WO2008020739A2

WO2008020739A2 - METHOD FOR EXTRACTING α-SPECIFIC TROMBIN-LIKE ENZYME (ANCISTRON-B) FROM AGKISTRODON BLOMHOFFII USSURIENSIS VENOM

Info

Publication number: WO2008020739A2
Application number: PCT/MN2006/000001
Authority: WO
Inventors: Georgiy Volkov; Alexiy Savchuk; Vitaly Karbovskyy
Original assignee: Wisdom Asset Company
Priority date: 2006-08-16
Filing date: 2006-08-30
Publication date: 2008-02-21
Also published as: WO2008020739A3

Abstract

The invention relates to producing bioactive substances and can be used for biotechnological processes, in medicine and in the pharmacological industry, in particular to producing medicinal and diagnosis preparations from snake venom. The inventive method consists in carrying out affinity chromatography using a sodium chloride step gradient, wherein, at the first stage, 10mM Tris HCl with a pH of 7.4 is used in the form of a buffer solution and said solution having a sodium chloride concentration of 2M is used in the form of an eluent. The albumin peak exhibiting trombin-like activity is collected, concentrated and, with a view to substitutea buffer, is applied to a column with Sephadex G 25 which is balanced by a 50mM Tris HCl buffer with a pH of 7.4 and a 0.13 M NaCl content.

Description

Title of invention:

The method of isolation of the α-specific thrombin-like enzyme (ANCISTRON-B) from Agistrodop blotchoffii and syriepsis venom

IPC: A 61 K 35/58 ⁸

FIELD OF THE INVENTION

The invention relates to the industry of bioactive substances and can be used in biotechnological industries, medicine and the pharmaceutical industry, namely the production of medicines and diagnostic preparations from snake venom.

BACKGROUND OF THE INVENTION An α-specific thrombin-like enzyme cleaves fibrinopeptides A from fibrinogen, thereby participating in the formation of desAA fibrin. The only source of thrombin-like enzymes are snake venoms. The composition of the poison in different snakes varies, this is due to the species specificity of the poisons, as well as to the habitat and living conditions.

A known method for producing thrombin-like enzymes from raw venom of snakes of the genus Agististop (Solovyov D.A., Ugarova T.P. Isolation and characterization of α-specific thrombin-like enzymes from venom of common thyroid muzzle . -1993- t.58 -N8.C.1221-1233) in which the source of raw materials is the venom of snakes Agstistopod halus halus and Agstistopot blothoffiii. The method includes dissolving the poison in a buffer solution and subsequent purification using sorbent-cybacron agarose. The protein yield of the active fraction is 1.1-5.1%.

There is a method of obtaining thrombin-like enzymes from snake venom Triteresis oxypavepsis by fractionation of poisoned buffer with Sephadex G-IOO, chromatography on SM-Touorerl 650M in two stages and final purification on Mono Q gel. (Nose S., Shimohigashi Y. et al. Rurifiсtiop apd сhаrаsterizatiop оf а soаgulapt epzume, okіpakho-bip II, frоm Тrеtеrіsіpіs іkіpаvepsis (Ніtehаbесепесепе) fibrinopeptides A apd B., Toxisop. -1994. -V 32, -P.1509-1520) The protein yield of the active fraction is 5.15%.

A known method for producing thrombin-like enzymes from crude venom of snakes of the genus Agistrodop sakhatilis, in which raw poison dissolved in a buffer, is subjected to gradual fractionation on Q-Sepharose, Sephadex G-75 and Mono Q gel (Koh. Y., Chung K., Kim. D. Вісхемисal сhаrаsterizаtiop оf а thrоmpіp-likе уеу аn а a frіpоlūtis sеrеpe rоroteas frоm аpak (Agkistrodop sakhatillis) vіpom., Tohisv. 5.5. %

A known method for producing thrombin-like enzymes from Agistrodop asiatica poison, in which the poison dissolved in the buffer is chromatographed on DEAE-Sepharose, Sephacryl S-200 and CM-Sepharose (Huapg Q., Teng M., Niu L. Purifacitum aprophota -slottipg ezproms frive-raspé spake (Agkistrodop asitis) vepom., Tohisop. -1999. -V.37, N7. -P.999-1013)

A method is known for producing thrombin-like enzymes from Agistrodop asiatica poison, in which the poison dissolved in the buffer is subjected to separation of a DEAE Sephadex A-50 anion exchanger, then chromatographed twice on Sephadex G-200 (Quapg C, Hopg J. et al. -Like rripsyler of Agkistrodop asipis vpom apd its comraritiop with bvipe thrombi., Thromb.Diath.hayemorrth -1971. -V.26 -P.224-234).

A known method of producing thrombin-like enzymes from crude venom of snakes of the genus Agistrodop asites, in which the venom is dissolved in a buffer (pH 7.2), is subjected to sequential chromatography on Sephadex G-75, an anion exchanger

DEAE-Sephadex A-50 with a gradient of sodium chloride 0.01-0.6M, sorbent

Heparin-Sepharose CL-6B (gradient of sodium chloride NaCl 0.1-0.3; 0.5M) and, in a separate case, additionally on Sephadex G-100. (Komori Y., Nikai T. et al. And the program is stud оf сlоttiпg fаtоrs frоm thе vеpom оf Аgкistrоdop асіtіs сoіlеsted іп сіпа іпап Тaiwап.,

Comp.Biochem.Physiol. -1987. -V.88B, N2. -P643-649).

A known method of producing thrombin-like enzymes from the raw poison of snakes Віthrоs аtrox, which consists in isolating a thrombin-like fraction that can cause fibrinogen coagulation. (Butlemap WH, Weiss LJ., Thеrombi-likе аbout frоm thаtors аtroh аmіpobеzamidіpe-substitutеd аgarose. J Віl.Сhem. 1976, Mag. -25. -251 (6). -P.1663-1669)

A known method is characterized by the fact that the poison dissolved in the buffer solution is subjected to stepwise chromatography on Sephadex G-IOO, on a SM-Touorerl 650M cation exchanger with a linear gradient of sodium chloride concentration and a full gradient volume, which provide the most complete identification and separate elution of thrombin-like enzymes, then separate rechromatography of these enzymes, setting linear gradients of sodium chloride concentration from elution conditions of the corresponding thrombus-like enzyme in the previous e while increasing the total volume of these gradients to levels which provides additional separation of adjacent thrombin enzyme to give the desired product in a homogeneous condition after the separate cleaning enzyme thrombin on the sorbent ABA-TSK. (RU 2271219 Cl (2004123274/15, 07.28.2004) 10.03.2006 Bull.7., Method for producing thrombin-like enzymes from snake venom).

A method for purification of thrombin-like proteases from snake venom is proposed, in which the protease in the form of a crude product is subjected to preliminary purification using affinity chromatography on basic ion exchangers and the obtained fraction is subjected to chromatography on weak cation exchangers or separated by adsorption on glass in the main region. (WO 97/32015 (PCT / EP97 / 00755) Method of perfection of thrombose of neck of perfume of from spike of vopom).

The closest to the achieved positive result is the method of obtaining thrombin-like enzymes from snake venom, which is characterized by the fact that in chromatographic purification of snake venom, identification and displacement of fractions containing thrombin-like enzyme, followed by isolation of the target product by chromatography on Sercharos, equilibrated with TPIC-HC1 buffer , while the poison of the snake Agistrodop halus in a 0.05M sodium chloride solution is chromatographed on a column filled with Serhasrul S-300 HR, after identification and mixing of stocks with a thrombin-like enzyme they are concentrated by ultrafiltration with selection by molecular weight SC, the resulting solution is chromatographed on a column with DEAE Serharose FF, balanced with acetate buffer solution, fractions with a thrombin-like enzyme are collected and mixed, re-concentrated by ultrafiltration, then the concentrate is chromatographed on a SP Serharose FF gel column, the fractions containing the target product are combined, diluted with distilled water to 12-15 s in a thrombin time test, sucrose, sodium azide are added up to 0.01%, poured into vials and freeze-dried. (RU 2 262 947C2 (2002120163/15, 07.24.2002) A method for producing a thrombin-like coagulating enzyme).

The disadvantage of the prototype is both a low level of profitability of the production process, a low level of purity and a large investment of time.

SUMMARY OF THE INVENTION

The basis of the invention is the task to develop a method for the isolation of a thrombin-like enzyme (ANCISTRON-B) from Agistrodop Yothoffii poison, allowing, by means of affinity chromatography and the use of carriers with higher chromatographic properties and physico-chemical stability, to increase the profitability of the production process, to shorten the time for thrombin isolation enzyme and increase its purity.

The problem is solved in that affinity chromatography is carried out using a stepwise gradient of sodium chloride, using 1OmM Tris HCl with pH 7.4 as a buffer solution containing 0.5M NaCl and the indicated solution with a concentration of sodium chloride 2M as eluent in the first stage. The protein peak containing thrombin-like activity is collected, concentrated, and applied in order to change the buffer onto a column with Serhadex G 25 balanced with 5OmM Tris HCl buffer with pH 7.4 containing 0.13 M NaCl.

The implementation of the invention

The problem is solved by the proposed method, when the parameters of the process of isolation of a thrombin-like enzyme are in the range specified in the claims. When the parameters are changed in the direction of increasing or decreasing, the characteristics of the resulting preparation worsen. The invention is illustrated by examples 1-3 of a specific implementation. Example 1

5 ml of 1OmM Tris HCl buffer with pH 7.4 is added to 100 mg of Far Eastern thyroid muzzle venom and mixed until the venom is completely dissolved at 4 ^° C. The dissolved venom is centrifuged at 5000 rpm at 4 ^° C for 15 minutes. The precipitate is discarded and the supernatant is used for affinity chromatography. The solution of the poison is applied to a column (C 10/10, volume 7 ml) with an affinity sorbent Vlie Serharose FF, previously equilibrated with 50 ml of 1OmM Tris HCl buffer with a pH of 7.4 (column equilibration rate of 2 ml / min, poison application rate 0.5 ml / min ) After application, 150 ml of 1OmM Tris HCl buffer with a pH of 7.4 and 50 ml of 1OmM Tris HCl buffer with a pH of 7.4 containing 0.5 M NaCl at a rate of 2 ml / min are passed through the column. The optical density was recorded continuously using a flow monitor REC 102 (Parmacia biotech). Under these conditions, part of the proteins (over 45%) is not adsorbed on the sorbent, and Ancistron binds to Vlie Serharose FF and elute with mM Tris HCl containing a buffer with a pH of 7.4 with a content of 2M NaCl at a rate of 2 ml / min. The protein peak containing thrombin-like activity is collected (100 ml), concentrated to 30 ml and, with the aim of changing the buffer, it is applied to a column with Serhadex G 25 (column size 2.5x27cm, volume 120ml), equilibrated with 5OmM Tris HCl buffer with pH 7.4 containing 0.1 ZM NaCl at a rate of 5 ml / min. The optical density was recorded continuously using a flow monitor REC 102 (Parmacia biotech). The fraction containing Antsistron (48 ml) is collected, packaged in vials according to activity (BSA (2 mg / ml) is used as a stabilizer) and dried by lyophilization. Characteristic Yield, mg 4.2 mg (8.4%)

Biological activity 35.7 NSh / mg

The degree of purification, time 20.5

Example 2. To 110 mg of Far Eastern mollusk venom add 5.1 ml of 1OmM Tris HCl buffer with a pH of 7.4 and move until the poison is completely dissolved at 4 ⁰ C. Dissolved venom centrifuged at 5000 rpm at 4 ⁰ C for 15 minutes The precipitate is discarded and the supernatant is used for affinity chromatography. The solution of the poison is applied to a column (C 10/10, volume 7 ml) with an affinity sorbent Vlie Serharose FF, previously equilibrated with 50 ml of 1OmM Tris HCl buffer with a pH of 7.4 (column equilibration rate of 2 ml / min, poison application rate 0.5 ml / min ) After application, 165 ml of 1OmM Tris HCl buffer with a pH of 7.4 and 55 ml of 1OmM Tris HCl buffer with a pH of 7.4 containing 0.5 M NaCl at a rate of 2 ml / min are passed through a column. The optical density was recorded continuously using a flow monitor REC 102 (Parmacia biotech). Under these conditions, part of the proteins (over 45%) is not adsorbed on the sorbent, and Ancistron binds to Vlie Serharose FF and elute with mM Tris HCl containing a buffer with a pH of 7.4 with a content of 2M NaCl at a rate of 2 ml / min. The protein peak containing thrombin-like activity is collected (110 ml), concentrated to 32 ml and, in order to change the buffer, it is applied to a column with Serdex G 25 (column size 2.5x27 cm, volume 120 ml), balanced with 5OmM Tris HCl buffer with pH 7.4 containing 0.1 ZM NaCl at a rate of 5 ml / min. The optical density was recorded continuously using a flow monitor REC 102 (Parmacia biotech). The fraction containing Antsistron (45 ml) is collected, packaged in vials according to activity (BSA (2 mg / ml) is used as a stabilizer) and dried by lyophilization. Characteristic

Yield, mg 4.4 mg (8.6%)

Biological activity 35.0 NIH / mg

The degree of purification, times 19.7

Example 3

4.8 ml of 1OmM Tris HCl buffer with a pH of 7.4 is added to 97 mg of Far Eastern mollusk poison and transferred until the poison is completely dissolved at 4 ⁰ C. The dissolved poison is centrifuged at 5000 rpm at 4 ⁰ C for 15 minutes. The precipitate is discarded and the supernatant is used for affinity chromatography. The solution of the poison is applied to a column (C 10/10, volume 7 ml) with an affinity sorbent Vlie Serharose FF, previously equilibrated with 50 ml of 1OmM Tris HCl buffer with pH 7.4 (speed column balancing 2 ml / min, the rate of deposition of poison 0.5 ml / min). After application, 170 ml of 1OmM Tris HCl buffer with a pH of 7.4 and 45 ml of 1OmM Tris HCl buffer with a pH of 7.4 containing 0.5 M NaCl at a rate of 2 ml / min are passed through a column. Optical density was recorded continuously using a flow monitor REC 120 (Parmacia biotech). Under these conditions, part of the proteins (over 45%) is not adsorbed on the sorbent, and Ancistron binds to Vlie Serharose FF and elute with mM Tris HCl containing a buffer with a pH of 7.4 with a content of 2M NaCl at a rate of 2 ml / min. The protein peak containing thrombin-like activity is collected (89 ml), concentrated to 26 ml and, with the aim of changing the buffer, it is applied to a column with Serhadex G 25 (column size 2.5x27 cm, volume 120 ml), balanced with 5OmM Tris HCl buffer with pH 7.4 containing 0.13 M NaCl with 5 ml / min. The optical density was recorded continuously using a flow monitor REC 102 (Parmacia biotech). The fraction containing Antsistron (50 ml) is collected, packaged in vials according to activity (BSA (2 mg / ml) is used as a stabilizer) and dried by lyophilization. Characteristic

Yield, mg 3.9 mg (7.9%)

Biological activity 36.2 NIH / mg

The degree of purification, times 21.2

Claims

Claim

A method for isolating an α-specific thrombin-like enzyme (ANCISTRON-B) from Agistrodop blotchoffii and syriepsis poison, including dissolving the poison in a buffer solution followed by centrifugation, affinity chromatography using a stepwise gradient of sodium chloride, collecting fractions containing the biological activity of an α-specific enzyme and lyophil-specific characterized in that the affinity chromatography is carried out on a column with the carrier Blie Serharose FF, using, in the first stage, 1OmM Tris HCl with p 7.4, containing 0.5 M NaCl and the indicated solution with a concentration of sodium chloride 2M as an eluent and a protein peak containing thrombin-like activity, are collected, concentrated, and applied to a column with Serhadex G 25 equilibrated with 5OmM Tris HCl buffer with pH to change the buffer 7.4 with a content of 0.13 M NaCl.