U.S. patent application number 10/701671 was filed with the patent office on 2004-09-23 for process for the preparation in pure form of the protease activating blood clotting factor vii, its proenzyme or a mixture of both proteins by means of ion-exchange chromatography.
This patent application is currently assigned to Aventis Behring GmbH. Invention is credited to Feussner, Annette, Roemisch, Juergen, Stoehr, Hans-Arnold.
Application Number | 20040186277 10/701671 |
Document ID | / |
Family ID | 7917489 |
Filed Date | 2004-09-23 |
United States Patent
Application |
20040186277 |
Kind Code |
A1 |
Roemisch, Juergen ; et
al. |
September 23, 2004 |
Process for the preparation in pure form of the protease activating
blood clotting factor VII, its proenzyme or a mixture of both
proteins by means of ion-exchange chromatography
Abstract
A process for the preparation in pure form of the protease
activating blood clotting factor VII and/or its proenzyme is
described in which these are obtained from biological fluids or
those obtained in the case of preparation by genetic engineering
a,) by anion- or cation-exchange chromatography at a pH below the
isoelectric point of the protein to be isolated or b) by a
combination of anion- or cation-exchange chromatography with
affinity chromatography and/or fractional precipitation at pHs of
between 2.5 and 9.0, preferably between 2.5 and 7.2, the affinity
chromatography being carried out using calcium
phosphate/hydroxyapatite a hydrophobic matrix, a chelate matrix, a
matrix which is coated with an immobilized monoclonal or polyclonal
antibody directed against the protein to be isolated, or its F(ab)
or F(ab).sub.2 fragments. A pharmaceutical preparation and a
reagent are moreover described which contain the said protease and
its proenzyme.
Inventors: |
Roemisch, Juergen; (Marburg,
DE) ; Feussner, Annette; (Marburg, DE) ;
Stoehr, Hans-Arnold; (Wetter, DE) |
Correspondence
Address: |
FINNEGAN, HENDERSON, FARABOW, GARRETT & DUNNER
LLP
1300 I STREET, NW
WASHINGTON
DC
20005
US
|
Assignee: |
Aventis Behring GmbH
|
Family ID: |
7917489 |
Appl. No.: |
10/701671 |
Filed: |
November 6, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10701671 |
Nov 6, 2003 |
|
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09632627 |
Aug 4, 2000 |
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6677440 |
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Current U.S.
Class: |
530/383 ;
435/226 |
Current CPC
Class: |
A61P 9/10 20180101; C12N
9/6424 20130101; A61K 38/00 20130101; A61P 7/04 20180101 |
Class at
Publication: |
530/383 ;
435/226 |
International
Class: |
C12N 009/64; C07K
014/745 |
Foreign Application Data
Date |
Code |
Application Number |
Aug 6, 1999 |
DE |
19937219.5 |
Claims
1. A process for the preparation in pure form of the protease
activating blood clotting factor VII and/or its proenzyme, wherein
they are obtained from biological fluids or those obtained in the
case of preparation by genetic engineering a) by anion- and/or
cation-exchange chromatography at a pH below the isoelectric point
of the protein to be isolated or b) by a combination of anion- or
cation-exchange chromatography with affinity chromatography and/or
fractional precipitation at pHs of between 2.5 and 9.0, preferably
between 2.5 and 7.2, the affinity-chromatography separation
processes being carried out using calcium phosphate/hydroxyapatite
a hydrophobic matrix, a chelate matrix, a matrix which is coated
with an immobilized monoclonal or polyclonal antibody directed
against the protein to be isolated, or its F(ab) or F(ab).sub.2
fragments.
2. The process as claimed in claim 1, wherein the preparation in
pure form is carried out in the presence of one or more protein
stabilizers which are selected from solubilizers, preferably
hydroxyproline, detergents, preferably Tween.RTM. or Triton.RTM.,
proteins, preferably albumin, gelatin, fibronectin, vitronectin or
similar proteins, reductants, preferably dithiothreitol,
mercaptoethanol or cystienee, and/or proteinase inhibitors such as
aprotinin, .alpha.-2-antiplasmin, C1-esterase inhibitor, the
inter-.alpha.-trypsin inhibitor, the antithrombinIII/heparin or
synthetic inhibitors.
3. The process as claimed in claim 2, wherein, in the preparation
in pure form, further protein stabilizers which are employed are
complexing agents of divalent ions, preferably EGTA, EDTA or
citrate and/or divalent ions, preferably calcium ions, and/or amino
acids, preferably glutamate, arginine, lysine or glycine, and/or
sugars, preferably glucose, arabinose, mannose or mannitol, and/or
alcohols, preferably ethylene glycol or polyethylene glycol.
4. The process as claimed in claims 1, wherein, for the
chromatographic separation of the protease activating factor VII
from its proenzyme by means of stepwise elution, a substance is
immobilized on the support material which has bonds of different
strength to the protease on the one hand and to the proenzyme on
the other hand, and the different eluates are then collected
separately from one another and the respective protein is isolated
from them.
5. The process as claimed in claim 1, wherein the fractional
precipitation of the protease and/or its proenzyme from its
solution is carried out by addition of polyethylene glycol from a
concentration of at least 10% by weight or ammonium sulfate from a
concentration of at least 15% by weight.
6. A pharmaceutical preparation, which comprises the protease
activating blood clotting factor VII and/or its proenzyme together
with one or more protein stabilizers as set forth in claims 2 and
3, for assisting blood clotting in the case of a tendency to
bleeding, in the case of absence of factors of the endogenous
clotting pathway, as FEIBA or for the prophylaxis and/or therapy of
syndromes associated with thrombotic complications, in inherited or
acquired deficiency states of the protease or its proenzyme, for
assisting wound healing alone or as a constituent of a fibrin
adhesive, a web or in combination with growth factors, for
subcutaneous or intramuscular, intravenous or topical
treatment.
7. The use of the pharmaceutical preparation of claim 6 for the
coating of surfaces of articles, consisting of plastic or metals,
to be implanted in the body, such as synthetic heart valves, blood
vessels or cannulas inserted for taking blood or for artificial
feeding.
8. A reagent comprising the protease activating blood clotting
factor VII and/or its proenzyme together with one or more protein
stabilizers as set forth in claims 2 and 3 for use in biological
test systems and for antigen detection.
Description
[0001] The invention relates to a process for the preparation in
pure form of the protease activating blood clotting factor VII, its
proenzyme or a mixture of both proteins, and of pharmaceutical
preparations which contain the proteins mentioned individually or
as a mixture.
[0002] German patent application 19 903 693.4 has already disclosed
a protease for the activation of blood clotting factor VII, a
process for its production, for its detection and for its
inactivation, and pharmaceutical preparations which contain this
protease. This protease, first isolated from plasma, occurs there
together with a nonactivated form, which is designated below as
"proenzyme". The protease activates blood clotting factor VII and
accelerates clotting, as has been shown by numerous experiments. In
the further investigation of the biological properties of this
protein, identified as serine protease, it emerged that
single-chain plasminogen activators, such as prourokinase, are also
effectively activated. Moreover, inactivation of factors V and VIII
in vitro was observed. In addition to the sequenced regions already
described in German patent application 19 903 693.4, N-terminal
sequencings of protease fractions were carried out. The following
amino acid sequences characterize the FVII-activating protease:
IYGGFKSTAGKHP; LLESLDPDXTPD; EFHEQSFRVEKI; SKFTXAXPXQFK; where X
means not identified. The sequences of the protease mentioned
elucidated up to now show that they agree 100% with sequences of
the protease published by Choi-Miura (Choi-Miura et al., J.
Biochem. 1996; 119: 1157 to 1165).
[0003] The investigations until now have especially concentrated on
the protease in its activated form. The inactive form of the
protease present in the plasma as a proenzyme was only recently
discovered by means of a protein band pattern in the SDS-PAGE after
reduction of the sample. Since, on the activation of the protease,
cleavage and thus activation take place at a site of the primary
structure typical for serine proteases two or more bands are
visible on electrophoresis. On reduction of the chains which are
connected by disulfide bridges, the individual bands become
recognizable in accordance with their lower molecular weight, the
proenzyme remaining as a large individual chain. This also became
clear in more complex solutions after transfer of the proteins to
membranes and subsequent Western blotting using suitable
antibodies.
[0004] For therapeutic reasons, there is now an interest in having
available both the protease in its activated form and the
proenzyme, in addition to the mixture of the two proteins
mentioned. Whereas the activated protease can be used for the rapid
activation of blood clotting factor VII or the single-chain
plasminogen activators in order to influence acute syndromes, the
proenzyme form of the protease is especially to be chosen as a
preferred agent for medium- to longer-term prophylaxis or treatment
of inherited or acquired deficiency states or alternatively for
increasing the plasma level beyond the physiological extent.
However, it is to be taken into account here that the stabilization
of an activated protease is difficult, since, for example,
self-degradation can take place or the molecule can be unstable on
account of its structural conditions. Previous studies have shown
that the protease activating factor VII can be isolated and
stabilized in its proenzyme form only under special
circumstances.
[0005] The previous investigations have shown that the biological
activities of this protease can be increased by calcium and/or
heparin or substances related to the latter. This property has
already been previously used in order to adsorb the protease on
immobilized heparin and to obtain an enriched fraction. Moreover,
it is already known that anion-exchange chromatography is also
suitable for the purification of the protease. The combination of
both purification steps is suitable for obtaining the protease in
enriched form. The use of an aprotinin matrix can also be used for
the preparation in pure form of the activated protease.
[0006] A process for the preparation in pure form and simultaneous
stabilization of the protease activating blood clotting factor VII
and/or its proenzyme has now been found, in which they are obtained
from biological fluids or those obtained in the case of preparation
by genetic engineering by
[0007] a) anion- and/or cation-exchange chromatography at a pH
below the isoelectric point of the to be isolated or
[0008] b) a combination of anion- or cation-exchange chromatography
with affinity chromatography
[0009] and/or fractional precipitation at pHs of between 2.5 and
9.0, preferably between 2.5 and 7.2, the affinity chromatography
being carried out using calcium phosphate/hydroxyapatite
[0010] a hydrophobic matrix,
[0011] a chelate matrix,
[0012] a matrix which is coated with an immobilized monoclonal or
polyclonal antibody directed against the protein to be isolated, or
its F(ab) or F(ab).sub.2 fragments.
[0013] A particularly suitable method for the preparation in pure
form of the protease and/or its proenzyme is anion- and/or
cation-exchange chromatography. The use of these methods for the
preparation in pure form of the activated protease was admittedly
proposed earlier, but the process conditions previously used did
not produce completely satisfactory results. This is principally
due to the fact that the risk of the activation of the proenzyme on
contact with the surfaces of the matrices is very high. The object
was therefore to develop a process which makes possible the
preparation of the activated protease and of the proenzyme in a
pure and stable form.
[0014] Surprisingly, it was then shown that very low pHs, in
particular pHs between 2.5 and 7.2, damage neither the activated
form of the protease nor the proenzyme and can therefore be
employed to good effect in the adsorption and elution. By this
means, almost trouble-free handling of the protease activating
factor VII is made possible, since most other proteases circulating
in the plasma are not active or only very slightly active in the
acidic medium and the risk of proteolytic activation is thus
minimized. The danger of the self-degradation of the protease is
also decreased in this way. Since, as is known, extremely acidic
pHs could include the risk of denaturation and thus bring about a
loss in activity of the protease, the activity of the protease
obtained from a strongly acidic medium was measured, for example,
by a photometric determination of the extinction occurring in the
case of action on chromogenic substrates. It was shown in this case
that both the protease and its proenzyme can be handled without
loss of activity in the short term, up to a pH of 2.0. At a pH of
2.5 to approximately 7.2, the protease and its proenzyme can be
stored for several months, the highest stabilities being observed
at a pH of below 6.5.
[0015] In this case it was surprisingly found that anion- and/or
cation-exchange chromatography at the low pHs mentioned can be used
for the purification of the protease and of the proenzyme. This is
so remarkable because in this case adsorption is possible on the
exchanger matrices at pHs which lie below the isoelectric point of
the protease or of the proenzyme.
[0016] Up to now, there is still no scientific explanation of on
which interactions this adsorption is based. However, it is
possible by means of this adsorption in the acidic medium to remove
a large number of impurities which do not bind to the matrices at
these pHs. A considerable enrichment of the protease on the matrix
is thus achieved. After washing the matrix, the protease and/or the
proenzyme can be eluted by an increase in the ionic strength.
[0017] For the preparation in pure form of the abovementioned
proteins, the anion-and/or cation-exchange chromatography can be
combined with affinity chromatography and/or fractional
precipitation. Independently of whether the abovementioned
purification processes are employed individually or in any desired
combination, it is recommended additionally to add protease
inhibitors in all process steps, in order to block cleavage of the
proenzyme into the activated protease. Suitable protein stabilizers
which are added in this case are
[0018] solubilizers, preferably hydroxyproline,
[0019] detergents, preferably Tween.RTM. or Triton.RTM.,
[0020] proteins, preferably albumin, gelatin, fibronectin and
vitronectin or similar proteins,
[0021] reductants, preferably dithiothreitol, mercaptoethanol or
cystiene, and/or
[0022] proteinase inhibitors such as aprotinin,
.alpha.-2-antiplasmin, C1-esterase inhibitor, inter-.alpha.-trypsin
inhibitor, antithrombin III/heparin or synthetic inhibitors.
[0023] A particularly good stabilization can be achieved during the
preparation in pure form of the protease and of the proenzyme if,
as further protein stabilizers,
[0024] complexing agents of divalent ions, preferably EGTA, EDTA or
citrate and/or
[0025] divalent ions, preferably calcium ions, and/or
[0026] amino acids, preferably glutamate, arginine, lysine or
glycine, and/or
[0027] sugars, preferably glucose, arabinose, mannose or mannitol,
and/or
[0028] alcohols, preferably ethylene glycol or polyethylene
glycol,
[0029] are added.
[0030] The abovementioned process steps can also additionally be
combined with fractional precipitation of the protease and/or its
proenzyme from its solution, which is carried out by addition
of
[0031] polyethylene glycol from a concentration of at least 10% by
weight or
[0032] ammonium sulfate from a concentration of at least 15% by
weight.
[0033] It is particularly worthy of note that in the processes
described above the proenzyme form of the protease can also be
obtained in pure form. As a matter of fact, it was seen that
ion-exchange chromatography, under the said acidic conditions,
using a solution which especially contained the proenzyme, led to
an eluate which contained the proenzyme exclusively or at least to
a very greatly enriched extent. In this case, the nativity of the
proenzyme thus obtained can be determined with the aid of one of
the activity tests which are described in German patent application
196 26 531.3. i.e., for example, by the photometric determination
of the extinction occurring on action on chromogenic substrates or
by the single-chain formation occurring after reduction of the
sample, which can be detected by SDS-PAGE/Western blotting. This
shows that according to the invention the preparation of the
proenzyme is possible in a rapid and efficient manner and with a
high yield.
[0034] When using the abovementioned process steps, it is thus
possible to obtain both the purified protease activating factor
VII, its proenzyme or alternatively a mixture of the activated
protease and the proenzyme. A route which is particularly worthy of
recommendation for the preparation of a pure activated protease
consists in the chromatographic separation of the protease
activating factor VII from its proenzyme by means of stepwise
elution, in which a substance is immobilized on the support
material which has bonds of different strength to the protease on
the one hand and to the proenzyme on the other hand. Different
eluates can thus be obtained which contain either only the
activated protease or only the proenzyme.
[0035] To obtain the proenzyme, an inhibitor with strong affinity
for the activated protease is in this case immobilized on the
matrix. Serine protease inhibitors, in particular C1 esterase
inhibitor, .DELTA.-2 antiplasmin, antithrombin III (with admixture
of heparin to the application solution) or low molecular weight,
high-affinity inhibitors, which can also be of synthetic nature,
are especially suitable for this. The proenzyme which is not bound
or less firmly bound to the matrix is then found in the solution
flowing through the column. On the other hand, for the preparation
in pure form of the activated protease, an inhibitor is immobilized
on the matrix which only has a weak inhibitory potential and
reversibly binds the activated protease. In this case, after
washing off the proenzyme, the active protease bound to the matrix
is eluted and can then be obtained in pure form. A suitable support
material for this process variant is, for example, one treated with
aprotinin or low molecular weight, reversible inhibitors, which can
also be of synthetic nature.
[0036] Of course, antibodies or fragments thereof, which can differ
between the activated protease and the proenzyme, can also be used
for the preparation of the said proteins in pure form. For this,
monoclonal antibodies can especially be employed which can
recognize "neoepitopes" after activation of the protease, i.e., for
example, the activation or cleavage site of the protein.
[0037] Under the affinity-chromatography processes to be employed
according to the invention, adsorption on calcium
phosphate/hydroxyapatit- e is to be emphasized as a simple and
rapid method for the enrichment of the protease and/or of the
proenzyme. In this case, the solution which contains the protease
and the proenzyme is mixed with calcium phosphate at a pH of
between 2.5 and 9.0, preferably between 2.5 and 7.2. After
subsequent sedimentation, e.g. by centrifugation or by filtration,
the sediment, if appropriate after resuspending one or more times
in a buffer solution, is eluted with addition of, for example, 0.2
M sodium citrate. The protease and the proenzyme are then found in
the eluate.
[0038] The adsorption of the protease on hydrophobic matrices or on
hydrophobic ligands which are coupled to appropriate matrices can
also be used according to the invention. Examples are phenyl- or
octyl-Sepharoses.RTM. or a phenylalanine coupled to a matrix. The
elution of the bound protein is carried out in a manner known per
se using a buffered solution of low ionic strength, which can
contain phenylalanine, glycerol or ethylene glycol.
[0039] Since the protease and the proenzyme enter into an
interaction with cations, especially with calcium and magnesium
ions, which is confirmed by an increase in their activity in the
presence thereof, chromatography by means of so-called "chelate
matrices" suggests itself for enrichment thereof from corresponding
solutions. Chelate compounds with zinc, copper or nickel ions are
particularly suitable in this case. After the washing of the matrix
loaded with the protease, an imidazole buffer can also be employed
for the elution of bound proteins, if appropriate with a linear
gradient.
[0040] The process according to the invention can also contain an
affinity-chromatography purification step in which the support
material employed is a matrix on which monoclonal or polyclonal
antibodies directed against the said proteins, or their F(ab) or
their F(ab).sub.2 fragments or other substances suitable for the
reversible binding of the said proteins, are immobilized.
[0041] The protease obtained by the process according to the
invention, its proenzyme or the mixture of both proteins can be
stored without loss of activity in the acidic pH range at pHs of
approximately 2.5 to 7.2 with or without the addition of additional
stabilizers, while in the alkaline range addition of stabilizers is
necessary.
[0042] Therapeutically, the said activated protease, the proenzyme
or the mixture of both compounds can be used to assist blood
clotting in the case of a tendency to bleeding, in the case of
absence of factors of the endogenous clotting branch or as FEIBA
(=factor VIII bypassing activity), but also for the endogenous and
exogenous activation of plasminogen activators such as prourokinase
or single-chain tPA. This activity can also be employed in
combination with single-chain or double-chain plasminogen
activators or anticoagulants by use of the said protease for the
prophylaxis or therapy of thromboembolic disorders. Syndromes which
are associated with thrombotic complications, such as cardiac
infarct, angina pectoris, stroke or leg vein thromboses, can thus
be successfully treated.
[0043] A further subject of the invention is therefore a
pharmaceutical preparation which contains an amount of the protease
activating blood clotting factor VII and/or its proenzyme form
sufficient for the dissolution of fibrin-containing thrombi. This
preparation can also moreover contain single-chain plasminogen
activators and/or anticoagulants. Expediently, a proteinase
stabilizer or a reductant such as dithiothreitol, mercaptoethanol
or cystienee is additionally added to the preparation in order to
reduce the risk of polymer formation during processing or on
storage.
[0044] Fibrinolytic processes also play a part in wound-healing
processes. In this case, the said protease and/or the proenzyme can
be administered intravenously or locally, subcutaneously,
intradermally, intramuscularly or, in the case of injuries and
wounds, as a constituent of a fibrin adhesive or alternatively
topically or bound to a suitable carrier matrix, e.g. in the form
of a web or of a patch, where combination with growth factors can
be expedient. In general, a pharmaceutical preparation of this type
is used in liquid or lyophilized form, to which protein stabilizers
known per se can be added, i.e., for example, complexing agents,
divalent cations such as calcium, amino acids such as glutamate,
arginine, lysine or glycine and/or sugars such as glucose,
arabinose, mannose or mannitol.
[0045] Moreover, the protease and/or its proenzyme can also be
employed for the coating of articles, consisting of plastics or
metals, to be implanted in the body, such as synthetic heart
valves, blood vessels, but also cannulas inserted for taking blood
or for artificial feeding.
[0046] The invention is illustrated by the following examples:
EXAMPLE 1
Preparation in Pure Form by Means of Immobilized Monoclonal
Antibodies
[0047] Monoclonal antibodies which are directed against the
protease activating factor VII were coupled to BrCN-Sepharose.RTM..
30 ml of this mAb matrix were packed into a column and the resin
was equilibrated with 50 mM sodium citrate, 0.1 M sodium chloride
(NaCl), 0.1 M arginine.times.HCl, pH 6.0.
[0048] 100 ml of citrate plasma were pumped through the column and
the matrix was then washed with 50 mM sodium citrate, 1 M NaCl, 0.1
M arginine.times.HCl, pH 6.0. The column was then washed again with
the equilibration buffer, after which elution with 0.1 M glycine,
pH 2.5, followed. The eluate (about 30 ml) was collected in a
volume of 3 ml of a 200 mM sodium citrate solution, pH 5.5, with
stirring and then adjusted to a pH of 4.5.
[0049] The eluate solution was used for further analysis. An
SDS-PAGE with subsequent transfer to a PVDF membrane and detection
of the factor VII activator band was carried out using the
unreduced and using the reduced sample. Activity tests of the
proteins thus obtained were carried out according to the process
described in German patent application 199 26 531.3, namely the
activation of prourokinase and factor VII, with subsequent
detection of urokinase or activated factor VII. The amounts of
protease tested in this system, determined as protease antigen,
correspond to the expected theoretical activity, whereby the
activity of the isolated protease or of the proenzyme with respect
to the biological activity was shown.
EXAMPLE 2
Anion-Exchange Chromatography
[0050] A solution containing the proenzyme form of the factor
VII-activating protease which still contained contaminations by
other proteins was pumped onto a Mono Q Sepharose in a buffer
solution of 20 mM Na acetate, 0.1 M glycine, pH 4.5 and then washed
with the abovementioned buffer. The fraction passing through was
collected. Bound proteins were eluted using 20 mM Na acetate, 2 M
NaCl, pH 4.5. The eluate was diluted in a buffer of 5 mM Na
citrate, 50 mM NaCl, pH 6.0, and investigated in the test systems
mentioned in Example 1. Aliquots were stored at 4 to 8.degree. C.
or frozen at -20.degree. C.
[0051] After storage of the eluate solution at 6.degree. C. for
several days, the tests were repeated, the dilutions of the
(thawed) samples in each case being carried out shortly before the
test.
[0052] SDS-PAGEs/Western blots confirmed that the protease had been
isolated in its proenzyme form. After SDS-PAGE and staining of
proteins by means of Coomassie Blue, in addition to the protease a
number of contaminating proteins, which were also to be found in
the fractions flowing through, were visible in the starting
solution (before chromatography). The protease was represented as a
band corresponding to the proenzyme form (i.e. even after
reduction) in pure form. The activity tests (see Example 1)
confirmed the nativity of the protein in the sense of the
retainment of the biological activities.
Sequence CWU 1
1
4 1 13 PRT Unknown Organism Description of Unknown Organism Peptide
characterizing the FVII-activity protease 1 Ile Tyr Gly Gly Phe Lys
Ser Thr Ala Gly Lys His Pro 1 5 10 2 12 PRT Unknown Organism
Description of Unknown Organism Peptide characterizing the
FVII-activity protease 2 Leu Leu Glu Ser Leu Asp Pro Asp Xaa Thr
Pro Asp 1 5 10 3 12 PRT Unknown Organism Description of Unknown
Organism Peptide characterizing the FVII-activity protease 3 Glu
Phe His Glu Gln Ser Phe Arg Val Glu Lys Ile 1 5 10 4 12 PRT Unknown
Organism Description of Unknown Organism Peptide characterizing the
FVII-activity protease 4 Ser Lys Phe Thr Xaa Ala Xaa Pro Xaa Gln
Phe Lys 1 5 10
* * * * *