Note: Descriptions are shown in the official language in which they were submitted.
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PURIFICATION OF THROMBIN-LIKE PROTEASES FROM SNAKE VENOMS
The present invention relates to a process i:or purifying throm-
bin-like proteases from snake venoms.
Examples of sut:h proteases are batroxobin, c:rotalase and, in par-
ticular, ancrod. The latter is an anticoagulant which is isolated
from the venom of the snake Agkistrodon rhodostoma (Merck Index
1989, No. 664). A mult'iplicitx 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, US Patent
3,743,722, US Patent 3,879,369, German Offenlegungsschrift
2,428,955, German Offenlegungsschrift 2,734,427). These processes
are essentially based on chromatographic steps and yield the an-
crod in varying yield and purity.
The preparation of, highly pure ancrod from anake venom has been
unsuccessful until now. A mixture of enzyme's with ancrod as the
main component was always isolated which, depending on the prepa-
ration, 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 tea a process for, purifying thrombin-'like
proteases.from snake venoms, which consists in
(1) subjecting.a protease crude product to a prepurification by
affinity chromatography or chromatography on a basic ion ex-
changer,. '
(2) 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 ad:~orption on glass and
(3) subjecting the main component from step 2 to gel chroinatogra-
phy or purifying this component in the acidic range by chro-
matography.on glass;
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where, however, at least one of steps (2) and (3) comprises a separation by
adsorption or chromatography on glass.
More specifically, the invention as claimed relates t:o a process wherein the
above mentioned steps (1 ) to (3) are detailed as follows:
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 of
from 30 to 60 arm; 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 e) onto a size exclusion gel matrix
or
onto a matrix of glass beads; said glass beads having a pore diameter of from
90 to 110 nm and a particle size of from 30 to 60 arm; 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 thrombin-like proteases from snake venoms
in a purity of from 95 to 100%.
It is recommended for step (2) to carry out the purification by adsorption on
glass and step {3) with the aid of gel chromatography.
In a particularly preferred embodiment of the invention, purification both in
step
(2) and in step (3) is carried out by adsorption or chrornatography on glass.
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Agmatine-, a~ginine- or heparin-Sepharose is particularly suita-
ble for the prepurification by affinity chromatography.
Basic ion exchangers suitable for the prepurification are parti-
cularly DEAF-cellulose and DEAF-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, prefe-
rably of 6-8.5.
During the prepurification, approximately 7()-80~ of foreign pro-
teins and other constituents are removed from the crude enzyme.
If the second step of the purification is carried out using a.ca-
tion exchanger, those suitable are the follbwing weakly acidic .
exchangersa 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-phos-
phate 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 chlori-
de.
In the second purification step, the enzyme is concentrated to
approximately 90 0.
For gel chromatography as a purification step (3), suitable gels, in
particular,
are: SEPHACRYL S-100HR*, SUPERDEX*, SEPHADEX*, ULTRO-GEL* and
SUPEROSE*.
If chromatography on glass is selected for this purification step (3), in the
acidic
pH range from 4-6 basic foreign proteins are adsorbe(i on the glass surface
from
the ancrod solution, while ancrod can be eluted from the column directly in
the
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equilibration buffer in far aver 95% purity. The desired ionic strength of the
buffer
can be regulated by addition of a salt such as sodium r>hloride.
The novel process is very particularly suitable for the prepara-
tion of ancrod in pure form, which according to this purification
process is obtained in a purity of. clearly over 954.
Example 1
a. Prepurification
3 g of dried venom of the Malayan pit viper were dissolved in
50 ml of tris(hydroxymethyl)aminomethane(TRIS)-phosphate buffer
pH 8.5, insoluble cell constituents of the venom were centrifuged
off and the clear, yellow solution was applied to a chromatogra-.
phy column which had a diameter of 1.6 cm and was packed up to a
height of 30 cm with DEAE-SEPHAROSE*FF (Pharmacies). The thrombin-
like enzymes of the venom and the proteins having acidic charac-
ter were bound to the matrix. Chromatography was carried out at a
flow rite of 150 - 200 ml/h. By washing the column at room tempe-
rature with about 300 m1 of equilibration buffer (10 mM TRIS-
phosphate buffer pH 8.5) until the A28omm [sic] value of the eluate
had fallen below 0.5 and further washing with 400 ml of 35 mM
TRIS-phosphate buffer pH 6.0 until the Az$o~" [sic] value of the
eluate was < 0.4, about 70 - 80 ~ of the foreign proteins (based
on the optical density of the starting solution at 280 nm) were
eluted. The main fraction containing ancrocl was eluted in 85 %
yield in 150 - 200 ml of 150 mM TRIS-phosphate buffer pH 6Ø
b. Main purification
The eluate which contained the ancrod was e:oncentrated to 20 ml
by means of ultrafiltration on a membrane having a nominal sepa-
ration limit of 10000 Daltons and rebuffered using a 100 mM TRIS-
phosphate buffer pH 8Ø This solution was applied to a column
which had a diameter of 1.6 cm and was packed up to a height of
l5 cm with BIORAN-CPG'~glass (Schott, pore diameter: 25 - 35 nm,
particle size: 30. - 60 Eun). By washing the column at room tempera-
ture with 300 ml.of the 100 mM TRIS-phosphate buffer pH 8.0 and
at a flow rate of 250 ml per hour, about 60 °s of foreign proteins
(based on the optical density of the material applied at 280 nm)
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were eluted from the column.
For elution, a 0.5 M sod~,um chloride solution which was buffered
to a pH of 8.0 using 100~mM TRIS-phosphate was employed. The
eluate was automatical~.y collected in about 10 ml fractions. The
thrombin-like enzymes were eluted in one peak with a subsequent
tailing range. In order to obtain the main .component (corresponds
to ancrod) in a form which contained at most 5 % of thrombin-like
secondary components, in the transition from the main peak to the
tailing range individual fractions were investigated by means of
reverse-phase HPLC both for their specific fibrinogenase activity
and for their composition. Only fractions which had a specific
activity of greater than 1700 U/OD28ox"" and which contained less
than 10 % of secondary components in HPLC were combined with the
main peak (about 80 mi). The main component was obtained from the
BIORAN column in a purity of 96 % and a yield of ?2 %.
c. Fine purification
The eluate containing the main component ancrod was concentrated
to 2 ml by ultrafiltration on a YM 10 memMrane (Amicon) and the
concentrate obtained was applied to a column which had a diameter.
of 1.6 cm and was packed up to a height of 85 cm with SEPHARYL~'
S-100 HR. The column had been equilibrated beforehand using a
buffer of 100 mM sodium chloride and 100 mM sodium phosphate,
which had a pH of 6.9. Residual proteases and TRIS were separated
from ancrod by means of this gel chromatography. The yield in
this step was about 90 %.
Example 2
a. Prepurification
2.1 g of dried venom of the Malayan pit viper were dissolved in
50 ml of 35 mM TRIS-phosphate buffer pH 8.5, insoluble cell con-
stituents of the venom were centrifuged off and the clear, yellow
solution was applied to a chromatography column which had a dia-
meter of 1.6 cm, was.packed up to a height of 30 cm with DEAF-SE-
PHAROSE-FF~(Pharmacia) and was equilibrated with the buffer men-
tioned above. By washing the column at room temperature with 600
ml of equilibration buffer until the A28o [sic] value of the
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0480/01161 CA 02247016 1998-os-ZO
eluate was < 0.2, approximately 70 % of the foreign proteins
(based on the optical density of the starting solution at 280 nm)
were eluted. The main fraction was eluted in 90 - 100% yield
using 200 - 250 ml of 150 mM TRIS-phosphate buffer pH 6Ø
5
b. Main purification
The eluate was concentrated to 20 ml as in Example 1 and
rebuffered using a 50 mM Na phosphate buffer pH 8.5. This
solution was applied to the BIORAN-CPG glass column (diameter:
1.6 cm, height: 15 cm, pore diameter:~25 - 35 nm, particle size:
30 - 60 Nm). By washing the column at room temperature with 300 ml
of the 50 mM Na phosphate buffer pH 8.5 and at a flow rate of 250
ml per hour, about 60 % of foreign proteins (based on the optical
density of the material applied at 280 nm) were eluted from the
column.
For elution of the thrombin-like enzymes, a 1 M sodium chloride
solution which had been buffered to a pH of 8.0 using 50 mM Na-
phosphate was employed. Using 150 ml of buffer, about 80 % of the
units of enzyme applied were eluted.
c. Fine purification
The eluate was concentrated to 20 ml as above and rebuffered
using a 50 mM Na phosphate buffer which had a pH of 5Ø This so-
lution was applied to a column which also had a diameter of
1~6 cm and was packed up 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 secondary components were bound to the BIORAN glass, while
the main component ancrod was eluted from the column using the
equilibration buffer in a purity of approximately 100 % and in
about 80 - 90% yield (based on the units applied).
Example 3
a, b. Pre- and main purification
2.1 g of dried venom of the Malayan pit viper were prefractiona-
ted on DEAF-SEPHAROSE analogously to Example 2, and the eluate
was concentrated to 20 ml analogously to Example 1 and rebuffered
using a 40 mM TRIS-phosphate buffer pH 6.2. This solution was ap-
plied to a chromatography column which had a diameter of 1.6 cm,
[lacuna] packed up to a height of 20 cm with CM-SEPHAROSE-FF
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(Pharmacia) and was equilibrated with the abovementioned buffer.
By washing the column at room temperature with 300 ml of the
equilibration buffer and at a flow rate of 250 ml/h, about 60 %
of foreign proteins (based on the optical density of the material
applied at 280 nm) were eluted from the column.
For elution of the thrombin-like enzymes, analogously to Example
2 a 1 M sodium chloride solution which was buffered to a pH of
8.0 using 50 mM Na phosphate was employed. Using 150 ml of buf-
fer, about 80 % of the units of thrombin-like enzymes applied
were eluted.
c. Fine purification
The fine purification of ancrod was carried out analogously to
Example 2 on BIORAN-CPG glass (pore diameter about 100 nm; parti-
cle size 30 - 60 Eun) using a 50 mM phosphate buffer pH 5Ø, Ancrod
was eluted from the column in over 95% purity and in about 85%
yield (based on the units applied).
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