242310
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202111233287.9A Active CN113913415B (en) | 2021-10-22 | 2021-10-22 | Method for separating human urinary kallidinogenase and thrombin regulating protein |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN113913415B (en) |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
-
2021
- 2021-10-22 CN CN202111233287.9A patent/CN113913415B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
Also Published As
| Publication number | Publication date |
|---|---|
| CN113913415A (en) | 2022-01-11 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102532254B (en) | Method for separating and purifying recombinant human serum albumin (rHSA) from rice seeds | |
| CN108070032B (en) | Purification method of recombinant human collagen | |
| CN101613390A (en) | A kind of method of separating and purifying high-purity cordycepin | |
| CN108048433B (en) | Preparation method of human prothrombin complex | |
| CN101455287A (en) | Melittin purification method | |
| EP3805257A1 (en) | Method for preparing precursor of recombinant human insulin or analogue thereof | |
| CN113913415B (en) | Method for separating human urinary kallidinogenase and thrombin regulating protein | |
| CN101089017A (en) | Process of separating and purifying melittin | |
| CN115925890A (en) | Method for purifying anti-new coronavirus neutralizing antibody | |
| CN1336434A (en) | Prepn. and affinity chromatographic purification process of kallidinogen enzyme | |
| CN111635402B (en) | Separation and purification method of pyrroloquinoline quinone | |
| CN115948351B (en) | Method for separating and purifying CVB1 | |
| CN110256597B (en) | Method for reducing heavy metal residues in ganoderma lucidum polysaccharide by membrane method | |
| CN107163102A (en) | A kind of method of hydrophilic polypeptides purifying | |
| CN107446905B (en) | Method for purifying recombinant human lysozyme | |
| CN103539688B (en) | A kind of method of separation and Extraction Serine from Corynebacterium glutamicum fermented liquid | |
| CN110922471A (en) | Chromatographic separation method for improving purity of porcine insulin | |
| CN113735963A (en) | Method for removing pigment in purification process of recombinant human serum albumin | |
| CN113563424B (en) | Daptomycin purification method | |
| CN102146361B (en) | Method for separating and purifying transglutaminase | |
| CN112341535A (en) | Method for preparing insulin by separation and purification through ion exchange chromatography | |
| CN114605515B (en) | Separation and purification process of high-activity phytohemagglutinin | |
| CN112851775B (en) | Diphtheria toxin non-toxic mutant CRM197 protein, production method and application | |
| CN113717253B (en) | Purification method of daptomycin | |
| CN102617727A (en) | Thymalfasin compound and novel preparation method thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |