SU1512473A3 - Method of producing proteins of 3200-3400 molecular weight, suppressing blood coagulation - Google Patents

Abstract

This invention relates to medicine, in particular to medical biotechnology. The purpose of the invention is to improve the quality of the target product by preventing the tendency to bleed when used. To do this, the aortic or vasculature inner lining is homogenized in a solution of 100 mmol / l sodium chloride, 50 mmol / l tris (oxymethyl) aminomethane hydrochloride, PH 7.5 (TBS) containing sodium ethylenediamine tetraacetate, a trypsin inhibitor from soybeans and benzamidine. The homogenate is centrifuged for 60 minutes at 100,000 G. From the supernatant, the target product is precipitated by salting out with ammonium sulfate. After centrifugation at 10,000-12,000 G, the resulting precipitate is dissolved in a small volume of TBS, dialyzed against TBS containing benzamidine. The dialyzed fraction is purified using chromatography on hydroxyapatite, dialysis, ion exchange chromatography on sephatsel with diethylaminoethyl groups, and repeated dialysis. The resulting fraction is concentrated using an PM-10 ultrafiltration membrane. Further purification is carried out either by chromatography on Sephadex G-100 followed by dialysis, or by gel filtration on superose-12, ion exchange chromatography on Mono Q anion exchangers and repeated gel filtration on superose 12, followed by dialysis. The invention makes it possible to achieve a higher quality target product, which, unlike the antithrombin-III obtained in the prototype method, does not inactivate the coagulation factor Haitrombin blood and therefore does not lead to bleeding tendency when it is used. The target product is a mol. M. Protein. 32,000 and 34,000, and isoelectric point values at PH 4.4-4.6.

Description

The invention relates to medicine, in particular to medical biotechnology, and can be used to produce proteins with a mole, m. 32,000-34,000 suppressing blood clotting.

The aim of the invention is to improve the quality of the target product by preventing bleeding from using it.

platelets are centrifuged for 15 minutes at 10,000 tons. Thus, a platelet-free plasma (PFP) is obtained, which is used

The drawing shows the profile of the gel to determine the anticoagulants

15124734

platelets are centrifuged for 15 minutes at 10,000 tons. Thus, a platelet-free plasma (PFP) is obtained, which is used

filtration on Sephadex G-100, used at the final stage of purification of the target product in example 1.

The method is carried out as follows.

 The inner membrane (intima) of the aorta or vasculature tissue is homogenized in a solution of 100 mmol / l NaCl,

activity of fractions.

Immediately after collecting the aorta, the animals are thoroughly washed with TBS, the Intima is separated from the aorta and homogenized in a Bown MX 32 type homogenizer, in TBS with 2 mmol / l ELTA (TBSE), which contains 16 mg / ml soybean trypsin inhibitor and 1.57 g / l benz-50 mmol / l tris-HCl, pH 7.5 (TBS), amidine.

Ethylene Diamine Tetra Acetate containing sodium is a homogenate of 8 aorta, which contains (EDTA), a trypsin inhibitor from 20% (w / v) of solid beans and benzamidine. The homogenate is centrifuged for 60 minutes at 100,000 g. From the supernatant, the desired product is precipitated by salting out with ammonium sulfate. After centrifugation at 10,000-12,000 g, the resulting precipitate is dissolved in a small volume of TBS, dialyzed against TBS containing benzamidine. The lialized fraction is purified by chromatography on hydroxyapatite, dialysis, ion exchange chromatography on sephacel with diethylaminoethyl groups (LEAE - sephacel), and re-dialysis. The resulting fraction containing VAC-protein (vascular anticoagulant - a vascular anticoagulant), conn 12000 g. The resulting precipitate is dissolved in a small volume of TBS and dialyzed against TBS containing 1.57 g / L of benzamidine. The lylated fraction is fed to a column (1 x X 20 cm) with hydroxyapatite, which is balanced with TBS. Industrial Column35

It is centered using an Amicon PM-10 ultrafiltration membrane. Further purification is carried out either by chromatography on Sephadex G-100 followed by dialysis, or by gel filtration. The cells are eluted from the column with 200 ml of sodium on Superose-12, ion-exchange chromium. - phosphate buffer (pH 7.5) with linear

Wash TBS in an amount corresponding to 4 column volumes. VAC-Belgrade 0-500 mmol / l. The fractions containing UAS proteins are pooled and dialyzed against 50 mmol / L NaCl, 20 mmol / L Tris / HCl, pH 7.5 (Tris-Tris / Oxymethyl / -aminomethan). The same buffer is also used for equilibration columns (3x5 cm) containing LEAE-Sephacel. In this column, the dialysate is subjected to chromatography. The column is washed with a buffer used for equilibration, which is taken in an amount corresponding to 4 column volumes. Then, VAC proteins are eluted from the column using 200 ml of NaCl solution, 20 mmol / l Tris / HC1, pH 7.5 with a linear NaCl gradient of 50-300 mmol / l. Fractions containing VAC proteins are collected, dialyzed about

Mono Q anionite matte and repeated gel filtration on superose-1 followed by dialysis.

The target product is a protein with a mol.m., 32000-34000, which suppresses blood coagulation, but differs in properties from that obtained in the method of the prototype protein anticoagulant antithrombin-III.

Example 1. Within 30 minutes after the slaughter of the cattle take the aorta. Bovine blood is collected with the addition of sodium citrate threefold to a final concentration of 0.38 wt.% And centrifuged for 10 minutes at room temperature 2000 g. The resulting plasma containing a small amount

va, centrifuged for 60 minutes at 100,000 g.

Ammonium sulfate powder is added to the supernatant to 30% of the saturating concentration, stirred for 30 minutes and then centrifuged for 20 minutes at 12000 g. Ammonium sulphate powder is added to the supernatant obtained in this way up to 90% of the persistent concentration and stirred for 30 minutes.

and centrifuged for 20 minutes

at 12000 g. The resulting precipitate is dissolved in a small volume of TBS and dialyzed against TBS containing 1.57 g / L of benzamidine. The lylated fraction is fed to a column (1 x X 20 cm) with hydroxyapatite, which is balanced with TBS. Column flush

ki eluted from the column with 200 ml of sodium phosphate buffer (pH 7.5) with a linear

ki eluted from the column with 200 ml of sodium phosphate buffer (pH 7.5) with a linear

Wash TBS in an amount corresponding to 4 column volumes. VAC proteins are eluted from the column with 200 ml of sodium phosphate buffer (pH 7.5) with linear

gradient 0-500 mmol / l. The fractions containing UAS proteins are pooled and dialyzed against 50 mmol / L NaCl, 20 mmol / L Tris / HCl, pH 7.5 (Tris-Tris / Oxymethyl / -aminomethan). The same buffer is also used for equilibration columns (3x5 cm) containing LEAE-Sephacel. In this column, the dialysate is subjected to chromatography. The column is washed with a buffer used for equilibration, which is taken in an amount corresponding to 4 column volumes. Then, VAC proteins are eluted from the column using 200 ml of NaCl solution, 20 mmol / l Tris / HC1, pH 7.5 with a linear NaCl gradient of 50-300 mmol / l. Fractions containing VAC proteins are collected, dialyzed about

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tive 500 mmol / l NaCl, 20 mmol / l Tris / HCl, pH 7.5 are then concentrated using an AIR PK-10 ultrafiltration membrane. A 2 ml concentrate is fed to a column (3x80 cm) with a facosity of G-100, which was equilibrated with 500 mmol / l NaCl and 20 mmol / l tris / HC1, pH 7.5. The eluate is collected in fractions of 2 ml of PA, the active fractions containing VAC proteins with a mole, May.32000 and 34000, are dialyzed against 10% vol.% Glycerol TBS and stored at -70 ° C. All purification steps are carried out at 0- 4 C.

The determination of the anticoagulant activity of the fractions containing VAC proteins is carried out as follows.

In a siliconized glass cuvette, mix (900 rpm) 175 μl of the test fraction (or 175 μl of TBS as a control) along with 50 μl of PFP and 25 μl of the diluted bovine thromboplastin (BTR After incubation for 3 min at 37 ° C cause a coagula By adding 250 µl of buffer that contains 80 mmol / l NaCl, 20 mmol / l CaC and Yu mmol / l Tris / HC1, pH 7.5. Optical fibrin formation is recorded optically. The coagulation time of the control sample is 65 s. This test used during the purification for the study of various fractions for the presence of VAC-active In order to determine the yield of VAC proteins during purification, a unit of VAC activity is determined, such as the amount of VAC-proteins that prolongs the coagulation time during the test, up to 100 s,

In this example, the following results are obtained.

1. The supernatant after the first precipitation with ammonium sulfate (30% of total; en): 630 mg of protein,

19500 units VAC activity, specific activity: 31.0 U / mg;

yield 100%;

purification rate 1.0,

2. Sediment after second precipitation with ammonium sulfate (90% of saturation),

470 mg of protein; 19000 units VAC activity} specific activity 40.4 units / mg; purification rate of 1.3.

3, Fraction after hydroxyapatite chromatography:

206 mg of protein;

17-300 beats. VAC Activity |

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specific activity 84.0 units / mg-,

yield 89%

purification degree is 2.7.

4. Fraction after chromatography on DEAE-Sephatsel: 35.8 mg of protein;

13900 units VAC activity; specific activity of 388 units / mg; yield 71%; purification rate of 12.5.

5. Fraction after chromatography on Sephadex G-r100: 0.45 mg protein;

666 units VAC activity;

specific activity of 1480 units / mg;

3.4% of the income;

purification rate 47.7,

The amount of protein was determined by the method of Lowry et al. (1951).

PRI mme R 2. 15 minutes after birth, human umbilical cords are collected. Arteries Immediately washed with ice-cold TBS buffer, then homogenized in a Brown MX 32 type homogenizer, in TBSE, which contains 16 mg / ml trypsin inhibitor from soybeans and 1.57 g / l benzamidine.

The homogenate, which contains 10% (w / v) solids, is centrifuged for 60 minutes at 100,000 g. In the supernatant, ammonium sulfate powder is added to 30% of the saturation concentration, stirred for 30 minutes and then centrifuged for 20 minutes at 100,000 g.

The resulting precipitate is dissolved in a small volume of TBS and dialyzed against TBS containing 1.57 g / l of benzimidine. The dialyzed fraction is fed to a column (1x20 cm) with hydroxyapatite, which is equilibrated with TBS. The column was washed with TBS in an amount corresponding to 4 column volumes. VAC proteins rlyuyut 200 ml of sodium phosphate buffer (pH 7.5) with a linear gradient of 0-500 mmol / l from the column. The fractions containing 1e VAC proteins are pooled and dialyzed against 50 mmol / L NaCl, 20 mmol / L-Tris / HC, pH 7.5.

The same buffer is used to balance the column (3x5 cm) containing the DEAE-Sephacel. In this column, the dialysate is subjected to chromatography. The column is washed with a buffer used for equilibration, which is taken in an amount corresponding to 4 column volumes. Then vac proteins

eluirugotot nz column using 200 ml of NaCl solution in 20 mmol / l Tris / HC, pH 7.5, with a linear gradient of NaCl 50-300 mmol- / l. Fractions containing. VAC proteins are collected, dialyzed against 500 mmol / l NaCl, 20 mmol / l Tris / HC1, pH 7.5, and then concentrated with the aid of Amicon PM-10 ultrafiltration membrane. A concentrate of volume 2 is fed to a column (3x80 cm) with Sephadex G-100 which was equilibrated with 500 mmol / l NaCl and 20 mmol / l: tris / HC1, pH 7.5. Eltoate is collected in fractions of 2 ml each, the active fractions containing VAC proteins with mol.mae. 32,000 and 34,000, are dialyzed against 10% by volume glycerin TBS and stored at 70 ° C. All purification steps are carried out at 0-4 °. PRI me R 3. A human placenta obtained immediately after delivery is cooled to 4 ° C and freed from blood. The membrane material is removed and homogenized in TBS buffer with 7.4, which contains 1 ,, 57 mg / ml trypsin inhibitor from soybeans, 1.6 g / l benzamidine and 1 mmol / l EDTA, the homogenate, which contains 20% ( w / v) solids, centrifuged for 60 minutes at 100,000 g. Ammonium sulphate powder is added to the supernatant up to 30% of an unpleasant concentration, stirred for 30 minutes and then centrifuged for 10 minutes at 10,000 g.

The precipitate obtained is dissolved in a small volume of TBS and dialyzed with TIBS TBS containing 1.57 g / l benzamidine. The dial fraction was fed to columns (1x20 cm) with hydroxyapatite which was equilibrated with TBS. The column is washed with TBS in an amount corresponding to 4 volumes of the column layer. VAC proteins elute from the column with 200 ml of sodium phosphate buffer (pI 7.5) with a linear gradient of 0-500 mmol / l. The fractions containing VAC proteins are combined and dialyzed against 50 mmol / l NaCl, 20 mmol / l Tris / HC1 7.5.

Fractions containing VAC proteins are further purified by separation on a DEAE-sephacel column. The column was equilibrated with 20 mmol / l Tris / HC1 buffer with 50 mmol / l sodium chloride, pH 7.4. The bound VAC proteins were eluted same

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chromatography with sodium chloride, pH 7.4, then the following chromatographic operations are performed sequentially at room temperature; c, gel filtration of the fractions containing VAC proteins on the column with supernose-12 (trade product of the Swedish company Pharmacia, which is crosslinked agarose with a particle size of 10 µm), which was equilibrated with a buffer of 20 mmol / l of bis-tris / HC1 ,. 100 mmol / l sodium chloride, pH 6.3, and ion exchange chromatography of the eluate on Mono O (commercial product of the Swedish company Pharmacia, which is a 10-µm ball anion exchange resin), which was balanced by the buffer used at the previous stage, The elution was carried out using a linear gradient of sodium chloride in a 20 mmol / l bis-tris / HC1, pH 6.3 o VLC protein was eluted at 190 mmol / l sodium chloride. Then fractions containing VAC proteins are subjected to rechromatography at Superose-12. After dialysis against buffer, TBS fractions containing VAC proteins with mol. 32000 and 34000 mol are stored at -70 ° C,

Example4. Within 30 minutes after slaughter, aorta cells are taken from cattle.

Immediately after collecting the aorta, thoroughly washed with TBS. The intima is separated from the aorta and homogenized in a Brown MX 32 type homogenizer in TBSE, which contains 16 mg / ml trypsin inhibitor from soybeans and 1.57 g / l benzamidine.

The homogenate is centrifuged for 60 minutes at 100,000 g.

Ammonium sulphate powder is added to the supernatant to 30% of total, more concentrated concentration; it is stirred for 30 minutes and centrifuged for 12 minutes at 12000 g. The solid is suspended in a small volume of IBS and dialyzed against TBS containing 1.57 g / l of benzamidine. The dialyzed fraction is fed to a column (1x20 cm) with hydroxyapatite, which has been equilibrated with TBS. The column was washed with TBS in an amount corresponding to 4 column volumes. VAC proteins elute from the column with 200 ml of sodium phosphate buffer (pH 7.5) with a linear gradient of 0-500 mmol / l.

The fractions containing VAC proteins are further purified by separation on a DEAE-Sephacel column. The column was equilibrated with 20 mmol / l Tris / HC buffer, 50 mmol / l sodium chloride, pH 7.4,

Bound VAC proteins are eluted using 200 mmol / l Tris / HC1 buffer, 300 mmol / l sodium chloride pH 7.4. The following chromatographic operations were then carried out sequentially at room temperature: gel filtration of VAC proteins containing fractions on a column with supersoid-12, to the torus was equilibrated with a buler of 20 mmol / l of bis-tris / HC1, 100 mmol / l of sodium chloride, pH 6 , 3, and ion exchange chromatography of the eluate on Mono O anion exchanger, which was equilibrated with the buffer used at the previous stage. Elution was carried out using a linear gradient of sodium chloride at 20 mmol / l. Bis-tris / HC1, pH 6.3, VAC proteins elute at 190 mmol / L sodium chloride. Then the fractions containing VAC proteins are rechromatographed on superose 12. After dialysis against TBS buffer, fractions containing VAC proteins with mole, May, 32000 and 34000 are stored at -70 ° C. All purification steps are carried out at 0-4 C,

Using disc electrophoresis in a polyacrylamide gel in the presence of sodium dodecyl sulfate, it was established that the obtained VAC activity is represented by proteins with a molecular weight of 32000 and 34000,

Isoelectric focusing at pH 3.5–9.5 in thin layers of polyacrylamide gel showed that VAC proteins have an isoelectric point at pH 4.4–4.6,

The amino acid composition of both VAC proteins, as determined by acid hydrolysis, is presented in the table (Trp was not determined).

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According to their biological activity, VAC proteins can be characterized as follows:

- they inhibit the activation of pro-Yabin with the help of blood clotting factor Xa in the presence of negatively charged phospholipids and Ca ions

- they do not inhibit the t-biological and amidolytic activity of factors Xa and Pa,

- they inhibit in vitro activation of factor X along the internal pathway using factor 1Xa in the presence of negatively charged phospholipids and

and ions,

- they inhibit in vitro blood vessel coagulation induced in vitro;

- they force out factor Xa and protrambin from the complex with the negatively charged surface of phospholipids;

- they prolong the thrombin time determined by the modified method;

- they prolong the activated, partial thromboplastin time determined by the modified method;

- they prolong the prothrombin time;

- their activity is not affected by collagenase and / or elastase.

When stored in TBS containing 10 vol% glycerol, VAS activity is stable for at least 3 months at -70 ° C, at 0 ° C - at least 12 hours, at 37 C - at least 30 minutes. At 56 C, activity disappears within 2 minutes.

The antithrombin-III obtained in the prototype method leads to an increase in the tendency to bleeding, due to the fact that it binds to the activated blood clotting factors, resulting in their inactivation. The resulting

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Compiled by V, Gudoshnikov Editor A, LOLINICH Tehred L, Serdyukova:

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/ W t t 200 No. (ractions

Proofreader M, Sharoshi

Claims (1)

Claim The method of obtaining proteins with mol.m, 32000-34000, inhibiting blood coagulation, by precipitation of the target product, followed by purification by chromatography, concentration and desalination using ultrafiltration, characterized in that, in order to improve the quality of the target product by preventing the tendency to bleeding when its use, as the raw material 25, the inner membranes of the aorta or vascularized tissue are used; prior to precipitation, they are homogenized in solution; their homogenization in solution is 100 mmol / l of sodium chloride, 50 mmol / l of tris (oxymethyl) -aminomethane hydrochloride with a pH of 7.5, containing sodium ethylenediaminetetraacetate, soybean trypsin inhibitor and benzamidine, the homogenate is centrifuged for 60 min at 10Ό000 g, the target product is precipitated by ammonium sulfate salting out and purification is carried out by hydroxyapatite chromatography, dialysis, ion exchange chromatography on sefacel with diatylaminoethyl groups, repeated dialysis, then either chromatography on Sephadex G = 100 followed by dialysis, or gel filtration on soup erose-12, ion-exchange chromatography on Mono Q anion exchange resin and repeated gel filtration on superose-12, followed by dialysis.