RU2495123C2 - METHOD TO PRODUCE MODIFIED FORM OF TRYPTOPHANYL-tRNA-SYNTHETASE - Google Patents

Abstract

FIELD: biotechnologies.

SUBSTANCE: animal pancreas tissues are ground and extracted. The extract is separated from cake. Extrinsic nucleic acids are deposited and removed with the help of streptomycin sulfate. Admixtures are repeatedly deposited from supernatant by salting out with ammonium sulfate. Stagewise fractionating of the extract is carried out. At the first stage of fractionating the method of ion-exchange chromatography is used with application of DEAE-cellulose DE-52. Stepwise elution is carried out with a buffer solution containing 25 mM of tris-HCl at pH 7.5, 1mM of EDTA, 0.1 mM of 2-mercaptoethanol and stabiliser of modified form of TRNA-synthetase in the form of a complex of substrates of 0.2 mM of L-tryptophan and 1.0 mM of Mg-ATP. Elution is carried out until optical density value is achieved as not more than 0.1 p.u./ml at 280 nm. Extrinsic proteins are removed with a buffer solution containing 0.09M NH₄Cl. The cleaned fraction is washed with a buffer solution containing 0.16M NH₄Cl. The produced mixture is deposited with ammonium sulfate until 60% of saturation. The residue is centrifuged and dissolved with the buffer solution containing 0.1M NH₄Cl. A protease inhibitor is added to the solution as 1 mM of phenyl methyl sulphonyl fluoride (PMSF) or 5 mM of disopropylfluorophosphate (DFP). The second stage of fractionating is carried out by the method of ion-exchange chromatography using DEAE-cellulose DE-52 with stepwise elution using a linear gradient of ammonium chloride salt 0.01 -0.06M. Fractions are extracted with the modified form of tryptophanyl-TRNA-synthetase (TRNA synthetase). The target product is concentrated by salting out with ammonium sulfate until 100% of saturation.

EFFECT: invention makes it possible to increase yield of a modified form of tryptophanyl-TRNA-synthetase.

2 cl, 2 dwg, 1 tbl, 10 ex

Description

The invention relates to medicine and relates to the production of biologically active substances from the organs of slaughtered animals used for the production of diagnosticums and medicines.

Tryptophanyl tRNA synthetase (TRSase) [1] is one of the most important enzymes catalyzing the specific aminoacylation of the tRNA messenger, one of the critical stages of protein synthesis, with the tryptophan amino acid. The enzyme carries out one of the most important metabolic processes - the reaction of specific aminoacylation of tRNA. Enzymes of this class of eukaryotic origin consist of a central “nuclear” part, similar in primary and secondary structures to similar enzymes from prokaryotes, and an additional domain attached to the “core” from the N-terminus or C-terminus. Most likely, additional noncanonical regulatory functions of eukaryotic enzymes were “acquired” during evolution through modification of the enzyme by controlled addition or removal of the above domains. Mammalian tyrosyl tRNA synthetase (TyrRSase), for example, contains an additional domain with cytokine function that is not found in TyrRSases of lower development organisms such as Caenorhabditis elegans or yeast [2]. This domain stimulates the chemotaxis of mononuclear phagocytes and the production of tumor necrosis factor alpha, as well as the endothelial monocyte activating polypeptide II (EMAP II) [2]. The data presented demonstrate a close relationship of protein synthesis processes with signaling functions.

The mammalian TRSase, a close homologue of TyrRSase, has been found to be actively involved in the organization of intercellular signaling routes [3]. For mammalian cells and tissues, including humans, the presence of two forms of TRSase differing in molecular weight was revealed: the original native TRPCase (471aa) and mini-TRPCase (424aa), which were believed to arise by alternative splicing of mRNA [4]. In addition, an important fact has been established of the strong induction of the synthesis of TPCase and modified mini-TPCase under the action of the antiproliferative interferon-γ cytokine [5]. The great importance of these effects began to become clear after the demonstration of the presence of a completely new, not directly related to the main initial function of TRSase, namely, the presence of mini-TRSase potent inhibitory pathological angiogenesis activity in various in vitro and in vivo experiments. For example, mini-TRSase blocked the migration of human umbilical vein endothelial cells caused by endothelial growth factors. In addition, mini-TRSase blocked angiogenesis stimulated by endothelial growth factors in tests on adhesive molecules of chicken cells and in vivo mouse matrigel test [5]. However, despite the presence of such powerful activities in the modified form, the full-sized TRSase did not have cytokine activities [4, 5]. The modified TRSase also had a strong antiangiogenic effect when the drug was exposed to the retina of a neonatal mouse at the site of localization of blood vessels. All these data allow us to draw a clear conclusion that the endothelial cells are the direct target of mini-TRSases in the tissues of the tested organisms.

The central role of the catalyzed reaction is explained by the fact that all the accuracy of protein synthesis and the accuracy of the implementation of the genetic code, which allows reproducing all the species diversity in the offspring on the earth, is determined precisely at this stage of protein synthesis. In the future, all the processes of "assembly" of protein molecules from amino acids are carried out almost automatically.

The indicated properties of TRSases make the enzyme an important object of research and development with the prospect of practical application in medicine of a modified form of the enzyme.

A method of obtaining a modified form of TRSase is described, for example, in Methods Enzymol. 1979; 59: 234-57, Kisselev L.L. et al., Tryptophanyl-tRNA synthetase from beef pancreas.

There is also known a method of obtaining a modified form of TRSase, which can be indicated as a prototype described in Eur. J. Biochem., (1976), 61, 139-146. Epely S., Gros C., Labouesse J., and Lemaire G. Limited Proteolysis of Tryptophanyl-tRNA Synthetase from Beef Pancreas. The method includes a substantial modification of the basic basic enzyme isolation technique, which consists in eliminating the protease inhibitors at the extraction stage and introducing an additional stage of ion exchange chromatography with the simultaneous exclusion of the chromatography step on Sephadex G-100 and the presence of elevated substrate concentrations (L-tryptophan 2 × 10 at all stages of fractionation) ^-4 M, and Mg-ATP (1 × 10 ^-3 M)). After carrying out the extraction procedures and stepwise salt fractionation with ammonium sulfate (40% saturation and 60% saturation), the mixture was applied to a DE-52 ion exchange resin column and the fractions were washed off with a stepwise gradient of ammonium chloride salt (0.09 M; 0.16 M; 0 , 2 M) in buffer A (25 mM Tris-HCl, pH 7.5, 1 mM EDTA, 0.1 mM 2-mercaptoethanol, 0.2 mM L-tryptophan, and 1.0 mM Mg-ATP) (FIG. .one). The fraction taken at 0.14 M salt was subjected to another separation cycle with a linear salt gradient of 0.1-0.16 M ammonium chloride in buffer A. The yield was 0.002% -0.003%, i.e., 40-45 mg of the modified enzyme was obtained with a purity of 99% according to denaturing protein polyacrylamide gel electrophoresis (PAGE) (FIG. 2).

In the second approach, the enzyme already purified by the standard procedure was subjected to treatment with activated charcoal (5% aqueous suspension in buffer A, without L-tryptophan and ATP) to remove endogenous tryptophan, while the enzyme passed into a modified form under the influence of residual impurities of endogenous proteases into during the day at 8 ° C.

A mini-method for producing modified TRSase included the application of the preparation of the starting TRSase with a concentration of 15 mg / ml in 20 mM Tris-HCl, pH 7.5, and a volume of 0.3 ml per carbon filter. An activated carbon filter was incubated at 4 ° C for 30 min and washed with 1 ml of buffer A without L-tryptophan and ATP, then the diluted solution was incubated in the refrigerator at 8 ° C overnight. The original TRSase was taken in an amount of 5 mg. After the procedure, 4.5 mg was obtained - 90% yield.

A modified enzyme was also obtained by limited proteolysis of the original enzyme with elastase. To do this, the initial enzyme TRSase at a concentration of 2.5 mg / ml, pre-treated with activated carbon to remove endogenous tryptophan (see above), was incubated with low concentrations of elastase (0.12 mg / ml in Tris-HCl buffer, pH 7.5 ) for 20 min at 25 ° C in the presence of 0.1 mm EDTA, 1 mm 2-mercaptoethanol and substrates - 0.2 mm L-tryptophan, 5.0 mm ATP. The reaction was stopped by the addition of a protease inhibitor of 1 mM PMSF or 5 mM diisopropyl fluorophosphate (DFF). In the case of DPP, the addition was carried out with glasses and gloves under the draft and left under the draft at room temperature, with stirring on a rocking chair to completely destroy the DPF, which quickly decomposes in an aqueous medium.

Control of the formation of the modified form was carried out using denaturing electrophoresis in SDS page. 5 mg of the initial preparation of TRSase was taken for the experiment; after the limited proteolysis procedure in the presence of substrates, 4.0 mg was obtained, that is, the yield was 80%, the purity of the preparation was 98%.

The functional activity of the biologically active modified form was evaluated according to the standard MTT test (Lee WS, Harder JA, Yoshizumi M. et al. Progesterone inhibits arterial smooth cell proliferation // Nat.Med. - 1997. - No. 3. - P. 1005-1008. )

The present invention is to obtain a biologically active modified form of TRSase.

The problem is solved in an improved way, which consists in the following.

Prepare raw materials - the pancreas by removing particles of fat. After extraction and precipitation of the cake, impurities of nucleic acids with streptomycin sulfate are removed. The method consists in the non-use of protease inhibitors at the extraction stage, the use of two successive stages of ion-exchange chromatography, and the presence of elevated substrate concentrations (0.2 mM L-tryptophan and 1.0 mM Mg-ATP) at all stages of fractionation. Stepwise salt fractionation procedures are carried out with ammonium sulfate (40% saturation and 60% saturation), then the precipitate at 60% saturation was dissolved in the application buffer (buffer A containing 0.05 M NH ₄ Cl), the mixture was applied to a column with an ion exchange resin DE-52 and washed off the fractions with a stepwise gradient of ammonium chloride salts (0.09 M; 0.14 M; 0.2 M) in buffer A (Fig. 1), the fraction taken at 0.14 M salt was subjected to another cycle separation with a linear salt gradient of 0.1-0.16 M ammonium chloride, previously diluting the selected fractions to a concentration of 0.1 M NH ₄ Cl (FIG. 2). Control of the formation of the modified form was carried out using denaturing electrophoresis in SDS page. The yield was 0.002% - 0.003%, that is, 40-45 mg of a modified enzyme with a purity of 99% was obtained according to denaturing protein polyacrylamide gel electrophoresis (PAGE).

The functional activity of the biologically active modified form was evaluated according to the standard MTT test (Lee WS, Harder JA, Yoshizumi M. et al. Progesterone inhibits arterial smooth cell proliferation // Nat.Med. - 1997. - No. 3. - P. 1005-1008. )

In the second method, the enzyme already purified by the standard procedure was subjected to treatment with activated charcoal (5% aqueous suspension in buffer A, without L-tryptophan and ATP) to remove endogenous tryptophan, while the enzyme passed into a modified form under the influence of residual impurities of endogenous proteases into during the day at 8 ° C. The enzyme concentration of 5 mg / ml was incubated with stirring in the cold (+ 4 ° C) for 30 min in a buffer composition of 20 mm Tris-HCl, pH 7.5, 0.1 mm 2-mercaptoethanol in the presence of a 5% suspension of activated coal. The suspension was centrifuged in the cold (+ 4 ° C) at 10,000 g for 5 min, the supernatant was diluted with the same buffer to 2.0-2.5 mg / ml and left overnight at 8 ° C. From the taken 5.0 mg of the TRSase preparation, 4.5 mg of the modified form was obtained with a yield of 90% and a purity of 99%.

A mini-method for producing modified TRSase included the application of the preparation of the starting TRSase with a concentration of 15 mg / ml in 20 mM Tris-HCl, pH 7.5, and a volume of 0.3 ml per carbon filter. An activated carbon filter was incubated at 4 ° C for 30 min and washed with 1 ml of buffer A without L-tryptophan and ATP, then the diluted solution was incubated in the refrigerator at 8 ° C overnight. The original TRSase was taken in an amount of 5 mg. After the procedure, 4.5 mg was obtained - 90% yield.

A modified enzyme was also obtained by limited proteolysis of the original enzyme with elastase. To do this, the initial enzyme TRSase at a concentration of 2.5 mg / ml, pre-treated with activated carbon to remove endogenous tryptophan (see above), was incubated with low concentrations of elastase (0.12 mg / ml in Tris-HCl buffer, pH 7.5 ) for 20 min at 25 ° C in the presence of 0.1 mm EDTA, 1 mm 2-mercaptoethanol and substrates - 0.1 mm L-tryptophan, 1.0 mm ATP. The reaction was stopped by the addition of a protease inhibitor of 1 mM PMSF or 5 mM diisopropyl fluorophosphate (DFF). In the case of DPP, the addition was carried out with glasses and gloves under the draft and left under the draft at room temperature, with stirring on a rocking chair, to completely destroy the DPF rapidly decomposing in an aqueous medium. Control of the formation of the modified form was carried out using denaturing electrophoresis in SDS page. The functional activity of the biologically active modified form was evaluated according to the standard MTT test (Lee WS, Harder JA, Yoshizumi M. et al. Progesterone inhibits arterial smooth cell proliferation // Nat.Med. - 1997. - No. 3. - P. 1005-1008. )

The technical result of the invention: obtaining a pure biologically active modified form of the enzyme.

SUMMARY OF THE INVENTION

Development of a method for producing a modified form of tryptophanyl tRNA synthetase (TRS-basics) to detect its antiangiogenic and anti-oncogenic biological activity with subsequent tests in order to obtain a new drug for the treatment of a number of common (in particular, oncological diseases).

This is achieved by the fact that in the method of producing TRS-basics, including crushing the pancreas of cattle or pigs, extraction, fractionation with ammonium sulfate, chromatography on columns with ion-exchange resins, we carefully selected the raw materials - pancreas, namely, cattle iron (Cattle) is taken no later than 30-40 minutes after the slaughter of the animal. Carefully selected glands with a minimum amount of fat, the impurities of which inhibit the enzyme. For transportation to the treatment site, glands in an amount of about 2-3 kg (about 15 cattle glands, or 40 pig glands) were placed in ice, the glands were crushed by passing through a (previously washed and chilled) meat grinder for 5 min directly into a tub with chilled buffer A (25 mM Tris-HCl, pH 7.5, 1 mM EDTA, 0.1 mM 2-mercaptoethanol, 0.2 mM L-tryptophan, and 1.0 mM Mg-ATP). All further procedures were carried out in the cold at + 4 ° C (in a cold room or in a refrigerator). The extract was stirred for 20 min and the extract was separated from the cake by centrifugation at 7000-8000 g, streptomycin sulfate was added to the extract with stirring to a final concentration of 1%. After 20 minutes of incubation without stirring, the nucleic acid pellet was removed by centrifugation at 3000 rpm or 7000-8000 g. The precipitate was discarded. To obtain the modified form, protease inhibitors were not used at the extract stage.

The next step was the direct fractionation of TRS-basics using stepwise salting out with ammonium sulfate. Ammonium sulfate, previously ground to a powder state, was added slowly with moderate stirring in the cold to a final concentration of 40% saturation over 20 minutes. Left to form a precipitate for 20 minutes. Then the precipitate was precipitated by centrifugation at an average acceleration of 10,000-15,000 g. The supernatant was poured into a separate tub and added in the cold, as described above, ammonium sulfate to 60% saturation. At this stage, the suspension was left at 4 ° C in the refrigerator overnight to form a precipitate.

The next step is column chromatography using anion exchange resins - cellulose, such as diethylaminoethyl cellulose (DEAE cellulose, DE-52). It is recommended to activate the resin before use in order to increase the yield of the target product. To do this, regardless of the microstructure, the ion exchanger is treated as follows: first, it is mixed with 15 times (dry weight / volume) 0.5 M HCl for 30 min at room temperature, then it is washed with distilled water to a pH of 4. using a funnel or decantation. After this resin is mixed with a 15-fold amount of 0.5 M NaOH for 30 min at room temperature and washed with distilled water until the suspension gives a neutral reaction. The resin thus treated is balanced with a starting buffer solution. Part of the supernatant is carefully drained over the edge of the tub, being careful not to stir up the sediment. The remaining mixture is centrifuged at 15,000 g. The supernatant is discarded, the remaining liquid is carefully removed from the walls of the centrifuge cups with filter paper. The precipitate is then dissolved in 30-50 ml of buffer A containing 0.05 M ammonium chloride NH ₄ Cl. The solution was layered on a 150 ml DE-52 column and eluted to a minimum optical density (not higher than 0.1 pfu / ml at 280 nm). Impurity proteins were removed by elution with buffer A containing a stabilizer (0.09 M NH ₄ Cl). The released protein peak was discarded. After the optical density was reduced to a minimum (about 0.1 pfu / ml), the purified fraction of the initial and modified forms of TRCase was removed with buffer A containing an increased concentration of stabilizer (0.16 M NH ₄ Cl). The elution profile from the column is shown in figure 1. The central part of the peak was taken and the preparation of TPC-ase was concentrated with ammonium sulfate, bringing it to 60% saturation. For this, a saturated 100% solution of ammonium sulfate was added dropwise in the cold until a precipitate formed, continued to add up to 60% saturation, and left overnight in the refrigerator to form a precipitate. The mixture was centrifuged at 10000 g, the supernatant residues were removed with filter paper. The precipitate was dissolved in buffer A containing a stabilizer (0.1 NH ₄ Cl), and then the solution was applied to another ion exchange column with DEAE cellulose DE-52 with a volume of 50 ml, balanced with buffer A containing 0.1 M ammonium chloride and 1 mm protease inhibitor - phenylmethylsulfonyl fluoride (PMSF). Eluted with a linear gradient of NH ₄ Cl 0.1-0.16 M at a rate of 25 ml / hour. 2.5 ml fractions were collected. The elution profile is shown in figure 2. The enzyme appeared in the form of a large peak at the beginning, the subsequent peaks were either impurity proteins or products of a deeper cleavage of TRSase. Fractions with weak enzymatic activity, and a modified form according to denaturing polyacrylamide gel electrophoresis (PAGE). To 30 ml of the combined eluate was added dropwise with stirring 20 ml of a 100% saturation solution of ammonium sulfate. After the formation of the precipitate was centrifuged at 10000 g. The supernatant was discarded. The precipitate was dissolved in a minimum volume of buffer A, at a concentration of not less than 20 mg per ml and stored frozen in 1 ml aliquots at -70 ° C, or at -20 ° C in 30% glycerol. Received 40.5 mg of a modified form of the enzyme with a purity of 99%.

The technological scheme of the proposed method can be represented as follows: collection and preparation of cattle pancreas or pigs → grinding and extraction → precipitation of nucleic acid impurities and cell debris → step-wise salt fractionation with ammonium sulfate → fractionation on an anion-exchange chromatographic column with a stepwise gradient of ammonium chloride eluent → fractionation on the second ion-exchange column DE 52 → concentration of the TRSase preparation and assessment of its biological activity.

The yield of the drug is 0.0020-0.005% of the fresh organ of the animal. The described stages of the technological process in the proposed method are logically justified.

One of the main points in the procedure for isolating the modified form of TRSase is the choice of an object - the pancreas of cattle and pigs, in which, as it has been established, its content is significantly (almost 100 times) increased compared to other tissues (in particular, in the liver). At the stage of selection of the gland, its freshness is important (the time of slaughter of the animal before the selection of the gland should be minimal), the gland should be lean, thoroughly cleaned of fat.

All stages of fractionation prior to the addition of a protease inhibitor should be carried out in a minimum time. In the early stages of fractionation, it is desirable to avoid strong foaming of the extracts (in particular, at the stage of salt fractionation with ammonium sulfate), as this can lead to partial denaturation and inactivation of the modified forms of the enzyme, and may affect its output. During centrifugation in the early stages, particles of fat floating on the supernatant are removed by draining the supernatant into a tub through several layers of gauze. Excess fat can also lead to a decrease in biological activity and the release of modified forms of TRSase.

The presence of L-tryptophan and ATP in buffer A, which contribute to the formation of the modified form and prevent further degradation of the forms of TRSase with the loss of noncanonical additional functions, is very important for obtaining modified forms of TRSase.

At the second fractionation stage, on the DE-52 ion-exchange column, it is necessary to add a PMSF protease inhibitor at a concentration of 1 mM, since, due to the pancreas enrichment with proteases, the modified form of TRSase, otherwise, will be completely cleaved.

At the stage of ion-exchange chromatography, the procedure of activation of the ion-exchange resin can be carried out, otherwise the yield of the drug is significantly reduced, and the fractionation efficiency is sharply reduced. It is necessary to achieve a sufficient complete washing out of the non-specifically bound protein before collecting the desired fraction, and select the middle part of the peak for further work. At the end of the peak, partially proteolized forms of TRSase follow. It is advisable to immediately concentrate the collected fractions, since the enzyme is much less stable in dilute solutions.

Ammonium ion (NH ₄ +) stabilizes TRSase; therefore, the enzyme can be stored in the form of a precipitate in ammonium sulfate at -20 ° C for a long time (1-2 months) before the column stages. This is important when testing the effectiveness of new column methods and comparing them, since the previous stages, subject to the method, do not change the yield of the drug and its purity.

The further directions of increasing the efficiency of the methodology lie within the framework of the main stages and require their improvement or omission of some stages, by combining them into one, or by completely eliminating certain stages due to an increase in the efficiency of the previous ones.

Changes in the methodology can be adapted for the specific purposes of the further use of modified forms of the enzyme.

In addition to the main aminoacylating activity, TRSase has at least one more important function, namely, it takes part in a chain of biochemical processes that stimulate the body's defensive reactions to the introduction of viral infections (in particular, destroy specific viral RNAs). Through the formation of diadenosine triphosphate (Ap3A) and, possibly, other ApnA, TRSase can take part in the regulation of processes such as stimulation of protein secretion by the glands, enzyme activities involved in the regulation of the cardiovascular system and transmission of the nerve signal (through membrane ATPases). In this regard, it becomes partly clear the relatively high content of TRSase in the pancreas, which is a powerful secretory organ. It is necessary to more accurately establish a complex network of reactions that regulate the processes of life and the role and place of TRSases in them. The prospect of specific directions for the use of this enzyme in medicine opens up.

The invention can be illustrated by the following examples.

Example 1

Take 2 kg of gland no later than 30-40 minutes after slaughtering the animal, glands with a minimum amount of fat are carefully selected, the gland is additionally cleaned of fat. For transportation to the treatment site, the iron was placed in ice, the iron was crushed by passing through a (previously washed and chilled) meat grinder for 5 min directly into a tub with 2.2 L of chilled buffer A (25 mM Tris-HCl, pH 7.5, 1 mM EDTA, 0.1 mM 2-mercaptoethanol, 0.2 mM L-tryptophan 2 × 10 ^-4 M, and 1.0 mM Mg-ATP). All further procedures were carried out in the cold at + 4 ° C. The extract was stirred for 20 minutes and the extract was separated from the meal by centrifugation. To the obtained 2 L of the extract, 0.5 L of 5% streptomycin sulfate was added with stirring to a final concentration of 1% (interrupted by a thin stream). After 20 min incubation without stirring, the nucleic acid pellet was removed by centrifugation at 3000 rpm (5000 g). The precipitate was discarded. 690 g of ammonium sulfate (pre-ground to powder) was added to the supernatant, slowly with moderate stirring in the cold to a final concentration of 40% saturation for 20 minutes. Left to form a precipitate for 20 minutes. Then, the precipitate was precipitated by centrifugation at an average acceleration of 10,000-12,000 g. The supernatant was poured into a separate tub and to the obtained 3.2 L was added in the cold, as indicated above, 316 g of ammonium sulfate to a 60% saturation. The extract was left to precipitate overnight.

Part of the supernatant is carefully drained over the edge of the tub, being careful not to stir up the sediment. The remaining 1.5 L mixture was centrifuged at 15,000 g. The supernatant is discarded, the remaining liquid is carefully removed from the walls of the centrifuge cups with filter paper. The precipitate was then dissolved in 50 ml of buffer A containing 0.05 M ammonium chloride NH ₄ Cl. The solution was layered on a 150 ml DE-52 column and eluted to a minimum optical density (not higher than 0.1 pfu / ml at 280 nm). Impurity proteins were removed by elution with buffer A containing 0.09 M NH ₄ Cl. The released protein peak was discarded. After the optical density was reduced to a minimum (about 0.1 pfu / ml), the purified fraction of the initial and modified forms of TRCase was removed with buffer A containing 0.16 M NH ₄ Cl. The elution profile from the column is shown in figure 1. The central part of the peak in a volume of 80 ml was taken and a mixture of the initial (about 20%) and modified forms of TRSase (about 75% form and 5% form 24 + 14 kDa was precipitated, adding 54 ml of saturated (100% saturation) ammonium sulfate, until 60 % saturation To do this, a saturated 100% solution of ammonium sulfate was added dropwise in the cold until a precipitate formed, continued to add up to 60% saturation and left in the refrigerator overnight to form a precipitate.The mixture was centrifuged at 10,000 g, the remaining supernatant was removed with filter paper. The precipitate was dissolved in 40 ml buffer and A containing 0.1 M NH ₄ Cl. To the solution was added 40 μl of a 1 M solution of PMSF to inhibit proteases (at the rate of 1 μl per 1 ml of solution) and left for 20 min in a fume hood with stirring to completely dissolve the PMSF. the solution was applied to a DE-52 ion-exchange resin column with a volume of 50 ml balanced with buffer A containing 0.1 M NH ₄ Cl, 1 mM PMSF, the protein unbound on the column was washed off until the optical density in the eluate was not higher than 0.1 о.е ./ml at 280 nm, then the fractions were washed off with a linear gradient of ammonium chloride salt (0.1 M - 0.16 M salt). Eluted at a rate of 25 ml / hour. 2.5 ml fractions were collected. The elution profile is shown in figure 2. The pure modified form of the enzyme stood out as a large peak in the second half of the gradient. Subsequent peaks were either impurity proteins and a 40 × 40 kD form, or a proteolysed form of TRSase (24 + 14 kD). Fractions No. 22-35 were collected, having weak enzymatic activity and representing a homogeneous form according to denaturing polyacrylamide gel electrophoresis. To 30 ml of the combined eluate was added dropwise with stirring 20 ml of a 100% saturation solution of ammonium sulfate. After the formation of a precipitate, it was centrifuged at 10,000 g. The supernatant was discarded. The precipitate was dissolved in 2.5 ml of buffer A. The protein concentration was 15.2 mg / ml, the final volume was 3 ml, that is, 45.6 mg of the modified form of the TRSase enzyme was obtained. The modified form of the enzyme was stored in aliquots of 1 ml at -70 ° C. The yield of the modified form of the enzyme, based on a gland mass of 2 kg, was about 0.0023%, with a purity of 98%.

In the second approach, the enzyme already purified by the standard procedure was subjected to treatment with activated charcoal (5% aqueous suspension in buffer A, without L-tryptophan and ATP) to remove endogenous tryptophan, while the enzyme passed into a modified form under the influence of residual impurities of endogenous proteases into during the day at 8 ° C. The enzyme concentration of 5 mg / ml was incubated with stirring in the cold (+ 4 ° C) for 30 min in a buffer composition of 20 mm Tris-HCl, pH 7.5, 0.1 mm 2 - mercaptoethanol in the presence of a 5% suspension of activated coal. The suspension was centrifuged in the cold (+ 4 ° C) at 10,000 g for 5 min, the supernatant was diluted with the same buffer to 2.5 mg / ml and left overnight at 8 ° C. From the taken 5.0 mg of the TRSase preparation, 4.5 mg of the modified form was obtained with a yield of 90% and a purity of 99%.

A mini-method for producing modified TRSase included applying the preparation of the starting TRSase with a concentration of 15 mg / ml in 20 mM Tris-HCl, pH 7.5, and a volume of 0.1 ml per carbon filter. An activated carbon filter was incubated at 4 ° C for 30 min and washed with 1 ml of buffer A without L-tryptophan and ATP, then the diluted solution was incubated in the refrigerator at 8 ° C overnight.

The original TRSase was taken in an amount of 5 mg. After the procedure, 4.5 mg was obtained - 90% yield.

A modified enzyme was also obtained by limited proteolysis of the original enzyme with elastase. To do this, the initial enzyme TRSase at a concentration of 2.5 mg / ml, pre-treated with activated carbon to remove endogenous tryptophan (see above), was incubated with low concentrations of elastase (0.12 mg / ml in Tris-HCl buffer, pH 7.5 ) for 20 min at 25 ° C in the presence of 0.1 mm EDTA, 1 mm 2 - mercaptoethanol and substrates - 0.1 mm L-tryptophan, 1.0 mm ATP. The reaction was stopped by the addition of a protease inhibitor of 1 mM PMSF or 5 mM diisopropyl fluorophosphate (DFF). In the case of DPP, the addition was carried out with glasses and gloves under the draft and left under the draft at room temperature, with stirring on a rocking chair, to completely destroy the DPF rapidly decomposing in an aqueous medium. Control of the formation of the modified form was carried out using denaturing electrophoresis in SDS page. The yield was 4.5 mg of the modified enzyme, i.e. 90% with a purity of 99% according to denaturing protein polyacrylamide gel electrophoresis (PAGE).

The functional activity of the biologically active modified form was evaluated according to the standard MTT test (Lee WS, Harder JA, Yoshizumi M. et al. Progesterone inhibits arterial smooth cell proliferation // Nat. Med. - 1997. - No. 3. - P. 1005-1008. )

Example 2

Similar to example 1, except that the first salt fractionation was carried out at 50% saturation of ammonium sulfate. Received 40.3 mg of a modified form of the enzyme with a degree of purity of 99%.

The second method for producing modified TRSase is similar to the corresponding section of Example 1, except for incubation of the enzyme preparation freed from endogenous tryptophan for 2 days at 8 ° C. Of the 5 mg of the initial form taken for the experiment, 4 mg of the modified form was obtained, that is, the yield was 80%. The mini-method when using incubation at 8 ° C for 2 days - 3.8 mg of the modified form was obtained, that is, the yield was 76%.

Example 3

Similar to example 1, except that the first salt fractionation was carried out at 30% saturation of ammonium sulfate. Received 42.10 mg of the drug with a degree of purity of 90% (according to electrophoresis in denaturing PAAG).

The second and mini-method for producing modified TRSase is similar to Example 1, but the incubation in 5% activated carbon lasted 3 hours. The yield was 3.2 mg and 3.5 mg, or 64% and 70%, respectively.

Example 4

Similar to example 1, except that the second salt fractionation was carried out at 50% saturation of ammonium sulfate. 38.92 mg of the enzyme preparation was obtained (yield - 0.0019%) with a purity of 99%.

The procedure for obtaining the modified form was carried out as in example 1, but L-tryptophan and ATP were removed from the incubation buffer. In this case, 2.0 mg (yield 40%) of a modified TRSase with a purity of 50% was obtained.

Example 5

Similar to example 1, except that the second salt fractionation was carried out at 70% saturation of ammonium sulfate.

42.14 mg of the preparation was obtained (yield 0.0021%) with a purity of 90%.

The second and mini-method for producing modified TRSase is similar to Example 1, but the incubation in 5% activated carbon lasted 3 hours. The yield was 3.2% and 3.5% or 64% and 70%, respectively.

Example 6

Similar to example 2, except that the elution step was omitted by ion exchange chromatography at 0.09 M NH ₄ Cl. 38.09 mg (yield 0.0019%) of the preparation were obtained with a purity of 90%.

The preparation of the modified form by the limited proteolysis method in the presence of substrates was carried out as in case 1, but twice as high elastase concentrations — 0.24 mg / ml — were used. The yield was 3.0 mg or 60%, with a purity of 90%.

Example 7

Similar to example 2, except that the ionic strength of the solution during elution of the main fraction was increased to 0.16 M NH ₄ Cl, and the gradient of the NH ₄ Cl salt of the second ion-exchange column was from 0.1 M to 0.2 M. 35.10 mg of the preparation obtained (yield 0.00175%) with a purity of 90%.

The second and mini-method for producing modified TRSase is similar to Example 1, but the concentrations of the starting preparations of TRSase were diluted 5 times (1 mg / ml and 0.5 mg / ml, respectively). Yields were 3.8 mg and 3.5 mg or 76% and 70%, respectively.

Example 8

Similar to example 1, except that the step of adding an inhibitor of the protease PMSF was included after the step of removing impurity nucleic acids. Received 10.60 mg of the drug with a degree of purity of 80%. That is, the addition of a protease inhibitor at this stage dramatically reduces the yield of the modified form of TRSase.

The second and mini-method for producing modified TRSase is similar to Example 1, but after treatment with activated carbon, the PMSF protease inhibitor up to 1.0 mM was added to the mixture. The yield was 1.2 mg (24%) in a modified form of TRSase with a purity of 20%. That is, the addition of a protease inhibitor at this stage actually blocked the transition to the modified form.

Example 9

Similar to example 1, except that the stage of adding PMSF was excluded before applying to the second ion-exchange column with a linear gradient of elution. The yield was 22 mg with a purity of 75%. That is, the use of protease inhibitors in the last column stage is important to obtain a good yield and purity of the modified form of TRSase.

The second and mini-method for producing modified TRSase is similar to Example 8, but after treatment with activated carbon, elastase was added to the mixture at a concentration of 0.12 mg / ml, followed by incubation overnight at 8 ° C. The yield was 0.5 mg (i.e. 10%) in a modified form of TRSase with a purity of 10% with respect to the modified form. That is, the addition of elastase at this stage actually led to the complete proteolysis of TRSase.

Example 10

Similar to example 9, except that all peaks of the enzyme were selected during the second fractionation on an ion exchanger, obtained with a linear gradient of ammonium chloride salt. The yield of the modified form of the enzyme was 31.55 mg (0.0016%) with a purity of 80%.

The second and mini-method for producing modified TRSase is similar to Example 9, after treatment with activated carbon, elastase was added to the preparation after removal of activated carbon at a concentration of 0.12 mg / ml, but incubation was carried out at 8 ° C for 1 hour. The yield was 2.5 mg (i.e. 50%) in the modified form of TRSase with a purity of 80% with respect to the modified form.

A comparative analysis of the technical and economic indicators of the proposed and known method are shown in table 1.

Table 1 Way Yield (% of feedstock) The content of impurity proteins and proteolysis products Proposed 0.0025 one% Famous 0,00025 3%

The invention allows to obtain a preparation of high purity and biological activity for the inhibition of angiogenesis processes. The method is one of the first methods for producing preparative amounts of a modified form of TRSase with additional non-canonical antiangiogenic activity.

The method of obtaining the modified form of TRSase differs from the known one by a significantly higher yield of the preparation (10 times) due to the optimization of procedures and the use of buffers with substrates of the catalyzed reaction, which ensured the accumulation of the modified form of TRSase in the fractions deposited and eluted from the columns during fractionation. A series of experiments on the development of methods was carried out on two sources of raw materials - cattle (cattle) and pigs.

The method, representing one of the first methods for producing a modified biologically active form of TRSase, is significantly reduced in execution time by replacing several column stages by 1-2. An original scheme of obtaining a modified form was used, based on the use of natural endogenous impurity proteases, which, simulating the processes occurring in vivo in tissues, lead to the preparation of preparative amounts of a homogeneous modified preparation of TRSase.

Literature

1. Methods Enzymol. 1979; 59: 234-257, Kisselev L.L. et al., Tryptophanyl-tRNA synthetase from beef pancreas.

2. Nat Rev Mol Cell Biol. 2010, 11 (9): 668-674. Gum M., Yang X.L., Schimmel P. New functions of aminoacyl-tRNA synthetases beyond translation.

3. Nat Rev Cancer. 2011, 11 (10): 708-18. Kirn S., You S., Hwang D. Aminoacyl-tRNA synthetases and tumorigenesis: more than housekeeping.

4. Heart Vessels. 2011, 26 (1): 69-80. Zeng R, Chen YC, Zeng Z., Liu WQ, Jiang XF, Liu R., Qiang O., Li X. Effect of mini-tyrosyl-tRNA synthetase / mini-tryptophanyl-tRNA synthetase on ischemic angiogenesis in rats: proliferation and migration of endothelial cells.

5. Proc Nati Acad Sci USA. 2008, 105 (32): 11043-11049. Park S.G., Schimmel P., Kim S. Aminoacyi tRNA synthetases and their connections to disease.

Claims (2)

1. A method of obtaining a modified form of tryptophanyl tRNA synthetase (TRSase), including grinding animal tissue of the pancreas, extraction, separation of the extract from the cake, precipitation and removal of impurity nucleic acids using streptomycin sulfate, reprecipitation of impurities from the supernatant by salting out with ammonium sulfate and stepwise fractionation of the extract, characterized in that at the first stage of fractionation by ion exchange chromatography using DEAE-cellulose DE-52, a step is carried out elution with a buffer solution containing 25 mM Tris-HCl at pH 7.5, 1 mM EDTA, 0.1 mM 2-mercaptoethanol and a stabilized modified form of TRCase in the form of a complex of substrates 0.2 mM L-tryptophan and 1.0 mM Mg -ATP, while the elution is carried out until the optical density reaches no higher than 0.1 pu / ml at 280 nm, after which the impurity proteins are removed with a buffer solution containing 0.09 M NH ₄ Cl, after which the purified fraction is washed with a buffer solution containing 0.16 M NH ₄ Cl, the resulting mixture is precipitated with ammonium sulfate to achieve 60% saturation, centrifuged, the precipitate is dissolved in a buffer solution containing 0.1 M NH ₄ Cl, a protease inhibitor is added to the solution, then the second fractionation step is carried out by ion exchange chromatography using DE-52 DEAE cellulose with stepwise elution using a linear gradient of ammonium chloride salt 0.01 - 0 , 16M, fractions with a modified form of tryptophanyl tRNA synthetase were selected and the target product was concentrated by salting out with ammonium sulfate to achieve 100% saturation. 2. The method according to claim 1, characterized in that as an inhibitor of proteases using 1 mm phenylmethylsulfonyl fluoride (PMSF) or 5 mm diisopropyl fluorophosphate (DFF).

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