Title of Invention	A METHOD OF PURIFYING A THROMBIN LIKE PROTEASES FROM SNAKE VENOMS
Abstract	A process for purifying thrombin-like proteases from snake venoms which comprises removing impurities from the protesses in three chromatographic steps is described.

Full Text

The present invention relates to a process for purifying thrombin—like proteases from snake venoms. Examples of such proteases are batroxobin, crotalase and, in particular, ancrod. The latter is an anticoagulant isolated from the venom of the snake Agkistrodon rhodostoma (Merck index 1989, No. 664). A large number of methods for preparing it from snake venom has been described (GB-B 1,094,301, GB-B 1,177,506, GB-B 1,293,793, US-A 3,743,722, US-A 3,879,369, DE-A-2,428,955, DE-A-2,734,427). The processes are essentially based on chromatographic steps and the resulting ancrod varies in yield and purity. There has as yet been no successful preparation of highly pure ancrod from snake venom. There has always been isolation of a mixture of enzymes with ancrod as the main component more or less contaminated with foreign proteins depending on the preparation. We have now found a way of preparing thrombin-like proteases in highly pure form from snake venoms. Accordingly the present invention provides a method of purifying a thrombin-like protease from snake venom selected from the group consisting of ancrod and strongly basic proteases, characterized in that it comprises a) obtaining a solution comprising the protease; b) loading the protease solution onto an affinity chromatography matrix or an anion exchange resin; c) eluting the protease in an eluate solution from the affinity chromatography matrix or anion exchange resin; d) loading the eluate solution from step c) onto a matrix f glass beads; said glass beads having a pore diameter of from 25 to 35 nm and a particle size or from 30 to 6|um; said eluate being applied to the matrix of glass beads in a basic solution at a pH value of from 7.5 to 9.0 whereby the glass beads adsorb the thrombin-like protease from Snake venom; e) eluting the protease in an eluate solution from the matrix of glass beads; f) loading the eluate solution from step c) onto a size exclusion gel matrix or onto a matrix of glass beads; said glass beads having a pore diameter of from, 25 to 35 nm and a particle size of from 30 to 60 um; said eluate being applied to the matrix of glass beads in an acidic solution; and g) eluting and recovering the purified protease. The invention furthermore relates to tnromiDin-iiKe pjr^teases trom snake venoms in a purity of from 95 to 100 %. It is advisable to carry out stage b by purification by adsorption onto glass and stage c by gel chromatography. In a particularly preferred embodiment of the invention, purification is by adsorption or chromatography on glass both in stage b and in stage c. Particularly suitable for the preliminary purification by affinity chromatography is agmatine-, arginine- or heparin-Sepharose. Basic ion exchangers suitable for the preliminary purification are, in particular, DEAE-cellulose and DEAE-Sepharose. Buffers which may be mentioned for the ion exchange chromatography are, in particular, tris phosphate and sodium phosphate buffers. The ion exchange chromatography is carried out at a pH of 5-9, preferably 6-8.5. In the preliminary purification, about 70-80% of foreign proteins and other constituents are removed from the crude enzyme. If the second step in the purification is carried out with a cation exchanger, the following weakly acidic exchangers are suitable: CM-SEPHAROSE, pH 5-9, and AMBERLITE CG50, pH 5-9. Chromatography on glass means that ancrod and related thror:i)in-like enzymes, and highly basic proteases, are bound at a pH of 7.5-9.0, preferably 8.0-8.5, to the glass matrix. About 60 % of foreign proteins which are, in particular, acidic are washed off the column unbound with the equilibration buffer (preferably tris phosphate or sodium phosphate buffer). Ancrod is fractionally eluted from the glass in a purity of more than 90 % by increasing the ionic strength of the buffer by adding sodium chloride to 0*3-1.0 M, In the second purification step, the enzyme is concentrated to about 90 %. Particularly suitable for the gel chromatography as purification step c ^.re: SEPHACRYL S-IOOHR, SUPERDEX, SEPHADEX, ULTROGEL and SUPEROSE. If chromatography on glass is chosen for this purification step c, basic foreign proteins are adsorbed from the ancrod solution to the glass surface in the acidic pH range 4-6, while ancrod can be eluted from the column in a purity of much greater than 95 % directly in the equilibration buffer. The required ionic strength of the buffer can be controlled by adding a salt such as sodium chloride* The novel process is very particularly suitable for purifying ancrod which results from this process in a purity of distinctly more than 95 %• Example 1 a. Preliminary purification 3 g of dried venom from the Malayan pit viper were dissolved in 50 ml of tris(hydroxymethyl)aminomethane (TRIS) phosphate buffer pH 8.5, insoluble cellular constituents of the venom were removed by centrifugation, and the clear yellow solution was loaded onto a chromatography column which had a diameter of 1.6 cm and was packed to a height of 30 cm with DEAE-SEPHAROSE-FF (supplied by Pharmacia). The thrombin-like enzymes in the venom, and the acidic proteins, were bound to the matrix. The chromatography was carried out with a flow rate of 150-200 ml/h. About 70-80 % of the foreign proteins (based on the optical density of the initial solution at 280 nm) were eluted by washing the column at room temperature with about 300 ml of equilibration buffer (10 mM TRIS phosphate buffer pH 8.5) until the A280nin of the eluate had fallen below 0.5, and washing further with 400 m.l of 35 mM TRIS phosphate buffer pH 5.0 until the A280run of the eluate was b. Kain purification The eluate containing ancrod was concentrated to 20 ml by ultrafiltration using a membrane with a nominal separation limit of 10,000 daltons, and the buffer was changed to 100 mM TRIS phosphate buffer pH 8.0. This solution was loaded onto a column which had a diameter of 1.6 cm and was packed to a height of 15 cin with BIORAN CPG glass (supplied by Schott^ pore diameter: 25 - 35 nm, particle size: 30 - 60 um). About 60 % of foreign proteins (based on the optical density of the loaded material at 230 nm) were eluted from the column by washing the column at room temperature with 300 ml of the 100 mM TRIS phosphate buffer pH 8.0 at a flow rate of 250 ml per hour. A 0.5 M sodium chloride solution buffered to pH 8.0 with 100 mM TRIS phosphate was employed for elution. The eluate was automatically collected in fractions of about 10 ml. The thrombin-like enzymes were eluted in a peak followed by a tailing region. In order to obtain the main component (corresponding to ancrod) in a form containing no more than 5 % of thrombin-like additional components/ individual fractions in the transition from the main peak to the tailing region were examined both for their specific fibrinogenase activity and for their composition by a reverse phase HPLC. Only fractions which had a specific activity of more than 1700 U/OD280nm and showed less than 10 % of additional components in the HPLC were combined with the main peak (about 80 ml). The main component was obtained from the BIORAN column in a purity of 96 % and a yield of 72 %. c. Final purification The eluate containing the main component ancrod was concentrated to 2 ml by ultrafiltration using a YM 10 membrane (supplied by Arrdcon) and the resulting concentrate was loaded onto a column vjhich had a diameter of 1.6 cm and was packed to a height of 85 cm with SEPHACRYL S-100 HR. The column had previously been equilibrated with a buffer of 100 mM sodium chloride and 100 mM sodium phosphate of pH 6.9. Remaining proteases and TRIS were removed from the ancrod in this gel chromatography. The yield in this step was about 90 %. Example 2 a. Preliminary purification 2.1 g of dried venom from the Malayan pit viper were dissolved in 50 ml cf 35 mM TRIS phosphate buffer pH 8.5, insoluble cellular constituents of the venom were removed by centrifugation, and the clear yellow solution was loaded onto a chromatography column v.:.ich had a diameter of 1.5 cm and was packed to a height of 3 0 cm V7ith DEAE-SEPHAROSE-FF (supplied by Pharmacia) and was equilibrated with the abovementioned buffer. About 70 % of the foreign proteins (based on the optical density of the initial solution at 280 nm) were eluted by washing the column at room temperature with 600 ml of equilibration buffer until the A280 of the eluate was b. Main purification The eluate was concentrated to 20 ml as in Example 1, and the buffer was changed to 50 rrd!-i Na phosphate pH 8.5. This solution was loaded onto a BIORAN CPG glass column (diameter: 1.6 cm, height: 15 cm, pore diameter: 25 - 35 nm, particle size: 30 - 60 |am). About 60 % of foreign proteins (based on the optical density of the loaded material at 280 nm) were eluted from the column by washing the column at room temperature with 300 ml of the 50 mM Na phosphate buffer pH 8.5 at a flow rate of 250 ml per hour. AIM sodium chloride solution buffered to pH 8.0 with 50 mM Na phosphate was employed to elute the thrombin-like enzymes. About 80 % of the loaded enzyme units were eluted with 150 ml of buffer. c. Final purification The eluate was concentrated to 20 ml as above and the buffer was changed to 50 mM Na phosphate pH 5.0. This solution was loaded onto a column which likewise had a diameter of 1.6 cm and was packed to a height of 15 cm with BIORAN CPG glass which, however, had a pore diameter of 90 - 110 nm and a particle size of 30 - 60 ^m. At pH 5.0, only basic proteins and the additional components were bound to the BIORAN glass, while the main component ancrod was eluted from the column with the equilibration buffer in a purity of approximately 100 % and in a yield of about 80 -90 % (based on the loaded units). ExaiTiple 3 a, b- Preliminary and main purification 2.1 g of dried venom from the Malayan pit viper were initially fractionated on DEAE-SEPHAROSE as in Example 2, the eluate was concentrated to 20 ml as in Example 1, and the buffer was changed to 4 0 niM TRIS phosphate pH 6.2. This solution was loaded onto a chromatography column which had a diameter of 1.6 cm and was packed to a height of 20 cm with CM-SEPHAROSE-FF (supplied by Phani'.acia) and was equilibrated with the abovementioned buffer. About 60 % of foreign proteins (based on the optical density of the loaded material at 280 nm) were eluted from the column by washing the column at room temperature with 300 ml of the equilibration buffer at a flow rate of 250 ml/h. A 1 M sodium chloride solution buffered to pH 8.0 with 50 mM Na phosphate was employed as in Example 2 to elute the thrombin-like enzymes. About 80 % of the loaded units of thrombin-like enzymes were eluted with 150 ml of buffer. c. Final purification The final purification of ancrod was carried out as in Example 2 on BIORAN CPG glass (pore diameter about 100 nm; particle size 30 - 60 um) with a 50 mM phosphate buffer pH 5.0.- Ancrod was eluted from the column in a purity of more than 95 % and a yield of about 85 % (based on the loaded units). WE CLAIM: 1. A method of purifying a thrombin-like protease from snake venom selected from the group consisting of ancrod and strongly basic proteases, characterized in that it comprises a) obtaining a solution comprising the protease; b) loading the protease solution onto an affinity chromatography matrix or an anion exchange resin; c) eluting the protease in an eluate solution from the affinity chromatography matrix or anion exchange resin; d) loading the eluate solution from step c) onto a matrix of glass beads; said glass beads having a pore diameter of from 25 to 35 nm and a particle size or from 30 to 6 um; said eluate being applied to the matrix of glass beads in a basic solution at a pH value of from 7.5 to 9.0 whereby the glass beads adsorb the thrombin-like protease fix)m Snake venom; e) eluting the protease in an eluate solution from the matrix of glass beads; f) loading the eluate solution from step c) onto a size exclusion gel matrix or onto a matrix of glass beads; said glass beads having a pore diameter of from 25 to 35 nm and a particle size of from 30 to 60 um; said eluate being applied to the matrix of glass beads in an acidic solution; and g) eluting and recovering the purified protease. 2. The method as claimed in claim 1, wherein the purification in step f) is carried out using a size exclusion gel matrix. 3. The method as claimed in claim 1, wherein the eluate solution is loaded onto a matrix of glass beads in step f). 4. The method as claimed in claim 1 in which the snake venom is from a snake of the genus Agkistrodon. 5. The method as claimed in claim 4 in which the snake venom is from Agkistro rhodostoma 6. The method as claimed in claim 1 wherein the purified product is ancrod. 7. A method of purifying a thrombin-like protease from snake venom as herein described and exemplified.

Documents:

355-mas-1997-abstract.pdf

355-mas-1997-claims filed.pdf

355-mas-1997-claims granted.pdf

355-mas-1997-correspondnece-others.pdf

355-mas-1997-correspondnece-po.pdf

355-mas-1997-description(complete)filed.pdf

355-mas-1997-description(complete)granted.pdf

355-mas-1997-form 1.pdf

355-mas-1997-form 26.pdf

355-mas-1997-form 3.pdf

355-mas-1997-form 4.pdf

355-mas-1997-form 5.pdf