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Method for large-scale production of thrombin regulating protein

A technology for regulating proteins and thrombin, which is applied in the preparation methods of thrombomodulin and peptides, chemical instruments and methods, etc., can solve the problems of unsuitable large-scale industrial production and low yield, and achieves close connection, high biological activity, and solution. The effect of active retention problems

Pending Publication Date: 2021-01-26
YANGZHOU AIDEA BIOTECH
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In summary, it can be seen that the current purification methods for extracting TM from human urine are low in yield and not suitable for large-scale industrial production. Looking for a separation and purification technology suitable for industrial scale-up production, to achieve large-scale, high-purity urinary protein TM production has become an urgent problem to be solved

Method used

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  • Method for large-scale production of thrombin regulating protein
  • Method for large-scale production of thrombin regulating protein

Examples

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Effect test

preparation example Construction

[0042] The preparation method of the thrombin affinity chromatography column adopts the following steps:

[0043] (1) Thrombin preparation, after dissolving the purchased thrombin freeze-dried powder in pure water, use a dialysis bag at 4°C to dialyze to remove the freeze-dried protective agent, etc.;

[0044] (2) Matrix preparation, take CNBr-activated agarose gel Focusose 4FF and wash with water;

[0045] (3) Coupling, flushing with coupling buffer (coupling buffer is 0.1mol / L NaHCO 3 , containing 0.5mol / L NaCl, pH 8.3) swell CNBr-activated agarose gel Focus4FF, quickly transfer the agarose gel Focus4FF to the coupling buffer containing thrombin to obtain the agarose-thrombin conjugate Combined complex;

[0046] (4) Blocking active groups, step (3) gained agarose-thrombin coupling complex, remove unreacted thrombin by suction filtration, then transfer to the coupling buffer (coupling buffer) containing glycine (0.1M) Liquid is 0.1mol / L NaHCO 3 , containing 0.5mol / L of Na...

Embodiment 1

[0051] Weigh 5g of thrombin regulatory protein crude product, add benzamidine hydrochloride with a final concentration of 10mM, stir and dissolve with 10 times pure water for 10min, centrifuge at 4000rpm for 10min to take the supernatant, adjust the pH to 6.0, the conductance is lower than the conductance of the equilibrium solution 2- 3mS / cm, load the QAE-Sepharose column that has been equilibrated, the equilibration buffer is pH 6.0 0.10M phosphate buffer, containing 0.05M NaCl, 10mM benzamidine hydrochloride, wash with the equilibration solution after loading 4 times the column volume, then wash with the washing solution (pH6.0 0.15M phosphate buffer, containing 0.2M NaCl, 10mM benzamidine hydrochloride) to wash 5 times the column volume, and finally use the eluent pH5.0 0.20M acetic acid Salt buffer, containing 0.4M NaCl, 10mM benzamidine hydrochloride elution 5 times the column volume.

[0052] Collect the eluate, concentrate it with a 30kD ultrafiltration membrane, adjus...

Embodiment 2

[0057]Weigh 5g of thrombin-regulated protein crude product, add mannitol at a final concentration of 10g / L, stir and dissolve in 10 times pure water for 10min, centrifuge at 4000rpm for 10min to take the supernatant, adjust the pH to 9.0, and the conductivity is lower than the conductivity of the equilibrium solution by 2-3mS / cm, load the equilibrated DEAE-Sepharose column, the equilibration buffer is pH9.0 0.25MTris buffer, containing 0.1MNaCl10g / L mannitol, wash 4 times the column volume with the equilibration solution after loading the sample, and then Wash 5 times column volume with washing solution (pH9.0 0.25M Tris buffer, containing 0.3MNaCl, 10g / L mannitol), and finally use eluent pH6.0 0.05M phosphate buffer, containing 0.5MNaCl, 10g / L Mannitol was eluted for 5 column volumes.

[0058] Collect the eluate, concentrate it with a 30kD ultrafiltration membrane, adjust the conductance to be lower than the conductance of the AF Red-650ML balance solution, the pH is 8.0, an...

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Abstract

The invention discloses a method for large-scale production of thrombin regulating protein. The method for large-scale production of the thrombin regulating protein comprises the following steps of: performing chromatography on a thrombin regulating protein crude product through an anion exchange chromatographic column, an affinity chromatographic column A, a composite affinity chromatographic column B, a thrombin affinity chromatographic column and a hydrophobic chromatographic column, collecting elution peaks, and performing ultrafiltration and concentration to obtain a thrombin regulating protein refined product. According to the method disclosed by the invention, the thrombin regulating protein crude product is taken as a raw material, a plurality of commercial production type affinitychromatographic columns, hydrophobic chromatographic columns and the like are screened for condition optimization, a chromatographic filler which has a purification effect on target protein and is simple and convenient to operate and easy to amplify is determined, and finally the method for large-scale production of the thrombin regulating protein is determined, so that the purification multipleis improved, and large-scale production of the thrombin regulating protein refined product is facilitated.

Description

technical field [0001] The invention relates to the technical field of protein separation and extraction, in particular to a method for large-scale production of thrombin regulatory protein. Background technique [0002] Human soluble thrombin modulin (TM) mainly exists in human plasma and human urine. It is a glycoprotein composed of 557 amino acid residues, with a molecular weight of about 60,300D and an isoelectric point of about 3.8. Discovered in experiments by N.L.Esmon in 1981, its structure includes an amino-terminal plant agglutination-like domain, 6 repeated endothelial growth factor (endothelial growth factor, EGF)-like domains, and 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 disorders of the coagulation system. [0003] In 1993, it was reported that TM could be isolated and purified from urine. For example, th...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/745C07K1/22C07K1/20C07K1/18C07K1/16
CPCC07K14/7455
Inventor 侯晓彦宗扬许冬志傅和亮沈小宁薛云杰陈珏敏
Owner YANGZHOU AIDEA BIOTECH
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