CN112266415A - Method for large-scale production of thrombin regulatory protein - Google Patents
Method for large-scale production of thrombin regulatory protein Download PDFInfo
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Images
Classifications
-
- 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
- C07K14/745—Blood coagulation or fibrinolysis factors
- C07K14/7455—Thrombomodulin
Abstract
The invention discloses a method for producing thrombin regulatory protein in a large scale. The method for producing the thrombin regulating protein on a large scale comprises the steps of subjecting a crude thrombin regulating protein product to anion exchange chromatography column, affinity chromatography column A, compound affinity chromatography column B, thrombin affinity chromatography column and hydrophobic chromatography column chromatography, collecting elution peaks, and performing ultrafiltration and concentration to obtain a refined thrombin regulating protein product. The method takes the crude thrombin regulating protein product as a raw material, screens various commercial production type affinity chromatography columns, hydrophobic chromatography columns and the like for condition optimization, determines the chromatography filler which has a purification effect on target protein, is simple and convenient to operate and easy to amplify, finally determines the method for producing the thrombin regulating protein in a large scale, improves the purification multiple, and is beneficial to the large-scale production of refined thrombin regulating protein products.
Description
Technical Field
The invention relates to the technical field of protein separation and extraction, in particular to a method for producing thrombin regulating protein in a large scale.
Background
Human soluble thrombin regulatory protein (TM), which is mainly present in human plasma and human urine, is a glycoprotein consisting of 557 amino acid residues, has a molecular weight of about 60,300D, an isoelectric point of about 3.8, and was originally found experimentally in 1981 by n.l. espon, and has a structure including an amino-terminal plant coagulation-like domain, 6 repetitive Endothelial Growth Factor (EGF) -like domains, a serine/threonine-rich region (hydrophobic region), a transmembrane region, and a short intracellular tail. Clinically TM is mainly used for Disseminated Intravascular Coagulation (DIC) caused by coagulation system disorders.
In 1993, it was reported that TM could be isolated and purified from urine. For example, Yamamoto et al, which uses QAE Sephadex, anti-TM monoclonal antibody column, thrombin as ligand affinity column and Sephadex G-200, have complicated processes because of the need to prepare monoclonal antibodies. Further, in the purification of TM as in D.E.Jackson et al, a combination of DEAE Sepharose, thrombin as a ligand affinity column and reverse phase HPLC was used, in which the reverse phase HPLC yield was low and which was not suitable for large-scale purification, and in addition, Aoki et al (Japanese patent application laid-open No. Sho-63-30423) and Kunihiro et al (Japanese patent application laid-open No. Sho-63-218399) reported a method for purifying TM from fresh urine, using ion exchange chromatography, thrombin-bound affinity column and gel permeation, and as a result, it was confirmed that this method was not suitable when the TM content was low. In conclusion, it can be seen that various purification methods for extracting TM from human urine at present are low in yield and are not suitable for large-scale industrial production, and a separation and purification technology suitable for industrial scale-up production is found to achieve large-scale and high-purity production of urine protein TM, which is a problem to be solved urgently.
Disclosure of Invention
The invention aims to provide a method for producing thrombin regulatory protein on a large scale aiming at the defects in the prior art, and is suitable for industrial amplification production of thrombin regulatory protein.
In order to realize the aim, the method for producing the thrombin regulating protein in a large scale adopts the technical scheme that:
a process for preparing the thrombin regulating protein in large scale includes such steps as passing the coarse thrombin regulating protein through anionic exchange chromatography column, affinity chromatography column A, composite affinity chromatography column B, thrombin affinity chromatography column and hydrophobic chromatography column, collecting the eluted peaks, ultrafiltering and concentrating.
The crude product of the thrombin regulating protein is a crude product which is precipitated by ammonium sulfate and has specific activity of not less than 1.0ug/E280, and is subjected to ultrafiltration concentration before sample loading, pH is regulated to 6.0-9.0, and the electric conductivity is 0.2-2 mS/cm.
After the balance of the anion exchange chromatography column equilibrium buffer solution, loading a thrombin regulating protein crude product, washing by a washing solution, eluting by the eluent, collecting the eluent of the sample, wherein the equilibrium buffer solution is 0.05-0.25mol/L phosphate buffer solution or Tris buffer solution containing 0-0.1M NaCl, the pH value is 6.0-9.0, the washing solution is 0.05-0.25mol/L phosphate buffer solution or acetate buffer solution containing 0.1-0.3mol/L NaCl, the pH value is 6.0-9.0, the eluent is 0.10-0.30mol/L phosphate buffer solution or acetate buffer solution containing 0.1-0.5M NaCl, and the pH value is 5.0-6.0.
Preferably, the equilibration solution is 0.10mol/L phosphate, contains 0.05M NaCl, has a pH of 6.0, and the eluent is 0.20M acetate buffer, contains 0.4M NaCl, has a pH of 5.0. The washing solution was 0.15mol/L phosphate buffer solution containing 0.2mol/L NaCl and having a pH of 6.0.
Adding protein protective agent into the sample solution of the thrombin regulating protein crude product and the equilibrium buffer solution in the purification process of the anion exchange chromatography column. Preferably, the protein protectant is 10-20mM benzamidine hydrochloride or 10-25g/L mannitol/glycine.
The anion exchange chromatographic column is a weak anion exchange resin chromatographic column or a strong anion exchange resin chromatographic column.
Ultrafiltering the eluate after anion exchange chromatography column chromatography with 10-30kDa ultrafiltration membrane, adjusting pH to 5.0-8.0, adjusting the conductance to 2-3mS/cm lower than that of the equilibrium solution, loading the well-balanced affinity chromatography column A for column chromatography purification, wherein the equilibrium solution is 0.02-0.2mol/L acetate or phosphate buffer solution, the phosphate buffer solution contains 0-0.5mol/L NaCl, the pH is 4.0-7.0, and collecting the sample penetration solution.
Preferably, the equilibration solution is 0.10mol/L acetate buffer, containing 0.1mol/L NaCl, and has a pH of 6.0.
The affinity chromatographic column A ligand is a composite affinity column of reactive dyes, and the reactive dyes are Blue sepharose 6Fast Flow and AF Red-650 ML. The filler contains partial hydrophobic and ionic properties under the premise of leading affinity effect.
Ultrafiltering a sample penetration liquid of the affinity chromatographic column A by a 10-30kDa ultrafiltration membrane, adjusting the pH to 5.0-8.0, adjusting the conductance to be 2-3mS/cm lower than that of a balance liquid, chromatographically purifying a compound affinity chromatographic column B with well-balanced sample application balance liquid, flushing the sample application liquid with the balance liquid with the pH of 5.0-8.0 after sample application is finished, continuously flushing the sample application liquid, finally eluting the sample application liquid with an eluent, and collecting a sample eluent, wherein the balance liquid is 0.05M phosphate buffer solution containing 0-0.1M NaCl; the washing solution is 0.05M acetate buffer solution with the pH value of 4.0-6.0, and contains 0-0.2mol/L sodium chloride; eluent pH of 4.0-6.0, 0.05M acetate buffer solution, and eluent containing 0.1-0.5M NaCl.
Preferably, the equilibration solution is 0.05M phosphate buffer, pH7.0, containing 0.05M NaCl, the wash solution is 0.05M phosphate buffer, pH6.0, containing 0.1M NaCl, and the eluent is 0.05M acetate buffer, pH5.0, containing 0.3M NaCl.
The ligand of the compound affinity chromatographic column B is an affinity chromatographic column of benzamidine.
Adopting a balanced thrombin affinity chromatographic column, loading a sample eluent after the chromatography of the compound affinity chromatographic column B, adjusting the pH value to 5.0-8.0 before loading, flushing the sample by using a flushing liquid after loading, finally eluting the sample eluent by using the eluent, and collecting the sample eluent, wherein the balancing liquid is pH5.0-8.0, 0.02-0.2M acetic acid or Tirs buffer salt, contains 0-0.08MNaCl, the flushing liquid is pH5.0-8.0, 0.02-0.2M acetic acid or Tirs buffer salt, contains 0.05-0.15MNaCl, and the eluent is pH5.0-8.0, 0.02-0.2M acetic acid or Tirs buffer salt, contains 0.2-1.0 MNaCl.
Preferably, the equilibrium solution is 0.05MTris, pH7.0, containing 0.05MNaCl, the wash solution is 0.05MTris, pH7.0, containing 0.15MNaCl, and the eluent is 0.05MTris, pH7.0, containing 1.0 MNaCl.
The thrombin affinity chromatographic column ligand is thrombin, and the thrombin is recombinant thrombin derived from human plasma, bovine plasma, porcine plasma and cell expression.
The thrombin affinity chromatographic column is filled with agarose-thrombin coupled compound, and the agarose-thrombin coupled compound is packed in the column by a wet method to obtain the affinity chromatographic column.
Loading the sample eluate after thrombin affinity chromatography column chromatography with a well-balanced hydrophobic chromatography column, adjusting pH to 5.0-8.0 before loading, washing with washing solution after loading, eluting with eluent, and collecting the elution peak, wherein the balance solution is pH5.0-8.0, 0.02-0.20M acetic acid or phosphate buffer solution containing 0.8-1.2M ammonium sulfate, the washing solution is pH5.0-8.0, 0.02-0.20M acetic acid or phosphate buffer solution containing 1.5-2.0M MNaCl, and the eluent is pH5.0-8.0, 0.02-0.20M acetic acid or phosphate buffer solution containing 0.5-1.4 MNaCl.
Preferably, the equilibrium solution is 0.05MPB, pH6.0, containing 1.0M ammonium sulfate, the rinse solution is 0.05MPB, pH6.0, containing 2.0M NaCl, the eluent is 0.05MPB, pH6.0, containing 0.8M NaCl.
The ligand of the hydrophobic chromatographic column is hydrophobic chromatographic packing of phenyl, butyl sulfide and octyl, and the main framework of the packing is any one of agarose, cellulose or polymer.
Compared with the prior art, the method has the advantages of simple and convenient steps, low cost, high yield, large-scale industrial production and capability of obtaining the thrombin regulating protein with high biological activity and stable quality, and has the following specific advantages:
1. most of foreign proteins can be removed through multi-step affinity chromatography, target protein degradation fragments without biological activity can be removed, and the purity of the target protein is greatly improved.
2. The purification steps are closely connected, the purification speed is high, and the industrial amplification is facilitated.
3. The protein protective agent is added in the early stage of the purification of the crude product, so that the problem of activity maintenance in the early protein purification process is well solved, degradation is prevented, and the yield is improved.
Drawings
FIG. 1 is a flow chart of the present invention for preparing thrombin regulatory proteins.
FIG. 2 is a SEC-HPLC detection profile of a refined thrombin regulating protein product in example 3 of the present invention.
Detailed Description
The present invention is further illustrated by the following description in conjunction with the accompanying drawings, which are to be construed as merely illustrative and not limitative of the remainder of the disclosure, and on reading the disclosure, various equivalent modifications thereof will become apparent to those skilled in the art and fall within the limits of the appended claims.
A method for producing thrombin regulating protein in a large scale comprises the following steps:
1. the crude product of the thrombin regulating protein is a crude product which is precipitated by ammonium sulfate and has specific activity not less than 1.0ug/E280, and is concentrated by ultrafiltration before loading, the pH is regulated to be 6.0-9.0, the electric conductivity is 0.2-2mS/cm, and a protein protective agent is added, wherein the protein protective agent is 10-20mM benzamidine hydrochloride or 10-25g/L mannitol/glycine;
2. loading an anion exchange chromatographic column, wherein the anion exchange chromatographic column is a weak anion exchange resin chromatographic column or a strong anion exchange resin chromatographic column, the anion exchange chromatographic column equilibrium buffer solution is 0.05-0.25mol/L phosphate buffer solution or Tris buffer solution, contains 0-0.1M NaCl, has the pH of 6.0-9.0, the eluent is 0.10-0.30mol/L phosphate buffer solution or acetate buffer solution, contains 0.1-0.5M NaCl, has the pH of 5.0-6.0, preferably, the equilibrium solution is 0.10mol/L phosphate, contains 0.05M NaCl, has the pH of 6.0, the eluent is 0.20M acetate buffer solution, contains 0.4M NaCl, and has the pH of 5.0; adding a washing step before elution of the eluent, wherein the washing liquid is 0.05-0.25mol/L phosphate or acetate buffer solution, the buffer solution contains 0.1-0.3mol/L NaCl, the pH value is 6.0-9.0, preferably, the washing liquid is 0.15mol/L phosphate buffer solution, the pH value is 6.0, and the washing liquid contains 0.2mol/L NaCl; adding a protein protective agent into the buffer solution, wherein the protein protective agent is 10-20mM benzamidine hydrochloride or 10-25g/L mannitol/glycine;
3. and (3) loading an affinity chromatographic column A, wherein a ligand of the affinity chromatographic column A is a composite affinity column of which the ligand is a reactive dye, and the reactive dye is Blue sepharose 6Fast Flow or AF Red-650 ML. The filler contains partial hydrophobic and ionic properties under the premise of leading affinity effect. After anion exchange chromatography column chromatography, the eluent is ultrafiltered by a 10-30kDa ultrafiltration membrane, the pH is adjusted to 5.0-8.0, the conductance is 2-3mS/cm lower than that of the equilibrium liquid, the eluent is purified by an affinity chromatography column A column chromatography, after the sample is finished, the eluent is washed by the equilibrium liquid with the pH of 5.0-8.0, the equilibrium liquid is 0.02-0.2mol/L acetate or phosphate buffer solution, the phosphate buffer solution contains 0-0.5mol/L NaCl, the pH is 4.0-7.0, and the sample penetration liquid is collected. Preferably, the equilibrium solution is 0.10mol/L acetate buffer solution, contains 0.1mol/L NaCl, and has pH of 6.0;
4. and (3) loading a compound type affinity chromatographic column B, wherein the ligand of the compound type affinity chromatographic column B is an affinity chromatographic column of benzamidine. Ultrafiltering the sample penetrating liquid after chromatography of the affinity chromatographic column A through a 10-30kDa ultrafiltration membrane, adjusting the pH to 5.0-8.0, adjusting the conductance to be 2-3mS/cm lower than that of the equilibrium liquid, chromatographically purifying the sample penetrating liquid by using a composite affinity chromatographic column B with well-balanced sample penetrating liquid, flushing the sample penetrating liquid with the equilibrium liquid with the pH of 5.0-8.0 after the sample penetrating is finished, continuously flushing the sample penetrating liquid with a flushing liquid, finally eluting the sample penetrating liquid with an eluent, and collecting the sample eluent, wherein the equilibrium liquid is 0.05M phosphate buffer solution containing 0-0.1M NaCl; the washing solution is 0.05M acetate buffer solution with the pH value of 4.0-6.0, and contains 0-0.2mol/L sodium chloride; eluent pH of 4.0-6.0, 0.05M acetate buffer solution, and eluent containing 0.1-0.5M NaCl.
Preferably, the equilibration solution is 0.05M phosphate buffer, pH7.0, containing 0.05M NaCl, the wash solution is 0.05M phosphate buffer, pH6.0, containing 0.1M NaCl, the eluent is 0.05M acetate buffer, pH5.0, containing 0.3M NaCl;
5. adopting a well-balanced thrombin affinity chromatographic column, wherein a ligand of the thrombin affinity chromatographic column is thrombin, and the thrombin is recombinant thrombin derived from human plasma, bovine plasma, porcine plasma and cell expression; the balanced thrombin affinity chromatographic column is characterized in that a penetrating fluid after the chromatography of the compound affinity chromatographic column B is loaded, the pH value is adjusted to be 5.0-8.0, the balanced fluid is pH5.0-8.0, 0.02-0.2M acetic acid or Tirs buffer salt, 0-0.08MNaCl is contained, the flushing fluid is pH5.0-8.0, 0.02-0.2M acetic acid or Tirs buffer salt, 0.05-0.15MNaCl is contained, the eluent is pH5.0-8.0, 0.02-0.2M acetic acid or Tirs buffer salt, and 0.2-1.0MNaCl is contained; preferably, the equilibration solution is 0.05MTris, ph7.0, containing 0.05MNaCl, the wash solution is 0.05MTris, ph7.0, containing 0.15MNaCl, the eluent is 0.05MTris, ph7.0, containing 1.0 MNaCl;
6. adopting a well-balanced hydrophobic chromatographic column, wherein a ligand of the hydrophobic chromatographic column is a hydrophobic chromatographic packing of phenyl, butyl sulfide and octyl, and a main framework of the packing is any one of agarose, cellulose or polymer; adjusting pH of eluate after thrombin affinity chromatography column chromatography to 5.0-8.0, loading into hydrophobic chromatography column, wherein the balance solution is pH5.0-8.0, 0.02-0.20M acetic acid or phosphate buffer solution containing 0.8-1.2M ammonium sulfate, the washing solution is pH5.0-8.0, 0.02-0.20M acetic acid or phosphate buffer solution containing 1.5-2.0MNaCl, the eluate is pH5.0-8.0, 0.02-0.20M acetic acid or phosphate buffer solution containing 0.5-1.4 MNaCl; preferably, the equilibrium solution is 0.05MPB, pH6.0, containing 1.0M ammonium sulfate, the rinse solution is 0.05MPB, pH6.0, containing 2.0M NaCl, the eluent is 0.05MPB, pH6.0, containing 0.8M NaCl, the elution peak is collected, ultrafiltered and concentrated to obtain the refined thrombin regulating protein.
The method takes a TM crude product as a raw material, screens various commercial production type affinity chromatography columns, hydrophobic chromatography columns and the like for condition optimization, determines a chromatography filler which has a purification effect on target protein, is simple and convenient to operate and easy to amplify, and finally determines a method for producing thrombin regulating protein in large scale.
The thrombin affinity chromatographic column is filled with agarose-thrombin coupled compound, and the agarose-thrombin coupled compound is packed in the column by a wet method to obtain the affinity chromatographic column.
The preparation method of the thrombin affinity chromatographic column comprises the following steps:
(1) preparing thrombin, namely dissolving purchased thrombin freeze-dried powder with pure water, and dialyzing by using a dialysis bag at 4 ℃ to remove a freeze-drying protective agent and the like;
(2) preparing a matrix, and washing CNBr activated agarose gel Focurose4FF with water;
(3) coupling, using a buffer flush of coupling buffer (0.1mol/L NaHCO in coupling buffer)3Containing 0.5mol/L NaCl and having a pH value of 8.3) swelling the CNBr-activated sepharose Focure 4FF, and quickly transferring the sepharose Focure 4FF into a coupling buffer solution containing thrombin to obtain an agarose-thrombin coupling compound;
(4) blocking active groups, filtering the agarose-thrombin coupled compound obtained in the step (3) to remove unreacted thrombin, and transferring the thrombin-blocked agarose-thrombin coupled compound into a coupling buffer (NaHCO with the coupling buffer of 0.1 mol/L) containing glycine (0.1M)30.5mol/L NaCl, pH 8.3) to block all residual active groups;
(5) washing, namely washing the agarose-thrombin coupled complex obtained in the step (4) by sequentially using an acetate-sodium acetate buffer solution containing NaCl (0.1mol/L acetate-sodium acetate, 0.5mol/L NaCl and pH 4.0) and a Tris-HCl buffer solution containing NaCl (0.1mol/L Tris-HCl, 0.5mol/L NaCl and pH 8.0), and removing the redundant ligand which is not coupled after coupling;
(6) inactivating thrombin, dissolving AEBSF in 20mM Tris-HCl, pH7.5 buffer solution, adding the AEBSF into the agarose-thrombin coupling compound obtained in the step (5), gently shaking for 1.5-2.5 hours, and washing with pure water to obtain thrombin affinity filler;
(7) and (4) filling the column, namely filling the column by adopting a wet method, taking an empty column tube, filling a bottom sieve plate, filling the empty column tube with pure water, opening a lower-end outflow tube to enable the water to naturally flow out, discharging bubbles at the bottom of the column, pouring the buffer solution suspended filler obtained in the step (6) into the column tube at one time, connecting a pipeline, and storing the mixture in a refrigerator at 4 ℃.
Example 1:
weighing 5g of thrombin-regulated protein crude product, adding benzamidine hydrochloride with the final concentration of 10mM, stirring and dissolving with 10 times of pure water for 10min, centrifuging at 4000rpm for 10min to obtain a supernatant, adjusting the pH to 6.0, wherein the conductance is 2-3mS/cm lower than that of a balance solution, loading the balanced QAE-agarose gel column, the balance buffer solution is a phosphate buffer solution with the pH of 6.00.10M and contains 0.05MNaCl and 10mM benzamidine hydrochloride, washing the column with the balance solution for 4 times of the column volume after loading, washing the column with a washing solution (pH 6.00.15M phosphate buffer solution containing 0.2M NaCl and 10mM benzamidine hydrochloride) for 5 times of the column volume, and finally eluting the column with an eluent with pH 5.00.20M acetate buffer solution containing 0.4MNaCl and 10mM benzamidine hydrochloride for 5 times of the column volume.
Collecting the eluent, concentrating with 30kD ultrafiltration membrane, adjusting the conductivity to be lower than that of Blue sepharose 6Fast Flow equilibrium liquid, adjusting pH to be 6.0, loading Blue sepharose 6Fast Flow chromatographic column with the equilibrium liquid being pH6.0, 0.10M phosphate buffer solution containing 0.1MNaCl, and collecting the sample penetration liquid after the loading is finished.
Adjusting pH of the sample to 6.0 by penetrating the sample, loading the balanced benzamidine affinity chromatography column, balancing the benzamidine affinity chromatography column by using a phosphate buffer solution with pH7.0 and 0.05M and a balance solution containing 0.05M NaCl in advance, washing the column by using the balance solution after the loading is finished by 4 times of column volume, washing the column by using a washing solution with pH6.0 and 0.05M containing 0.1M NaCl in a phosphate buffer solution with pH6.0 and 0.05M containing 0.1M NaCl in 5 times of column volume, finally eluting the column by using an eluent with pH5.0 and 0.05M containing 0.3M NaCl in 6 times of column volume, and collecting a sample eluent.
The thrombin affinity chromatographic column is balanced by 5-7 times of column volume in advance with a balance solution, the balance solution is pH7.0, 0.05M Tirs buffer salt and contains 0.05MNaCl, the thrombin affinity chromatographic column is loaded, after the loading is finished, the balance solution is used for washing 4 times of column volume, then the balance solution is used for washing 5-6 times of column volume with pH7.05M Tirs buffer salt and 0.15MNaCl, and then the balance solution is used for eluting 5-10 times of column volume with pH7.00.05MTris and 1.0MNaCl, and elution peaks are collected.
Loading thrombin affinity chromatography eluent by using a well-balanced hydrophobic chromatography column, adjusting the pH value to 6.0, wherein the balance liquid is phosphate buffer solution with the pH value of 6.00.05M and contains 1.0M ammonium sulfate, washing 3-5 times of column volume by using the balance liquid after loading is finished, washing 5-6 times of column volume by using washing liquid with the pH value of 6.00.05MPB and 2.0MNaCl, finally eluting 5 times of column volume by using the pH value of 6.00.05MPB and the eluent with the pH value of 0.8MNaCl, collecting an elution peak, and performing ultrafiltration and concentration on the elution peak by using a 10kDa ultrafiltration membrane to obtain a thrombin regulating protein refined product.
Example 2:
weighing 5g of thrombin-regulated protein crude product, adding 10g/L mannitol with final concentration, stirring and dissolving with 10 times pure water for 10min, centrifuging at 4000rpm for 10min to obtain supernatant, adjusting pH to 9.0, wherein the conductance is 2-3mS/cm lower than that of the equilibrium solution, loading the balanced DEAE-agarose gel column, the equilibrium buffer solution is pH 9.00.25MTris buffer solution containing 0.1MNaCl10g/L mannitol, washing the column with the equilibrium solution for 4 times of column volume after loading is finished, washing the column with washing solution (pH 9.00.25MTris buffer solution containing 0.3M NaCl and 10g/L mannitol) for 5 times of column volume, and finally eluting with pH 6.00.05M phosphate buffer solution containing 0.055 MNaCl and 10g/L mannitol for 5 times of column volume.
Collecting eluate, concentrating with 30kD ultrafiltration membrane, adjusting the conductivity to be lower than that of AF Red-650ML equilibrium solution, adjusting pH to 8.0, loading Blue sepharose 6Fast Flow chromatographic column, wherein the equilibrium solution is pH 8.00.10M phosphate buffer solution containing 0.5MNaCl, and collecting sample penetration solution after loading.
Adjusting the pH value of the penetrating liquid to 6.0, loading a balanced benzamidine affinity chromatographic column, balancing the benzamidine affinity chromatographic column by using a phosphate buffer solution with the pH value of 7.00.05M and a balance liquid containing 0.05MNaCl in advance, flushing 4 times of column volume by using the balance liquid after the loading is finished, flushing 5 times of column volume by using a flushing liquid containing 0.1MNaCl in a phosphate buffer solution with the pH value of 6.00.05M, finally eluting 6 times of column volume by using an acetate buffer solution with the pH value of 5.00.05M and an eluent containing 0.3MNaCl, collecting the eluent, and loading the thrombin affinity column.
The thrombin affinity chromatographic column is balanced by 5-7 times of column volume in advance with a balance solution, the balance solution is pH8.0, 0.20M Tirs buffer salt and contains 0.08MNaCl, the sample is loaded, after the sample loading is finished, the balance solution is used for washing 4 times of column volume, then the balance solution is used for washing 5-6 times of column volume with pH8.0, 0.20M Tirs buffer salt and washing liquid containing 0.15MNaCl, the elution solution is used for eluting 5-10 times of column volume with pH8.00.20MTris and elution solution containing 0.5MNaCl, and elution peaks are collected.
Loading thrombin affinity chromatography eluent by using a well-balanced hydrophobic chromatography column, adjusting the pH to 8.0, wherein the balance solution is phosphate buffer solution with the pH of 8.00.02M and contains 1.2M ammonium sulfate, washing 3-5 times of column volume by using the balance solution after loading is finished, washing 5-6 times of column volume by using washing solution with the pH of 8.00.02MPB and 1.5MNaCl, finally eluting 5 times of column volume by using the pH of 8.00.02MPB and the eluent with the pH of 0.5MNaCl, collecting an elution peak, and performing ultrafiltration concentration on the elution peak by using a 10kDa ultrafiltration membrane to obtain a thrombin regulating protein refined product.
Example 3:
weighing 50g of thrombin-regulated protein crude product, adding benzamidine hydrochloride with the final concentration of 10mM, stirring and dissolving for 30min by 10 times pure water, centrifuging for 10min at 4000rpm, taking supernatant, regulating pH to 7.0, wherein the conductance is 2-3mS/cm lower than that of a balance solution, loading the balanced QAE-agarose gel column, the balance buffer solution is phosphate buffer solution with the pH of 7.00.10M and contains 0.05M NaCl and 10mM benzamidine hydrochloride, washing the column with the balance solution for 4 times of the column volume after loading is finished, washing the column with washing solution (pH 7.00.15M phosphate buffer solution, containing 0.2M NaCl and 10mM benzamidine hydrochloride) for 5 times of the column volume, and finally eluting the column with washing solution of pH 6.00.2M acetate buffer solution, containing 0.4M NaCl and 10mM benzamidine hydrochloride for 5 times of the column volume.
Collecting the eluent, concentrating with 30kD ultrafiltration membrane, adjusting the conductivity to be lower than that of Blue sepharose 6Fast Flow equilibrium liquid, adjusting pH to 7.0, loading Blue sepharose 6Fast Flow chromatographic column with the equilibrium liquid being pH7.00.10M phosphate buffer solution containing 0.1MNaCl, and collecting the sample penetration liquid after the loading is finished.
Adjusting the pH value of the penetrating liquid to 6.0, loading a balanced benzamidine affinity chromatographic column, balancing the benzamidine affinity chromatographic column by using a phosphate buffer solution with the pH value of 7.00.05M and a balanced liquid containing 0.05MNaCl in advance, flushing 4 times of column volume by using the balanced liquid after the loading is finished, flushing 5 times of column volume by using a flushing liquid containing 0.1MNaCl in a phosphate buffer solution with the pH value of 6.00.05M, finally eluting 6 times of column volume by using an acetate buffer solution with the pH value of 5.00.05M and an eluent containing 0.3MNaCl, and collecting the eluent.
The thrombin affinity chromatographic column is balanced by 5-7 times of column volume in advance with a balance solution, the balance solution is pH7.0, 0.05M Tirs buffer salt and contains 0.05MNaCl, the thrombin affinity chromatographic column is loaded, after the loading is finished, the balance solution is used for washing 4 times of column volume, then the balance solution is used for washing 5-6 times of column volume with pH7.05M Tirs buffer salt and 0.15MNaCl, and then the balance solution is used for eluting 5-10 times of column volume with pH7.00.05MTris and 1.0MNaCl, and elution peaks are collected.
Loading thrombin affinity chromatography eluent by using a well-balanced hydrophobic chromatography column, adjusting the pH value to 6.0, wherein the balance liquid is phosphate buffer solution with the pH value of 6.00.05M and contains 1.0M ammonium sulfate, washing 3-5 times of column volume by using the balance liquid after loading is finished, washing 5-6 times of column volume by using washing liquid with the pH value of 6.00.05MPB and 2.0MNaCl, finally eluting 5 times of column volume by using the pH value of 6.00.05MPB and the eluent with the pH value of 0.8MNaCl, collecting an elution peak, and performing ultrafiltration and concentration on the elution peak by using a 10kDa ultrafiltration membrane to obtain a thrombin regulating protein refined product.
Claims (12)
1. A method for producing thrombin regulatory protein in a large scale is characterized in that: comprises the steps of subjecting a thrombin regulating protein crude product to anion exchange chromatography column, affinity chromatography column A, compound affinity chromatography column B, thrombin affinity chromatography column and hydrophobic chromatography column chromatography, collecting elution peaks, and performing ultrafiltration and concentration to obtain a thrombin regulating protein refined product.
2. The method for modeling thrombin regulatory protein according to claim 1, wherein: the crude product of the thrombin regulating protein is a crude product which is precipitated by ammonium sulfate and has specific activity of not less than 1.0ug/E280, and is subjected to ultrafiltration concentration before sample loading, pH is regulated to 6.0-9.0, and the electric conductivity is 0.2-2 mS/cm.
3. The method for modeling thrombin regulatory protein according to claim 1, wherein: after the balance of the anion exchange chromatography column equilibrium buffer, loading a thrombin regulatory protein crude product, washing by a washing solution, eluting by the eluent, collecting the eluent of the sample, wherein the equilibrium buffer is 0.05-0.25mol/L phosphate buffer or Tris buffer containing 0-0.1M NaCl, the pH value is 6.0-9.0, the washing solution is 0.05-0.25mol/L phosphate buffer or acetate buffer containing 0.1-0.3mol/L NaCl, the pH value is 6.0-9.0, the eluent is 0.10-0.30mol/L phosphate buffer or acetate buffer containing 0.1-0.5M NaCl, and the pH value is 5.0-6.0.
4. The method for modeling thrombin regulatory protein according to claim 1, wherein: adding protein protective agent into the sample solution of the thrombin regulating protein crude product and the equilibrium buffer solution in the purification process of the anion exchange chromatography column.
5. The method for modeling thrombin regulatory protein according to claim 1, wherein: ultrafiltering the eluate after anion exchange chromatography column chromatography with 10-30kDa ultrafiltration membrane, adjusting pH to 5.0-8.0, adjusting the conductance to 2-3mS/cm lower than that of the equilibrium solution, loading the well-balanced affinity chromatography column A for column chromatography purification, wherein the equilibrium solution is 0.02-0.2mol/L acetate or phosphate buffer solution, the phosphate buffer solution contains 0-0.5mol/L NaCl, the pH is 4.0-7.0, and collecting the sample penetration solution.
6. The method for modeling thrombin regulatory protein according to claim 1, wherein: the affinity chromatographic column A ligand is a composite affinity column of reactive dyes, and the reactive dyes are Blue sepharose 6Fast Flow and AF Red-650 ML.
7. The method for modeling thrombin regulatory protein according to claim 1, wherein: ultrafiltering a sample penetration liquid of the affinity chromatographic column A by a 10-30kDa ultrafiltration membrane, adjusting the pH to 5.0-8.0, adjusting the conductance to be 2-3mS/cm lower than that of a balance liquid, chromatographically purifying a compound affinity chromatographic column B with well-balanced sample application balance liquid, flushing the sample application liquid with the balance liquid with the pH of 5.0-8.0 after sample application is finished, continuously flushing the sample application liquid, finally eluting the sample application liquid with an eluent, and collecting a sample eluent, wherein the balance liquid is 0.05M phosphate buffer solution containing 0-0.1M NaCl; the washing solution is 0.05M acetate buffer solution with the pH value of 4.0-6.0, and contains 0-0.2mol/L sodium chloride; eluent pH of 4.0-6.0, 0.05M acetate buffer solution, and eluent containing 0.1-0.5M NaCl.
8. The method for modeling thrombin regulatory protein according to claim 1, wherein: the compound affinity chromatographic column B is a compound affinity chromatographic column taking benzamidine as a ligand.
9. The method for modeling thrombin regulatory protein according to claim 1, wherein: adopting a balanced thrombin affinity chromatographic column, loading a sample eluent after the chromatography of the compound affinity chromatographic column B, adjusting the pH value to 5.0-8.0 before loading, flushing the sample by using a flushing liquid after loading, finally eluting the sample eluent by using the eluent, and collecting the sample eluent, wherein the balancing liquid is pH5.0-8.0, 0.02-0.2M acetic acid or Tirs buffer salt, contains 0-0.08MNaCl, the flushing liquid is pH5.0-8.0, 0.02-0.2M acetic acid or Tirs buffer salt, contains 0.05-0.15MNaCl, and the eluent is pH5.0-8.0, 0.02-0.2M acetic acid or Tirs buffer salt, contains 0.2-1.0 MNaCl.
10. The method for modeling thrombin regulatory protein according to claim 1, wherein: the thrombin affinity chromatographic column ligand is thrombin, and the thrombin is recombinant thrombin derived from human plasma, bovine plasma, porcine plasma and cell expression.
11. The method for modeling thrombin regulatory protein according to claim 1, wherein: loading the sample eluate after thrombin affinity chromatography column chromatography with a well-balanced hydrophobic chromatography column, adjusting pH to 5.0-8.0 before loading, washing with washing solution after loading, eluting with eluent, and collecting the elution peak, wherein the balance solution is pH5.0-8.0, 0.02-0.20M acetic acid or phosphate buffer solution containing 0.8-1.2M ammonium sulfate, the washing solution is pH5.0-8.0, 0.02-0.20M acetic acid or phosphate buffer solution containing 1.5-2.0M MNaCl, and the eluent is pH5.0-8.0, 0.02-0.20M acetic acid or phosphate buffer solution containing 0.5-1.4 MNaCl.
12. The method for modeling thrombin regulatory protein according to claim 1, wherein: the ligand of the hydrophobic chromatographic column is hydrophobic chromatographic packing of phenyl, butyl sulfide and octyl, and the main framework of the packing is any one of agarose, cellulose or polymer.
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