Purification method of natural thrombin regulatory protein

Abstract

The invention discloses a purification method of natural thrombin regulatory protein. The method includes the following steps: (1) selecting a composite chromatography column with hydrophobic groups and anisole as a ligand, and then preparing a balancing solution and an eluent; balancing the composite chromatography column by phosphate with 3-5 times of column volume until the conductivity and pHvalue are consistent with that of the balancing solution; (2) taking a thrombin regulating protein solution intermediate with a concentration of 0.005-10 mg / mL, adjusting the pH value of the thrombinregulating protein solution intermediate to 4.0-8.0, sampling and performing chromatography purification on the composite chromatography column, using the balancing solution with a pH value of 4.0-8.0for flushing after the sampling is completed, then using a flushing liquid with a pH value of 4.0-8.0 for flushing, eluting with eluent, and collecting the eluent; (3) ultrafiltering the eluent obtained in the step (2) to obtain a concentrated solution; (4) pouring the concentrated solution obtained in the step (3) to a freeze-drying plate for freeze-drying to obtain a thrombin regulatory proteinfinished product. The method is simple in operation, high in yield, and high in purity.

Description

technical field [0001] The invention relates to a method for purifying natural thrombin regulatory protein. Background technique [0002] Soluble thrombin modulin (Thrombomodulin, 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. TM was originally discovered in experiments by N.L.Esmon et al. in 1981. Its structure includes an amino-terminal plant agglutination-like domain, six repeated endothelial growth factor (endothelial growth factor, EGF)-like domains, a serine / threonine enrichment region (hydrophobic region), a transmembrane region and a short intracellular tail. TM is known for its powerful blood coagulation blocking effect. It blocks the blood coagulation activity of thrombin by specifically binding to thrombin. At the same time, TM can significantly promote the protein C (Protein C, PC) of thrombin. Activation, an important co...

AI Technical Summary

Problems solved by technology

[0003] At present, a lot of work has been done on the separation and purification of human thrombin regulatory protein from natural sources in various literatures. For example, Yamamoto et al. used QAE cross-linked dextran, anti-TM monoclonal antibody columns, and thrombin in the process of purifying TM. Affinity column and cross-linked dextran G-200 as a ligand, but because this method requires the preparation of monoclonal antibodies, the cost is high and the process is relatively complicated

D.E.Jackson and others used a combination of DEAE agarose gel, thrombin as a ligand affinity column and reverse HPLC when purifying TM. In this method, reverse HPLC has low yield and is not suitable for large-scale purification.

In addition, the prior art discloses a U.S. patent, the application number is US456178, the publication number is US5202421A, the publication date is 1993-04-13, and the name is Anticoagulant substance obtained from urine and process for the preparation thereof. The prior art also discloses A European patent, the application number is EP96117071.9, the publication number is EP0770626A2, the publication date is 1997-05-02, and the name is Method for purifying thrombomodulin. These two patents disclose the method of purifying TM from fresh urine, using Ion-exchange chromatography, thrombin-conjugated affinity columns and gel permeation were used, and it turned out that this method is not applicable when the TM content is very low

[0004] In summary, it can be seen that ion exchange chromatography, monoclonal antibody affinity column and thrombin affinity column are indispensable means for extracting and purifying TM at present. Among them, ion exchange chromatography is simple to operate and low in cost. The disadvantage is that the target protein The purification factor of the affinity chromatography column is low, and the affinity chromatography column can increase the purification factor of the target protein by about 10-100 times in one step, but the synthesis process of this type of affinity chromatography column is complicated, the cost is high, the loading capacity is low, and the macromolecular ligands are prone to fall off. drop, risk of contaminating the target protein

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