RU2346983C1 - Method of production of plasminogen activator - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: invention relates to production of highly-purified highly-active plasminogen-activating enzyme preparations, which may be used in biochemistry and medical science. Method implies the following: Morikrasa enzyme preparation is dissolved in tris-chloride buffer with pH 7.2-7.5 to obtain concentration of at least 2.5%. Resulting solution is fractionated by means of ion-exchange chromatography on DEAE-Toyoperl sorbent. Proteinase is eluted with NaCl concentration gradient of 0.1-1.5 M. Then, resulting solution of plasminogen activator is concentrated and demineralised by ultrafiltration using a membrane permeable for peptides with molecular weight up to 10 kDa. Plasminogen activator is purified by gel chromatography on Sephadex G 50 in 0.1 M ammonium-acetate buffer with pH 6.3-6.5, which contains 1.0-1.2 M of sodium chloride.

EFFECT: simplified method of production and shorter process time.

3 dwg, 1 tbl

Description

The invention relates to biotechnology, in particular to the production of highly purified highly active enzyme preparations that can be used in biochemistry, medicine, for the treatment of thromboembolic diseases, as well as for research purposes.

In the blood plasma there are numerous proteinases that are involved in the process of blood coagulation. The main active enzyme in blood coagulation is thrombin, the substrate of which is fibrinogen. In case of damage to the walls of blood vessels, prothrombin is quickly converted with the help of activator proteinases to thrombin, which, in turn, converts fibrinogen into fibrin (thrombin splits small fragments from the fibrinogen molecule; in this case, fibrin monomer molecules are formed, which polymerize to form fibrin). Fibrin has a mesh structure such as a bandage, which closes the damage, forming a clot. Plasmin is a proteinase, opposite in action to thrombin. In blood plasma, it is also in an inactive state in the form of plasminogen. When the blood vessel wall has recovered, plasminogen is converted into active plasmin and hydrolyzes the remnants of fibrin, i.e. dissolves an unnecessary clot with the formation of small peptides that are harmless to the body. When the healing process has passed, active thrombin and plasmin are inhibited by blood plasma inhibitors and destroyed by other proteinases. In a healthy body, these processes are balanced, while patients have an imbalance. With an excess of prothrombin and thrombin, there may be a tendency to thrombosis, with an excess of plasminogen and plasmin, a tendency to bleeding.

Plasminogen is a proenzyme widely distributed in the body. Its concentration in blood plasma reaches 0.2 g / l. The main function of plasminogen activators is the regulation of the liquid state of the blood (fibrinolysis).

The wide spread of diseases associated with thrombosis, deposits of fibrin masses in joints, hematomas, etc. requires an expansion of the arsenal of drugs that can dissolve blood clots formed in the body.

Currently, the main criteria that thrombolytic drugs must meet are identified:

- high activating effect on plasminogen - an inactive proenzyme from the blood, which turns after the introduction of the activating enzyme into plasmin, destroying blood clots;

- low proteolytic activity in relation to plasma proteins;

- resistance of the enzyme to the action of blood plasma inhibitors. Therapy of thrombosis develops in several ways:

1. search for plasminogen activators;

2. the search for fibrinolytic drugs containing proteinases other than plasminogen, and capable of hydrolyzing fibrin clots.

The first way is more natural and safe for the body. The second way is sometimes more effective, but fraught with side effects.

Known purified thrombolytic drugs currently used in medicine have several disadvantages. For example, streptokinases ("streptase", "cabicinase", "avelizin") cause allergic reactions, and with the introduction of high doses lead to hypotension. Urokinase from human urine ("abbokinase", "urocidan") does not cause allergic reactions, but is rapidly destroyed and excreted from the body. Staphylokinase is secreted by a strain of a pathogenic microorganism - Staphylococcus aureus, so the enzyme for use in medicine is obtained in the form of a recombinant protein. All of the above drugs are of high cost, so the search for new drugs that regulate the state of the blood is relevant.

It is known that many blood-sucking organisms, such as leeches, some bats and snakes after a bite, inject thrombin inhibitors (leech hirudin) or plasminogen activators into the bloodstream. The ingress of large quantities of such substances into the body can lead to a hemorrhagic effect - the blood stops clotting, and the victim may die from bleeding. In some snakes, on the contrary, both prothrombin activators and plasminogen activators are part of the poison. For example, there is a known method for producing a fibrinolytic enzyme from a southern moccasin snake venom, which is subjected to ion exchange chromatography on CM-cellulose, gel filtration on Sephadex G-100, affinity chromatography on p-aminobenzamidine-sepharose. The enzyme belongs to the class of metalloproteinases, is able to hydrolyze blood clots in vitro at a concentration of 100 μg / ml and above.

The disadvantages of the method include inaccessible raw materials, an expensive cleaning method. Moreover, snake metalloproteinase is not able to activate plasminogen with the formation of plasmin. In this case, a harmful effect on the hemostatic system is possible due to the formation of toxic products of thrombus hydrolysis or destruction of hemoglobin and other blood proteins.

Guan A.L., Retzios A.D., Henderson G.N., Markland F.S., Jr., "Purification and characterization of a fibrinolytic enzyme from venom of the southern copperhead snake (Agkistrodon contortrix contortrix)." Arch. Biochem. Biophys., 1991, 289, (2), 197-207.

There is a method of producing a substance with anticoagulant activity from plants growing in South Africa (from Aspilia afrikana), as well as those growing in the middle zone of the European part of Russia - meadowsweet or bark of peony roots. The raw materials are crushed and extracted with distilled water at a ratio of raw materials: extract 1: 5, heated in a boiling water bath, after which the precipitate is removed by centrifugation at 1800-2000 rpm, the supernatant is frozen at (-10) - (- 20) ° C and lyophilized are dried.

The method has the following disadvantages:

- a rare distribution area of a plant species,

- insufficient antiplatelet properties of the product,

- There are side effects from the use of product fractions.

Moreover, the preparations obtained by this method are more likely to be dietary supplements, rather than therapeutic agents. They are administered orally, the activities of the components of the fibrinolysis system increase by only 1.4-2 times, the mechanism of action is unknown.

Patent RU, No. 2143270, А61К 35/78, 1999.12.27.

A known method of obtaining plasminogen activator from the culture medium of a number of lines of monolayer cell cultures, including from a cell culture of a kidney of a pig embryo in the presence of peptone. The method consists in growing the culture for a long time at a temperature of 36.6-37.0 ° C, followed by the addition of distilled water to the culture medium and subsequent centrifugation at 7000-8000 rpm for 15-20 minutes and separating the supernatant. This method of obtaining plasminogen activator from cell culture leads to the production of a non-enzymatic activator, the structure of which has not been established.

The disadvantage of this method is its duration and obtaining the target product only with a narrow specificity of plasminogen activator. Auth test. USSR No. 1732516, A61K 38/48, C12N 9/68, 1995.08.20.

The processes for producing plasminogen activator in the form of a recombinant protein have the following disadvantages: upon receipt of recombinant proteins by gene expression in E. coli cells, the protein can form insoluble inclusion bodies. Expression in E. coli cells is also unsuitable for the production of glycosylated proteins. The main disadvantage of E. coli as a system for the expression of physiologically active proteins is the presence of E. coli endotoxin impurities in protein preparations. Removal of endotoxins is a complex process requiring large material costs.

Animal tissue cells grow much slower than bacterial, and are less susceptible to genetic engineering manipulations aimed at improving the technological characteristics of strains.

A known method of obtaining a plasminogen activator from human blood vessels. The method is a five-step process and includes precipitation of the protein from the extract with ammonium sulfate, affinity chromatography on arginine-sepharose, hydrophobic chromatography on phenyl-sepharose, gel filtration on Sephacryl G-200 and affinity chromatography on fibrin-sepharose. The method is very time-consuming, carried out on expensive sorbents, allows you to get a purified preparation of plasminogen activator with a yield of 38%. Given the inaccessible source of the enzyme and the complexity of purification, this method can only be used in laboratory conditions.

EP No. 0 124 613 (JP), C12N 9/72, A61K 38/00, publ. 1984.11.14.

A known method of obtaining a plasminogen activator from human colon tissue. Like the above method, it is a five-step process, including ligand exchange chromatography on Zn-Sepharose, affinity chromatography on concanavalin A-sepharose followed by dialysis and lyophilization and rechromatography on concanavalin A-sepharose. In other examples, affinity chromatography on fibrin-sepharose or benzamidine-sepharose is used instead of rechromatography on concanavalin-sepharose. This method is also not preparative due to the use of currently inaccessible starting material, a laborious and expensive cleaning process.

EP No. 0 241 448, (US) C12N 9/64, C12N 9/72, publ. 1987.10.14.

An interesting method for producing prolonged thrombolytic agents obtained by chemical modification of the fibrinolytic enzyme by acylation of the active center with an excess of 4-nitrophenyl ester of cis or trans isomers of 4- (N, N, N-trimethylamino -) - cinnamic acid. Acylation is carried out at a temperature of 0-25 ° C and a pH of 7.0-8.5 for 10-70 minutes in the absence of an organic solvent. The obtained acyl enzyme is separated by gel filtration or ultrafiltration, protein fractions are lyophilized.

The resulting complexes of proteinase activators with reversible inhibitors, when introduced into the blood, begin to be activated only when they enter plasminogen, since they have more affinity for them than modifying inhibitors. As a result, the chemical reagents release the active center of activators in favor of the natural plasminogen substrate and plasmin formation occurs.

Thrombolytic enzymes, which are modified in this method, are quite expensive, and after processing their cost increases.

Patent RU No. 2121362, А61К 38 / 02,1998.11.10.

Closest to the claimed is a method of obtaining a plasminogen activator from a culture fluid of human melanoma cells, comprising separating the cellular material from the culture fluid by centrifugation at 7000 g, ligand exchange chromatography of the culture fluid on a column with Zn-IDA agarose, affinity chromatography on concanavalin A agarose and gel filtration on Sephadex G150 Superfine. The result is a plasminogen activator purified in 263 times with a 46% yield. The purity of the obtained preparation is confirmed by the presence of one strip on the electrophoregram after SDS-PAGE electrophoresis. The disadvantages of the method include expensive raw materials that potentially contain oncogenic proteins. Thus, the possibility of the harmful effect of the drug when it enters the patient’s blood increases. The need to maintain cell culture, a multi-stage purification method, including chromatography on expensive affinity sorbents, the use of a buffer containing potassium thiocyanate (assigned to the category of poisons) in a high concentration (1.6 M -129 g / l), a long isolation process also inhibit the preparation of an activator plasminogen in amounts sufficient for use as a thrombolytic drug in medicine.

US patent No. 4752603, C12N 9/64, A61K 38/00, publ. 19888.06.21.

The objective of the invention is to increase the yield of plasminogen activator, simplifying and cheapening the method of its production, expanding the range of plasminogen activators.

The problem is achieved in that in the method for producing a plasminogen activator from an enzyme-containing raw material, including fractionating it using ion exchange chromatography, the Moricrase collagenolytic enzyme preparation is used as the enzyme-containing raw material, the latter is dissolved in Tris-chloride buffer with a pH of 7.2-7, 5 to a concentration of not less than 2.5% in the solution, followed by fractionation of the solution on a DEAE-Toyoperl sorbent, elution of the proteinase in a 0.1-1.5 M NaCl concentration gradient, concentration and desalting by ultrafiltration of the obtained activator solution on a membrane passing peptides with a molecular weight of less than 10 kDa, and purification of the plasminogen activator by gel chromatography on Sephadex G50 in 0.1 M ammonium acetate buffer with a pH of 6.3–6.5 containing 1.0– 1.2 M sodium chloride.

The preparation of collagenolytic enzymes "Morikraz", developed by the NGO "Trinita" in conjunction with the chemical faculty of Moscow State University M.V. Lomonosov, characterized and isolated from hepatopancreas of the Far Eastern crab Paralithodes camtshatica (PC) according to the developed original method.

Rudenskaya G.N., Isaev V.A., Shmoilov A.M., Karabasova M.A., Shvets S.V., Miroshnikov A.I., Brusov A.B. "Preparation of proteolytic enzymes from kamchatka crab Paralithodes camchatica hepatopancreas." Appl. Biochem. Biotechnol., 88, 175-83, 2000.

Moricrase is the total preparation of king crab proteinases, containing collagenolytic serine proteinase, trypsin, elastase, metalloproteinase, carboxypeptidase and aminopeptidase. Due to the combined action of these enzymes, the drug easily hydrolyzes collagen, fibrin, azokasein and other proteins.

Interest in this source is associated with the disposal of waste from crab fisheries. An important advantage is the harmlessness of crab collagenase preparations for humans. Moricrase is produced in significant quantities, relatively inexpensive, is used in ointment compositions for the treatment of burns, ulcers, postoperative scars.

As shown by experimental data, Moricrase has thrombolytic activity, is able to activate plasminogen, however, the direct injection of Moricrase solution into the bloodstream is dangerous due to its high collagenolytic and proteolytic activity.

Isolated according to the proposed method from Moricrase metalloproteinase - an enzyme that is an effective plasminogen activator, has a slight total proteolytic activity and is practically not able to hydrolyze collagen. The thrombolytic effect of such an enzyme is due to the activity of endogenous plasmin, which is formed after plasminogen activation by the enzyme.

The essence of the method is as follows.

As a source of enzymes use a solution of the drug Morikraz (its concentration in the solution is not less than 2.5% or 2.5 g / l), which is fractionated by ion exchange chromatography. Fractionation is carried out at room temperature on a DEAE-Toyoperl sorbent in Tris-chloride buffer with a pH of 7.2-7.5. Proteinases elute in a concentration gradient of sodium chloride from 0.1 to 1-1.5 M.

Buffer replacement, desalination and drug concentration are carried out by ultrafiltration on a membrane that retains proteins with a molecular weight of at least 10 kDa.

To completely remove trypsin impurities, further purification of the metalloproteinase preparation from impurities of serine proteinases is carried out by gel chromatography on Sephadex G50 in 0.1 M ammonium acetate buffer with a pH of 6.3 - 6.5, containing 1.0 - 1.2 M sodium chloride.

Example.

On a 20 × 2 cm column with DEAE-Toyoperl equilibrated with Tris-HCl buffer at pH 7.5, 10 ml of Moricrase solution with a concentration of 25 mg / ml is applied. After washing the column with start buffer, elution is carried out in a NaCl concentration gradient of 0.1 - 1.0 M.

Fractions containing metalloproteinase are combined, concentrated by ultrafiltration on a cell with a membrane passing peptides with a molecular weight of less than 10 kDa.

The result is a purified metalloproteinase preparation with an activator content of about 84% (the activity of the drug for azocasein is 84% inhibited by an EDTA metalloproteinase inhibitor) containing a trace admixture of trypsin-like serine proteinase (Figure 1) Yield 50%, purification 2.5 times.

Figure 1 presents:

a) SDS-PAGE electrophoregram of the drug "Morikraz". On the right is a set of protein markers (BSA 66.2 kDa, ovalbumin 45 kDa, carbanhydrase 31 kDa, soybean trypsin inhibitor 21.5 kDa, lysozyme 14.4 kDa);

b) Kamchatka crab metalloproteinase preparation after ion exchange chromatography. On the left is a set of marker proteins (cellulase 94 kDa, BSA 66.2 kDa, ovalbumin 45 kDa, carbanhydrase 31 kDa, soybean trypsin inhibitor 21.5 kDa, lysozyme 14.4 kDa).

For further purification, the resulting preparation was applied to a Sephadex G50 column, equilibrated with 0.1 M ammonium acetate buffer with a pH of 6.4, containing 1 M NaCl.

The result is a metalloproteinase preparation with an activator content close to 100%, homogeneous according to the SDS page electrophoresis (FIG. 2), with a yield of 39% and a purification degree of 5.5 times.

Figure 2 presents the preparation of king crab metalloproteinases after ion exchange chromatography and gel chromatography. On the left is a set of marker proteins (cellulase 94 kDa, BSA 66.2 kDa, ovalbumin 45 kDa, carbanhydrase 31 kDa, soybean trypsin inhibitor 21.5 kDa, lysozyme 14.4 kDa).

The fibrinolytic activity of the drug and its ability to activate plasminogen is determined using standard fibrin plates prepared according to the following method: "Methods for the study of the blood fibrinolytic system." Ed. G.V. Andreenko. M.: Moscow State University, 1981. P. 43.

30 μl of human blood plasma or saline is applied to the surface of the fibrin plate, and then the drug solution is layered on top. Fibrin plates are incubated at a temperature of 37 ° C. The area of the lysis zone is defined as the product of two perpendicular diameters.

The activation of plasminogen by metalloproteinase is also investigated in vitro by incubating a solution containing plasminogen and metalloproteinase at a temperature of 37 ° C and a pH of 7.5 for 2 hours or more. Proteins from the solution precipitate TCA at a final concentration of 7-10% (w / v), wash the precipitate with acetone and dissolve in the sample buffer. Then carry out electrophoresis according to Laemmli [Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15; 227, (5259): 680-685] and stained with Coomassie Brilliant Blue gel to visualize protein bands.

The ability of the drug to lyse preformed human blood plasma clots is determined by the following method.

To 250 μl of human blood plasma add 50 μl of thrombin solution (2000 units per 0.2 g dry weight) with a concentration of 2 mg / ml and incubated for 5 minutes in a water bath at a temperature of 37 ° C.

To the resulting clot, add 500 μl of a solution of the enzyme preparation and incubated in a water bath at a temperature of 37 ° C until the clot is completely lysed. Saline is added to control tubes. Clot lysis is recorded visually.

Figure 3 presents the electrophoregram of the products of the interaction of plasminogen with metalloproteinase (incubation time - 2 hours). 1 - plasminogen without metalloproteinase, 2 - a set of protein markers (their molecular weights in kDa are indicated on the side), 3 - reaction mixture (MP - metalloproteinase).

The table shows the data characterizing the activator activity of the obtained product.

In the case of using the plasminogen activator obtained by the proposed method, complete lysis of plasma clots (which is equivalent to the complete restoration of the patency of a blood vessel) occurs within 4 hours 20 minutes under the influence of 500 μg of the enzyme. In the case of using the plasminogen activator obtained by the prototype method, the addition of 20 μg or more plasminogen activator to the plasma clot leads to incomplete lysis of the thrombus in 12 hours, and it is impossible to determine whether restoration of vascular patency occurs during incomplete lysis of the thrombus (since the prototype has a degree of lysis evaluated using the radiometric method, which does not allow us to establish whether the mechanical integrity of the thrombus is violated - that is, whether the patency of the vessels is restored).

Thus, with comparable activator activity of the obtained enzyme with the activity of the enzyme isolated in the prototype, the proposed method allows you to:

1) use more affordable raw materials (crab hepatopancreas is a by-product of the food industry, so our technique allows us to more fully utilize natural raw materials), and there is no need to cultivate cells (especially tumor cells),

2) reduce the cost of cleaning the drug (less expensive sorbents, fewer stages),

3) use drugs that are safe for the body.

Thus, the proposed method allows to obtain a new plasminogen activator from affordable, cheap and not containing oncogenic protein raw materials, to reduce the process time and reduce the cost of the preparation process.

Claims (1)

A method of obtaining a plasminogen activator from an enzyme-containing raw material, comprising fractionating it using ion exchange chromatography, characterized in that the Moricrase collagenolytic enzyme preparation is used as the enzyme-containing raw material, the latter is dissolved in Tris-chloride buffer with a pH of 7.2-7.5 to a concentration it in a solution of at least 2.5%, followed by fractionation of the solution on a DEAE-Toyoperl sorbent, elution of a proteinase in a concentration gradient of NaCl 0, l-1.5 M, concentration and desalination of the resulting solution of plasminogen activator by ultrafiltration on a membrane passing peptides with a molecular weight of less than 10 kDa and purification of plasminogen activator by gel chromatography on Sephadex G50 in 0.1 M ammonium acetate buffer with a pH of 6.3-6.5 containing 1.0- 1.2 M sodium chloride.

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