Plasminogen urokinase type modified activator, dna sequence, recombinant plasmid, strain-producer, method for preparing plasminogen urokinase type modified activator and pharmaceutical composition eliciting thrombolytic effect

FIELD: genetic and protein engineering, medicine, molecular biology, pharmacy.

SUBSTANCE: invention proposes a modified form of plasminogen urokinase type activator (activator) wherein amino acid sequence differs from that in the natural activator as result of replacing the sequence Arg-Arg-His-Arg-Gly-Gly-Ser in the composition of inhibitory loop for the sequence Arg-His-His-Ala-Gly-Gly-Ser and by replacing 24 N-terminal amino acids for the foreign sequence consisting of 16 amino acid residues. Invention proposes the constructed recombinant plasmid (pUABC 34) comprising DNA fragment that encodes a new activator. As result of transformation of E. coli K-12 JM109 cells with plasmid pUABC 34 the recombinant strain E. coli VKPM-8145 as a producer of a modified form of activator is obtained. This polypeptide is characterized by reduced sensitivity to effect of inhibitor PAI-1 and absence of some by-side effects in the complete retention of biological activity of the natural activator produced by the recombinant. This provides effective applying a new activator as a component of pharmaceutical compositions eliciting thrombolytic effect.

EFFECT: valuable medicinal properties of activator and pharmaceutical composition.

6 cl, 6 dwg, 8 ex

 

The invention relates to the field of biotechnology, genetic and protein engineering, i.e. to technology for proteins with modified natural or new properties.

More specifically, the present invention relates to a new modified plasminogen activator urokinase type (Maputo), which has improved thrombolytic properties.

The invention relates also to a DNA sequence which encodes a new Maputo, recombinant plasmids containing the coding Maputo DNA sequence, the Escherichia coli strain producer of the new Maputo, the way to get a new Maputo and to pharmaceutical compositions with thrombolytic effect, containing the new Maputo.

Natural and modified plasminogen activators (PAS) are used as thrombolytic agents for the treatment of diseases caused by blockage of blood vessels with fibrin, such as myocardial infarction, pulmonary embolism, venous thrombosis, thrombolytic disorders of the eye, in particular hemophthalmus, hyphema and other plasminogen Activators catalyze the conversion of plasminogen to plasmin, which, in turn, analyzes fibrin. One of the activators of plasminogen is urokinase.

It is known that the urokinase extracted from urine, presents three forms: low molecular weight (33 kDa), high molecular weight is (50-55 kDa) dvuhtsepochnyj and macromolecular odnozadachnoy (PUK), with a significant predominance of low molecular weight form. The greatest value for medicine are thrombolytic drugs on the basis of high-molecular-weight precursor (zymogen) urokinase - prourokinase, due to its low activity until it is activated by a blood clot. The PUK is synthesized in the cell ribosomes associated with the endoplasmic reticulum. Initially, the synthesis of polypeptide-predecessor (the so-called preproorexin), which in the process of co-translational secretion of cleaved signal peptide. Secretiruema of cells form from the derived signal peptide and is the PUK. The PUK enzymatic almost not active, and activates, like most serine proteases, by enzymatic gyrolite. Activators of prourokinase in the body are typically plasmin, kallickrein and cathepsin Century

The PUK predominantly activates fibrin-bound plasminogen, which has a different conformation compared with circulating plasminogen. Using prourokinase as a thrombolytic drug is particularly effective because of its extremely low activity before the activation of the immediate vicinity of the thrombus.

The PUK it is also called plasminogen activator urokinase type (AND THE UT).

The catalytic activity of prourokinase and the time of her life in the body are controlled by the interaction of prourokinase with activator inhibitor type I plasminogen PAI-1.

In the body, in addition to participating in the processes of thrombolysis, ABOUT performs other functions: participates in inflammatory processes and activates cell proliferation, thereby stimulating the healing process of damaged tissues and metastasis of tumors. Participation in these processes is mediated by the interaction with other proteins. Thus, the processes of proliferation are controlled by the interaction of the N-terminal domain of ABOUT with the cell receptor.

To use ABOUT as a thrombolytic drug, preferably while maintaining the level of biological activity to reduce the inhibition of PAI-1, which can be achieved by making changes to one of the binding sites of inhibitor, and reduce side effects associated with the interaction of ABUT with the receptor, which is achieved by modification of the amino-terminal segment ABOUT.

Known complete amino acid and nucleotide sequence of preproorexin (Holmes W.E. et al., Biotechnology 0: 923-929, 1985; SWISS-PROT: R).

Currently, the interaction of PAI with plasminogen activator urokinase type is well-studied process. Known three-dimensional structures of these proteins: ABOUT (Spraggont G.S et l., Structure 3: 681-688, 1995) and PAI-1 (J. Mottonen et al., Nature 355: 270-273, 1992). Formulated interaction ABOUT with the inhibitor PAI-1 (Y. Xue et al., Structure 6:627-636, 1998).

From the above findings indicate that the interaction between ABOUT and PAI is carried out in at least two areas: the active centre of ABOUT and the so-called inhibitory loop, representing a sequence with 198 204 amino acid natural ABOUT: ArgArgHisArgGlyGlySer and protruding from the globules of ABOUT close to the active centre. According to Spraggont G.S et al. (Structure 3: 681-688, 1995) this loop can form only 2 hydrogen bonds with the surrounding surface amino acids, which indicates low due inhibitory loop with the rest of APUC.

In view of the above, it seemed natural that the deletion of the inhibitory loop will reduce sensitivity to PAI, without affecting the styling and the enzymatic activity of ABOUT. However, in an attempt to obtain the modified ABUT with deletional loop (D.S. Adams, J Biol Chem 266:13 8476-8482,1991) has led to a sharp decrease in the output of the active ABOUT.

In U.S. patent No. 5728654 by analogy with data obtained on other serine of the protease - tissue plasminogen activator (tPA), theoretically predicted modifications in at least one of the provisions of the inhibitory loop ABOUT, which according to the authors of the patent can result in reduced sensitivity is lesti and even resistance to PAI-1 by reducing the positive charge in the field of inhibitory loop of the protease. However, the specific modified ABOUT in the patent is not received and is not described. Direct transfer of the results obtained for the tPA, the PUK is highly questionable, because it does not consider the effects of mutations on the whole spatial structure of the enzyme. tPA has a very unique structure of the active center, which distinguishes it from all serine proteases. And the available literature data suggest that the main binding site of PAI-1 is not inhibitory loop, and the active center of the enzymes. As a result, tPA differently interacts with inhibitors of the enzymatic activity.

Thus, significant differences in the structure of the active centers tPA and ABOUT and, as a consequence, differences in the degree of communication and the nature of the inhibitory interaction of the loop with the active center of the enzyme (i.e. differences in the influence area inhibitory loop to maintain the conformation of the active site) is not possible to theoretically predict the effect of mutations in the inhibitory loop ABOUT on its Association with PAI-1 and enzymatic (fibrinolytic and amylolyticus) activity.

Not to mention another important fact: proposed by the authors of U.S. patent No. 5728564 abrupt change of charges in inhibitory loop (this loop really is visibly out globules of APUC) can cause on the surface the displacement is new protein antigenic determinants and as a consequence, the development of immune reactions, especially in the case of repeated use. In addition, to reduce the sensitivity to PAI-1 should be very careful that the introduction of the drug did not cause hemorrhagic complications.

So, when receiving a pharmacologically acceptable modifications of natural APUT you must keep a fine balance between reduced sensitivity to the inhibitor PAI-1 and its ability to cause hemorrhagic and immunological complications and preservation of the functional activity of the enzyme, which can be achieved mainly by experimenting with subsequent correction of the ideas about the structure and function of such a complex protein molecule, which is ABOUT.

Previously the authors of this invention was obtained recombinant plasmid DNA pUABC22 encoding a modified plasminogen activator urokinase type (Maputo), and Escherichia coil - producer Maputo (RF Patent No. 2140453). In the patent are not given amino acid sequence Maputo and the nucleotide sequence of DNA, its coding. It is known that the Maputo encoded by this plasmid pUABC22, is a recombinant PUK, which differs from the natural prourokinase replacing the first 24 amino acids of the N-terminal domain with homology to epidermal rostovomu factor, 15 cogert the s amino acids. However, Maputo (hereinafter mapout), encoded by plasmid pUABC22 not contain modifications in the binding site with the inhibitor PAI-1.

The main objective of the present invention consisted in obtaining such a modified ABOUT, which would have reduced sensitivity to PAI-1, had no side effects while completely preserving the biological activity inherent in natural APUT, i.e. in General was characterized by a better than natural ABOUT, thrombolytic properties.

New modified plasminogen activator urokinase of the type claimed in accordance with the present invention, has improved thrombolytic svoistvami and has a deduced amino acid sequence of SEQ ID NO 1. In the sequence selected replaced compared to natural ABOUT amino acids: 2 amino acids inhibitory loops and 16 non-native amino acids N-terminal domain. This protein possesses fibrinolytic and amylolyticus activities inherent in natural APUT.

New modified APUC has a molecular mass of 43 kDa, identified by the method of polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate.

The decreased sensitivity of Maputo to the inhibitor PAI-1 is achieved at the expense of the produced amino acid substitutions in the binding site of the inhibitor activator is plasminogen I type PAI-1 ABOUT.

More specifically, the modification resulting in less sensitivity to PAI, is to replace the natural amino acid sequence inhibitory loop RRHRGGS (ArgArgHisArgGlyGlySer) sequence RHHAGGS (ArgHisHisAlaGlyGlySer).

This amino acid sequence corresponds to amino acids No. 178-184 numbering of amino acids natural human prourokinase, No. 198-204 in the case of its predecessor - preproorexin (Holmes W.E. et al., Biotechnology 0: 923-929, 1985; SWISS-PROT: P00749) and # 170-176 if the claimed sequence (SEQ ID NO 1). This replacement allows you to avoid violations of the protein folding while reducing sensitivity to PAI-1.

Due to these modifications achieves a higher enzymatic activity in the presence of PAI-1 than in the case of natural APUT. This allows you to achieve similar thrombolytic effect at lower doses of the drug. In addition, the new modified ABOUT may be particularly effective for patients with a high content of PAI-1 in blood.

New modified APUT contains additionally modified in comparison with the natural ABOUT the sequence of the amino-terminal domain with homology to epidermal rostovomu factor, namely, the 24 amino acid N-terminal domain, inducing cell proliferation, replaced by 15 alien amino acids.

Shadowedobject of the claimed group of inventions is the DNA sequence, which encodes a new modified ABOUT and has the nucleotide sequence SEQ ID NO 2.

In a given sequence of SEQ ID NO 2 selected nucleotides encoding the substituted amino acids. Selected codons can be replaced by any other similar codons determined replaced by the corresponding amino acids.

The DNA sequence encoding a new modified ABOUT receive using the method of site-directed mutagenesis.

Another object of the invention is recombinant plasmid DNA pUABC34 for the expression of new, modified ABOUT having the amino acid sequence of SEQ ID NO 1. Plasmid pUABC34 has a size 4539 P.N. and contains the following consistently connected structural elements:

the DNA fragment encoding the Maputo and having the nucleotide sequence of SEQ ID NO 2, containing 2 plots the recognition by the restriction enzyme NcoI, site recognition by the restriction enzyme NdeI and site recognition by restriction enzyme BelI;

- artificial intergenic sequence MGP gene and the tRNA Arg;

- gene resistance to ampicillin;

- part of the beginning of replication;

- lac-promoter.

Map of the plasmid is presented in figure 1. The card does not provide exhaustive information about the details of sequences, such as restriction sites present in the DNA, but the decree is installed on this figure the sites are sufficient for unambiguous detection of functional elements plasmids.

At the heart of plasmids pUABC34 is the technology of recombinant DNA, all methods which are well known to specialists in this field. For this BalI - BspEI fragment of plasmid pUABC22 size 4 TPN, containing the gene for resistance to the antibiotic, the plot started replication, gene arginine tRNA, artificial intergenic sequence IHL 14, and N - and C-terminal fragments of the coding region Maputo, and BspEI - BslI - fragment of plasmid pUABC22 size 0.33 KBP, representing a fragment of the coding region Maputo, not containing modifications of the sequence are ligated with a synthetic fragment "a", which represents the sequence encoding the modified binding site with PAI-1 (see figure 3). In turn, to obtain a synthetic fragment "And" synthesize 8 pairs of complementary oligonucleotides (see figure 2), phosphorylate them using phage polynucleotide kinase T4, pairs annealed and are ligated with the formation of the synthetic fragment size 150 BP

The next object of the claimed group of inventions is a strain of Escherichia coli producing Maputo. The strain obtained by transformation of cells of E. coli K-12 JM109 recombinant plasmid DNA pUABC34.

The strain is deposited in Russian national Collection of Industrial Microorganisms and has a collection number VKPM B-8145.

Another object of saleemhoussami invention is a method of obtaining new, modified ABOUT. How is that cultured cells of the producer strain of Escherichia coli VKPM B-8145 in conditions that ensure the expression of Maputo, and allot of them target product.

For this purpose, cells of the strain E. coli VKPM B-8145 containing plasmid pUABC34, grown to a density OD600=2.5 in LB-medium with ampicillin at 37°and intensive aeration, collected by centrifugation, treated with lysozyme and destroy with the help of ultrasound. Sedimentary fraction of the cell lysate was dissolved in denaturing conditions, and then spend reconstitute enzymatic activity Maputo.

Another object of the claimed group of inventions is a pharmaceutical composition having thrombolytic activity. The pharmaceutical composition includes a new Maputo as an active ingredient and a pharmaceutically acceptable carrier.

The pharmaceutical compositions can be obtained well-known in the art methods.

Pharmaceutical compositions based on the new Maputo can be used as a drug in any dosage form, mostly in the form of parenteral preparations such as solutions for injection.

In the preparation of pharmaceutical compositions in the form of solution for injection as carriers can be used: RAS is Writely, such as water, ethyl alcohol, macrogel (polyglycol), propylene glycol, ethoxylated isostearoyl alcohol, polioksidony isostearoyl alcohol, polyoxyethylenesorbitan ether fatty acids, polisher, such as mannitol, dextran and the like; pH regulators and buffers such as sodium citrate, sodium acetate or sodium phosphate, stabilizers such as sodium pyrosulfite, glutathione, etilendiamintetrauksusnoy acid, thioglycolate acid or tomalachka acid, etc. In this case, the drug may contain sodium chloride, glucose or glycerin in an amount sufficient to obtain an isotonic solution. These carriers can be added to obtain solutions for subcutaneous, intramuscular and intravenous injection method known in this technical field.

The number of Maputo the present invention, which shall contain the above-described pharmaceutical composition, varies according to the type of composition, method of administration, the dosing scheme and therefore cannot be precisely defined, and is selected in accordance with the need of a certain range. The composition may preferably contain about 0.1-10 wt.% Maputo by weight of the composition.

For a better understanding of the present invention, the description is accompanied by the following illustrations is:

Figure 1 presents a map of the recombinant plasmid DNA pUABC34 containing basic functional elements of the plasmid and the typical area of recognition by restrictase. The promoter of the lac-operon, designated as "P"; Maputo means the coding region of Maputo; ArR- gene resistance to ampicillin, ori - plot start replication; gene arginine tRNA (.li) labeled tRNA, artificial intergenic sequence MGP designated as IHL. To bind the location of functional elements to physical map (sequence) shows the plots of recognition by restrictase: NcoI, NdeI, Cloned, SacI and ScaI.

Figure 2 presents the structure of the oligonucleotides In1-In8, which have a nucleotide sequence of SEQ ID NO 3-10 and applied in the synthesis of artificial fragment "And" codesusa area of Maputo. Direction sequence specify the 3'- and 5'-ends; for clarity, the oligonucleotides are arranged in such a way that they are part of the synthetic fragment codesusa region Maputo; indicate which of the oligonucleotides are phosphorylated; illustrious Assembly of the two chains. Directly below the sequences of the oligonucleotides indicated coded amino acid sequence. Amino acids, non-natural, are underlined.

Figure 3 illustrates the construction of recombinant who lamidi pUABC34, coding Maputo. Letters a, b and C labeled DNA fragments, from which a plasmid pUABC34. A fragment of A synthetic fragment of the coding region Maputo size 0.15 TPN Fragment "B" is BalI - BspEI fragment of plasmid pUABC22 size 4 TPN. It contains gene stability to the antibiotic, the plot started replication, gene arginine tRNA, artificial intergenic sequence MGP, as well as N - and C-terminal fragments of the coding region Maputo. The fragment is a BspEI - BelI - fragment of plasmid pUABC22 size 0.33 TPN It contains a fragment of the coding region Maputo, does not contain modifications.

Figure 4 illustrates the calibration curve fibrinolytic test and the determination of the activity of Maputo. The horizontal axis represents the activity of the standard urokinase in IU/ml When the building was used points 12.5, 25, 50 and 100 IU/ml On the vertical axis and the diameter of the zone of lysis in mm Calibration points are marked square icons. Points used in the measurement of experimental samples Maputo, indicated by triangles.

Figure 5 illustrates the results amiloliticescoe test. The horizontal axis represents the response time in seconds. The vertical axis represents the optical density of the reaction mixture after stopping the reaction. The optical density of areeda at 405 nm. Value OD=0.1 corresponds to 0.28 mm concentration of hydrolyzed chromogenic substrate S2444.

Kinetic curves 3, 4 and 5 were obtained for concentrations of standard 625, 1250 and 2500 IU/ml, respectively. Triangular icons marked points used in the measurement of experimental samples of Maputo. Kinetic curves 1 and 2 were measured for concentrations of Maputo 20 and 10 µg/ml, respectively. All the icons correspond to the values of the background generated by plasmin (curve 6).

Figure 6 shows the results amiloliticescoe test in the presence of inhibitor PAI-1. The curves illustrate the effect of PAI-1 on the claimed modified APUT (mapout)containing modified amino acid sequences in the binding site of the inhibitor, and the well-known modified APUT (mapout, RF Patent №2140453), in which this modification is absent. The horizontal axis represents the response time in seconds. On the vertical axis measures the optical density of the reaction mixture after stopping the reaction at 405 nm. Value OD=0.1 corresponds to 0.28 mm concentration of hydrolyzed chromogenic substrate S2444.

Curve 1 - mapout without PAI; curve 2 - mapout without PAI, curve 3 - molar ratio mapout/RU=1:1; curve 4 - molar ratio mapout/RU=1:1; curve 5 - molar ratio mapout/RA=1:2, curve 6 - molar aspect] is the solution mapout/RA=1:2.

The sequence listing of nucleotides and amino acids that characterize the present invention, includes the following sequence:

SEQ ID NO 1 is the complete deduced amino acid sequence of new, modified ABOUT. In the sequence selected replaced compared to natural ABOUT amino acids.

SEQ ID NO 2 is a fragment of DNA encoding the new Maputo. Selected codons can be replaced by any similar, corresponding to the replaced amino acid.

SEQ ID NO 3, SEQ ID NO 10 are sequences of oligonucleotides In1-In10, respectively, used in the preparation of synthetic fragment "And" coding region Maputo.

Below are examples of specific implementation of the present invention the following examples. However, it should be borne in mind that these examples illustrate, but not limit the present invention.

Example 1. The synthetic fragment.

3 shows the Assembly of artificial fragment of the coding region of the oligonucleotide which is defined as a synthetic fragment "And". It replaces the natural BalI-BclI fragment. Synthetic oligonucleotides In1-In8 synthesize phosphoramidite method. Use of a nucleotide sequence designated as SEQ ID NO 3, SEQ ID NO 10 Lane is one sequences and are presented in figure 2.

5×10-10mol of each of the oligonucleotide was dissolved in 200 µg buffer solution composition: Tris-Hcl 25 mm, pH 7.5; MgCl210 mm; ATP, 0.5 mm; EDTA 0.1 mm. The oligonucleotides In2-In7 add polynucleotides phage T4 at a concentration of 0.1 unit/ál. Phosphorylation is continued for 1 hour at 37°C. Further polynucleotides inactivate at 90°C for 20 sec.

Phosphorylated oligonucleotides unite in pairs: In3 with In4, In5 with In6. To the phosphorylated oligonucleotide In2 add nefosfaurilirovanna oligonucleotide In1 (5·10-10mol), phosphorylated oligonucleotides bound In7 add nefosfaurilirovanna oligonucleotide In8 (5·10-10mol). Combined in pairs of oligonucleotides is heated at 90°C for 10 seconds, and then slowly (2 hours or more) is cooled to +20°in a metal container. Four received stitched pairs of the oligonucleotide is mixed, add DNA ligase of phage T4 (up to 10 m/ml), incubated for 20 hours at +15°and then inactivate the DNA ligase 60°for 20 seconds.

The reaction products are separated in a 5% polyacrylamide gel. After bromide staining with ethidium cut out from the gel product with a length of 150 BP and extracted it into a buffer solution (Tris-Hcl 20 mm, pH 7.5, 20 mm NaCl; 0.1 mm EDTA) by passive diffusion for 60 hours at 50°C. the Product (synthetic artificial fragment "is", encoding the modified binding site with PAI-1) concentrate butanol-1 to volume of from 100 to 400 µl, and then planted by adding 3 volumes of ethanol. The product is dissolved in a buffer solution of composition: Tris-Hcl 20 mm, pH 7.5; 0.1 mm EDTA and its concentration determined spectrophotometrically.

Example 2. The preparation of recombinant plasmid DNA pUABC34.

1. 10 μg of plasmid pUABC22 (RF Patent No. 2140453) hydrolyzing 20% restricts BalI and BspEI in the buffer of the following composition: Tris-Hcl 25 mm, pH 7.5; MgCl210 mm; 50 mm NaCl; 0.1 mm EDTA. The products of hydrolysis are separated in a 0.8% low-melting agarose (Clontech Lab. Inc.). After bromide staining with ethidium identify the fragment size 4 TPN, a piece cut out of the gel, subjected to incubation at 70°C for 15 minutes, remove the agarose from the sample by extraction with an aqueous solution of phenol and impurities phenol is removed by extraction with butanol-1.

The resulting product are planted by adding 3 volumes of ethanol and dissolved in a buffer solution: Tris-Hcl 20 mm, pH 7.5; 0.1 mm EDTA. Figure 3 fragment 4 TPN labeled "In".

2. 10 μg of plasmid pUABC22 hydrolyzing 20% restricts BelI and BspEI in the buffer of the following composition: Tris-Hcl 25 mm, pH 7.5; MgCl210 mm; 50 mm NaCl; 0.1 mm EDTA. The products of hydrolysis are separated in a 1.2% low-melting agarose (Clontech Lab. Inc.). After bromide staining with ethidium identify the product length 0.33 TPN, a piece cut out of the gel is; remove the agarose from the sample by extraction of aqueous phenol and impurities phenol is removed by extraction with butanol-1. The resulting product are planted by adding 3 volumes of ethanol. The resulting fragment was dissolved in buffer: Tris-Hcl 20 mm, pH 7.5; 0.1 mm EDTA. Figure 1 is a fragment 0.33 TPN designated as "C".

3. Both selection mixed with each other and a synthetic fragment "And"add MgCl2up to concentrations of 10 mm ATP to 0.5 mm and the DNA ligase of phage T4 - up to 10 units/ml After 20-hour incubation at +15°the resulting mixture was transformed into cells of E. coli K-12 JM109. For this purpose, cells of E. coli K-12 JM109 grown at 37°C in LB medium until reaching an optical density of 0.3 (at 600 nm). Cells are harvested by centrifugation and resuspending buffer (STOs3Soo 20 mm, KCl 100 mm, l210 mm, nl210 mm, glycerol 10%, pH 5.5) at 0°With the amount to 1/2 the volume of the original culture. Cells are again collected by centrifugation and resuspended at 0°in the same buffer amount to 1/20 of the original volume of the culture.

To 0.2 ml of the cell suspension add ligase mixture. After incubation for 40 min at 0°and subsequent incubation at 42°C for 2 min to cells add 1 ml of LB medium. Cells are incubated at 37°C for 40 min and plated on plates with ampicillin (0.1 mg/ml). From the obtained clones using restrict the th assay, clones, containing plasmids with 2 plots of recognition by the restriction enzyme NcoI and 2 plots of recognition by the restriction enzyme NdeI. The nucleotide sequence of one of the selected clones was confirmed by the method of Sanger and plasmid with confirmed nucleotide sequence designated as pUABC34.

Example 3. Getting a producer strain Maputo.

Cells of Escherichia coli JM109 transformed with the plasmid pUABC34 by the method of example 2 and get producing strains modified plasminogen activator urokinase type. The strain is deposited in Russian national Collection of Industrial Microorganisms and has a collection number VKPM B-8145.

Example 4. The preparation of recombinant modified plasminogen activator urokinase type.

Bacteria strain E. coli VKPM B-8145 containing plasmid pUABC34, grown to a density OD600=2.5 V for 16 h in 50 ml LB-medium with ampicillin (100 μg/ml) at 37°and intensive aeration. After cultivation, the cells are harvested by centrifugation for 30 min at 4000 rpm and suspended in 2.5 ml of buffer solution (Tris-Hcl 20 mm, pH 6.7, 30 mm NaCl), add 2.5 mg of lysozyme and incubated for 10 min at 4°C. To the resulting mixture are added Tris-HCl, pH 8.3, 50 mm EDTA to 20 mm, incubated for 30 min at 4°C, then treated with ultrasound for 1 minute, Sedimentary fraction of the cell extract is separated by centrifuge the management within 90 min at 5000 rpm, suspended in 1 ml of 2%Triton X-100 and centrifuged 5 min at 14000 rpm Residue suspended in 100 μl of a solution containing 200 mm EDTA and 500 mm Tris-HCl, pH 8.0, add 800 μl of denaturing solution (6 M guanidinium, 100 mm 2-mercaptoethanol), incubated for 20 min at 65°insoluble precipitate is removed by centrifugation.

The procedure of reconstitute enzymatic activity carried out as follows. The solution was diluted 20-fold with buffer: 100 mm Tris-HlCl, pH 9.0, 200 mm arginine-chloride, 5 mm restored glutathione, 1 mm oxidized glutathione, denaturing agent (1 M guanidinate) and incubated for 40 h at 4°C. the resulting mixture was subjected to dialysis against 1 l of a solution containing 100 mm NaCl, 20 mm Tris-HCl, pH 8.0, within 6 hours of Precipitated precipitate is removed by centrifugation and measured activity in the test for fibrinolysis.

Denaturirovannyj recombinant modified plasminogen activator presents mainly preferments form with a molecular mass of 43 kDa, which is confirmed by electrophoretic data and sonograficheskih analyses. Active Maputo is about 200000 units per 1 g of biomass.

Was investigated the specific activity of the new, modified ABOUT in comparison with natural ABOUT by determination of fibrinolytic activity by lysis of fibrin and determination amid the lytic activity after activation by plasmin. In addition, it was investigated the effect of the inhibitor PAI-1 in Maputo test amylolyticus activity. During the research as the standard used high molecular weight urokinase company Calbiochem with an activity of 100,000 IU/mg.

Example 5. Determination of fibrinolytic activity.

Determination of fibrinolytic activity carried out by the method (Gaffney, P.J. et al., Thrombos and Heamostas 45: 34-37, 1981). The Agarose (Clontech Lab. Inc.) diluted with water to obtain 1.2% of the suspension; the suspension is boiled to dissolve the agarose and cooled to a temperature of 37°C.

Fibrinogen (Sigma F 8630) dissolved in phosphate buffer (20 mm sodium phosphate, pH 7.5, 300 mm NaCl, 0.1% Triton X-10, 5% glycerol) to a concentration of 10 mg/ml at 37°C. Add cacodylate sodium (pH 7.4) to a concentration of 5 mm and thrombin (Sigma, T 3399) to a concentration of 0.08 u/ml Solutions of fibrinogen and agarose is mixed and poured into Petri dishes. After polymerization at room temperature, the Cup can be used or stored at +4°C.

Prepare a 0.2% solution of bovine serum albumin (BSA) in phosphate buffer: 10 mm phosphate, 150 mm NaCI, pH 7.4. The sample standard high molecular weight urokinase diluted in 0.2% solution of BSA in such a way as to obtain cultivation from 12.5 to 100 IU/ml Then prepare solutions Maputo with a concentration of 20 and 40 mg/ml

2 ml of each dilution of the standard high is the molecular urokinase (12.5, 25, 50 and 100 IU/ml) and the samples applied to the surface of fibrin gel. After 10 hours incubation at 30°measure the diameter of the zones Lissa for samples of standard and Maputo. The standard used to construct a calibration curve, which then determine the activity of Maputo. Square icons in figure 4 illustrate the calibration curve. Triangular icons marked points used in the measurement of experimental samples Maputo.

As follows from the results of fibrinolytic test activity Maputo is not different from the activity of the natural high molecular weight urokinase and is ~105IU/mg.

Example 6. Definition amylolyticus activity.

The sample standard high molecular weight urokinase dissolved in 0.2% solution of BSA in phosphate buffer (10 mm sodium phosphate, 150 mm NaCI, pH of 7.4) to a concentration of 5000 IU/ml of the Plasmin (Serva, P-32965) dissolved in phosphate buffer to a concentration of 1 u/ml 100 ál of the standard solution of urokinase incubated with 1 μl of plasmin solution for 1.0 to 1.5 hours at 37°C.

Prepare samples of Maputo in 0.2% solution of BSA in phosphate buffer with a concentration of 10 and 20 ág/ml 100 ál of solution samples Maputo incubated with 1 μl of plasmin solution for 1.0 to 1.5 hours at 37°C.

Prepare serial dilutions of the standard of high molecular weight urokinase, Akti is a new plasmin, using a 0.2% solution of BSA in phosphate buffer. Get solutions with a concentration of standard 625, 1250 and 2500 IU/ml

As a control, use a solution of 1 μl of plasmin solution in 0.2% solution of BSA in phosphate buffer.

Amylolyticus activity is estimated by the rate of hydrolysis of a chromogenic substrate S2444 (L-pyroglutamyl-glycyl-L-arginine-4-nitroanilide hydrochloride, Serva B-52338) in the presence of added enzymes. To 100 μl of the dilutions of the activated urokinase, control solution and test samples Maputo add 10 ál of 10 mm aqueous solution of a chromogenic substrate S2444 and incubated at 37°C for 30 minutes the Reaction is stopped by adding 500 μl of a solution of 10% acetic acid, the solution is stirred and measure the optical density at a wavelength of 405 nm. The results amiloliticescoe test are presented in figure 5. Kinetic curves 3, 4 and 5 were obtained for concentrations of standard 625, 1250 and 2500 IU/ml, respectively. Triangular icons marked points used in the measurement of experimental samples of Maputo. Kinetic curves 1 and 2 were measured for concentrations of Maputo 20 and 10 µg/ml, respectively. All the icons correspond to the values of the background generated by plasmin (curve 6).

The slope of the initial (linear) plot curves conclude speed gyrolite chromogenic substrate. The velocity of the hydrolysis, related to the concentration of the enzyme, characterized by the specific activity of the enzyme.

As follows from the results amiloliticescoe test activity Maputo is not different from the activity of the natural high molecular weight urokinase and is ~105IU/mg.

Example 7. To determine the sensitivity of Maputo to the inhibition of PAI-1.

The effect of the inhibitor PAI-1 on the activity of the new Maputo (mapout) determine in amiloliticheskoi test. For comparison, use a known mapout (RF Patent No. 2140453), from which the claimed Maputo characterized by the presence of modification of amino acid sequences in the binding site of the inhibitor PAI-1.

Prepare samples mapout and mapout 0.2% solution of BSA in phosphate buffer (10 mm sodium phosphate, 150 mm NaCl, pH 7,4) with a concentration of 20 µg/ml

The plasmin (Serva, P-32965) dissolved in phosphate buffer to a concentration of 1 unit/ml 100 ál of solution samples Maputo incubated with 1 μl of plasmin solution for 1.0 to 1.5 hours at 37°C.

As a control, use a solution of 1 μl of plasmin solution in 0.2% solution of BSA in phosphate buffer.

The inhibitor PAI-1 (Calbiochem) dissolved in 0.1% solution of BSA in phosphate buffer (Na phosphate 10 mm, NaCl 150 mm, pH 7.2-7.5) to a concentration of 500 µg/ml

To an aliquot of 10 µl of 10 mm aqueous solution of a chromogenic substrate S2444 add 0, 4, and 8 μl of a solution of PAI-1, which allows then to create a ratio IU the DN Maputo and PAI, 0, 1:1 and 1:2.

To the resulting dilutions PAI-1 in a solution of a chromogenic substrate S2444 add 100 μl of control solution or solutions of the test samples mapout and mapout and incubated at 37°C for 1, 2, 3, 4, 5, 6, 10 and 30 minutes the Reaction is stopped by adding 100 μl of a solution of 10% acetic acid, the solution is stirred and measure the optical density at a wavelength of 405 nm.

Comparative activity of new and known Maputo in the absence and in the presence of PAI-1 assess the nature of the kinetic curves presented on Fig.6. Curve 1 was obtained for mapout without PAI; curve 2 - for mapout without PAI. Curve 3 was obtained at a molar ratio of Maut and PAI equal to 1:1; and curve 5 is at a ratio of 1:2. Curve 4 is obtained at a molar ratio of Maut and PAI equal to 1:1; and curve 6 is at a ratio of 1:2.

Because in the course of the reaction is not only the hydrolysis of a chromogenic substrate, but also the gradual inactivation of enzymes, due to their interaction with PAI, and the reaction rate varies not only due to changes in substrate concentration, but also due to the gradual inactivation of the enzyme. This makes inadequate description of the system only the initial reaction rate, but requires a description of the change rate in time.

As follows from the data of Fig.6, in the absence of PAI integrated effect of the reaction on the initial the section of the curve, and the yield of the reaction plateau is identical for both enzymes mapout and mapout. When conducting the reaction in the presence of PAI activity as mapout and mapout decreases, but in significantly different ways.

So when the ratio mapout/RA equal to 1:1, the activity within the first 3 minutes is reduced 2.5 times, and after 30 min 2.6 times compared with the same quantities in the absence of PAI. The decrease in the activity of Maut in a similar situation occurs, respectively, only 1.3 and 1.2 times.

And when the ratio mapout/RA equal to 1:2, activity mapout within the first 3 minutes of reaction decreases to 3.8 times, and for 30 min at 4.3 times compared with the same quantities in the absence of PAI. The decrease in the activity of Maut in a similar situation occurs, respectively, only 1.8 and 1.6 times.

It should be noted that the curves for mapout in the presence of PAI (curves 3 and 5, 6) facing the saturation already at the 6th minute of the reaction at a low level, which indicates almost complete inactivation mapout by this time. When this curves reactions in the absence of PAI (curves 1 and 2) and curves reactions mapout with PAI (curves 4 and 6) to this time had not yet saturated, which indicates the presence in the reaction mixture of the active enzyme.

Example 8. Dosage form for injection.

For the preparation of dosage forms for injection using the following composition: modificirowan the th ABOUT - 23,5 mg of sodium chloride 180 mg

Modified APUT dissolved in water for injection, the solution is subjected to sterilization using a sterilizing filter Millipore" with the size of the pores is 0.22 mm Sterile solution is poured into 5 ml vials, frozen at a temperature of -46°C for 4 hours, and then carry out the freeze-dried at -40°With gradual increase of temperature up to 20°C for 48 hours.

The sequence listing is provided at the end of the description.

1. Modified plasminogen activator urokinase type (Maputo) with improved thrombolytic properties, having deduced amino acid sequence of SEQ ID No. 1.

2. A DNA fragment encoding a modified plasminogen activator urokinase type according to claim 1 and having the nucleotide sequence SEQ ID No. 2 or equivalent sequence due to the degeneracy of the genetic code.

3. Recombinant plasmid DNA pUABC34 for expression of a modified plasminogen activator urokinase type according to claim 1, having a size 4539 P.N. and contains the following consistently connected structural elements:

the DNA fragment encoding the Maputo and having the nucleotide sequence SEQ ID No. 2, containing 2 plots the recognition by the restriction enzyme NcoI, site recognition by the restriction enzyme NdeI and site recognition by restriction enzyme BclI;

artificial intergenic sequence MGP gene and the tRNA Arg;

the gene of resistance to ampicillin;

plot start replication;

lac-promoter.

4. The bacterial strain Escherichia coli VKPM B-8145 - producer of a modified plasminogen activator urokinase type.

5. A method of obtaining a modified plasminogen activator urokinase type according to claim 1, which consists in the fact that the cultured cells of the producer strain E. coli VKPM B-8145 in conditions that ensure the expression of Maputo, and allot of them target product.

6. Pharmaceutical composition having thrombolytic activity, including derived plasminogen activator urokinase type as the active ingredient and pharmaceutically acceptable carrier, characterized in that the active substance it contains a modified plasminogen activator urokinase type according to claim 1 in a therapeutically effective dose.



 

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