Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Hydrolase
Reexamination Certificate
1998-08-17
2001-03-13
Weber, Jon P. (Department: 1651)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Hydrolase
C435S814000, C435S815000
Reexamination Certificate
active
06200791
ABSTRACT:
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 which is isolated from the venom of the snake Agkistrodon rhodostoma (Merck Index 1989, No. 664). A multiplicity of methods for its preparation from snake venom have already been described (GB Patent 1,094,301, GB Patent 1,177,506, GB Patent 1,293,793, U.S. Pat. No. 3,743,722, U.S. Pat. No. 3,879,369, German Offenlegungsschrift 2,428,955, German Offenlegungsschrift 2,734,427). These processes are essentially based on chromatographic steps and yield the ancrod in varying yield and purity.
The preparation of highly pure ancrod from snake venom has been unsuccessful until now. A mixture of enzymes with ancrod as the main component was always isolated which, depending on the preparation, was contaminated with more or less foreign proteins.
A way has now been found to prepare thrombin-like proteases from snake venoms in highly pure form.
The invention relates to a process for purifying thrombin-like proteases from snake venoms, which consists in
a. subjecting a protease crude product to a prepurification by affinity chromatography or chromatography on a basic ion exchanger,
b. subjecting the fraction containing the thrombin-like enzymes thus obtained to chromatography on a weak cation exchanger or separating it in the basic range by adsorption on glass and
c. subjecting the main component from step b to gel chromatography or purifying this component in the acidic range by chromatography on glass,
where, however, at least one of steps b and c comprises a separation by adsorption or chromatography on glass.
The invention furthermore relates to thrombin-like proteases from snake venoms in a purity of from 95 to 100%.
It is recommended for stage b to carry out the purification by adsorption on glass and stage c with the aid of gel chromatography.
In a particularly preferred embodiment of the invention, purification both in stage b and in stage c is carried out by adsorption or chromatography on glass.
Agmatine-, arginine- or heparin-Sepharose is particularly suitable for the prepurification by affinity chromatography.
Basic ion exchangers suitable for the prepurification are particularly DEAE-cellulose and DEAE-Sepharose.
Buffers which may be mentioned for ion exchange chromatography are, in particular, tris-phosphate and sodium phosphate buffers.
Ion exchange chromatography is carried out at a pH of 5-9, preferably of 6-8.5.
During the prepurification, approximately 70-80% of foreign proteins and other constituents are removed from the crude enzyme.
If the second step of the purification is carried out using a cation exchanger, those suitable are the following weakly acidic exchangers: CM-SEPHAROSE, pH 5-9, and AMBERLITE CG50, pH 5-9.
Chromatography on glass means that ancrod and related thrombin-like enzymes as well as strongly basic proteases are bound to the glass matrix at pHs of 7.5-9.0, preferably 8.0-8.5. About 60% of especially acidic foreign proteins are washed from the column in unbound form with the equilibration buffer (preferably tris-phosphate or sodium phosphate buffer). Ancrod is eluted from the glass fractionally in over 90% purity by increasing the ionic strength of the buffer to 0.3-1.0 M by addition of sodium chloride.
In the second purification step, the enzyme is concentrated to approximately 90%.
For gel chromatography as a purification step c, suitable gels, in particular, are: SEPHACRYL S-100HR, SUPERDEX, SEPHADEX, ULTROGEL and SUPEROSE.
If chromatography on glass is selected for this purification step c, in the acidic pH range from 4-6 basic foreign proteins are adsorbed on the glass surface from the ancrod solution, while ancrod can be eluted from the column directly in the equilibration buffer in far over 95% purity. The desired ionic strength of the buffer can be regulated by addition of a salt such as sodium chloride.
The novel process is very particularly suitable for the preparation of ancrod in pure form, which according to this purification process is obtained in a purity of clearly over 95%.
REFERENCES:
patent: 3743722 (1973-07-01), Nolan
patent: 3879369 (1975-04-01), Nolan
patent: 4027012 (1977-05-01), Antonini
patent: 4137127 (1979-01-01), Stocker
patent: 5712117 (1998-01-01), Sprecher
patent: 24 28 955 (1975-03-01), None
patent: 27 34 427 (1978-02-01), None
patent: 1094301 (1967-12-01), None
patent: 1177506 (1970-01-01), None
patent: 1293793 (1972-10-01), None
Merck Index, 11 Ed. 1989, p. 664.
Bonilla, “Defibrinating Enzyme from Timber Rattlesnake (Crotalus H. Horridus) Venom: A Potential Therapeutic for Defibrination I. Purification and Properties”, Thromb. Res., 6(2), pp. 151-169, Feb. 1975.
Schwarz Margarete
Zahn Wolfgang
Keil & Weinkauf
Knoll Aktiengesellschaft
Weber Jon P.
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