Note: Descriptions are shown in the official language in which they were submitted.
CA 02083286 2001-02-09
1
RECOMBINANT FIBRINOGENASES, PREPARATION AND USE THEREOF
The present invention relates to novel proteins
with fibrinogenolytic properties, called fibrinogenases,
the preparation and use thereof for the prophylaxis and
therapy of diseases.
To date it has been possible to isolate from the
venom of the Malayan pit viper (Agkistrodon rhodostoma)
only one fibrinogen-cleaving enzyme having anticoagulant
properties (Biochem. J. 131 (1973) 799). This protein is
:LO called Arvin, Arvin or ancrod in the literature.
The possib:Le uses of this protein are limited
because signs of resistance may appear after 6 to 8 weeks
and are presumably attributable to the production of
ancrod-neutralizing antibodies. Hemorrhagic complications
also occur in a few cases.
We have now found, and prepared pure, other
proteins with fibri.nogenolytic properties.
The present invention relates to glycosylated,
partially glycosylated or non-glycosylated polypeptides
with the amino-acid sequences 1, 2, 3, 4 and 5 given in
the sequence listing, where Xaa and Xab are residues of
natural a-amino acids, and to the allelic variants
thereof which are identical in more than 95$ of the
amino-acid positions to the indicated sequences.
The residue Xaa is Asn, Gln, Ser, Thr, Gly, Asp,
Glu, Lys, Arg or Pro, but preferably Asn, Gln, Ser and
Thr-and, in particular, Asn and Gln. Xab is preferably
Phe, Tyr, Leu, Ile,. Ala, Val, Thr or Ser.
The present invention also relates to DNA sequen
ces which code for the abovementioned proteins, and to
vectors which contain these DNA sequences. Preferred DNA
sequences are depicted as sequences Nos . 6 to 9 in the
sequence listing.
The proteins according to the invention can be
prepared by known methods of genetic manipulation.
Thus, it is possible to isolate from the glandu-
lar tissue of a Malayan pit viper (Agkistrodon
20$2$
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rhodostoma) mRNA and to convert it into double-stranded
cDNA. -A cDNA library is set up after insertion of this
cDNA into a commercial cloning vector, eg. a gt 10. The
methods used for this can be found, for example, in
Maniatis et al., Molecular Cloning, CSH Press (1982). The
screening of such gene banks with radiolabeled oligo-
nucleotide probes or radiolabeled DNA fragments is also
now a widely used and described method. This method can
be used to isolate and characterize a cDNA clone which
has homology with the oligonucleotide probe or with
radiolabeled DNA fragments, and is described in DNA
cloning, Vol. I, IRL Press, 1985.
The cDNA which has been characterized in this way
can easily be obtained using restriction enzymes. The
fragments resulting from this can be used, where appro
priate in combination with chemically synthesized oligo-
nucleotides, adaptors or gene fragments, to clone the
sequences coding for the protein. The gene fragments or
synthetic DNA sequences are incorporated into cloning
vectors, eg. the commercial plasmids Ml3mp or pkk-223-3,
in a conventional manner. The genes or gene fragments can
also be provided with suitable control regions which have
been chemically synthesized or isolated from bacteria,
phages, eukaryotic cells or viruses thereof and which
make expression of the proteins possible.
The transformation or transfection of suitable
host organisms with the hybrid plasmids obtained in this
way is likewise known and described in detail (M. Wigler
et al., Cell 16 (1979) 777-785; F.L. Graham and A.J. van
der Eb, Virology 52 (1973) 456-467). The hybrid plasmids
can also be provided with appropriate signal sequences to
allow the polypeptides to be secreted into the medium.
Vectors which can be used for expression in
mammalian cells are those which place the gene to be
expressed, in this case the cDNA which codes for one of
the fibrinogenases described in the sequence listing,
under the control of the mouse metallothionein or viral
2~~~~$
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SV40 promoter {J. Page Martin, Gene, 37 (1985) 139-144).
The presence of the methionine start codon and of the
leader/prosequence of the gene for the appropriate
protein is necessary for expression. Clones which contain
copies of these vectors as episomes or integrated in the
genome are then isolated. Integration and expression of
the foreign gene on the basis of the bovine papilloma
virus are particularly advantageous. It is possible to
construct shuttle vectors in conjunction with prokaryotic
sequences which code for replication in bacterial cells
and for antibiotic resistance. The plasmid is initially
constructed and multiplied in bacterial cells and is then
transferred into the eukaryotic cells, eg. into the mouse
fibroblast cell line c127.
It is also possible to use other cell systems,
eg. yeast and other fungi, insect cells and animal and
human cells such as CHO, COS, L and 293 cells, in con-
junction with suitable expression vectors for the expres-
sion of the cloned cDNA.
These eukaryotic expression systems have the
advantage that they are able to secrete their products
efficiently and usually in native form. They also have
the ability to carry out post-translational modification
on their products.
Thus, on expression in eukaryotic cells, the
described fibrinogenases acquire glycoside side-chains.
These side-chains are absent in the polypeptides produced
in bacteria. The glycoside side-chains can also be
removed completely or partially using appropriate glyco-
sidases. Most eukaryotic proteins expressed in bacteria,
result as denatured inclusion bodies in the cell and must
be renatured by appropriate methods. In addition, bac-
teria are often incapable of eliminating the initiator
amino acid methionine from the finished protein. These
difficulties can be avoided by using secretion systems
(Donald Oliver, Ann. Rev. Microbiol. 39, (1985) 615-48;
John Ghrayeb et al. The EMBO Journal 3 {1984) 2437-2442.
- O.Z. 0050/41781
However, because of the degeneracy of the genetic
code, it is also possible to use other DNA sequences, eg.
chemically synthesized genes with different DNA sequen-
ces, for the expression of the described fibrinogenases.
Application of established methods of mutagenesis to the
cloned genes allows production of variants of these
fibrinogenases with a similar action.
The resulting polypeptides are purified from the
culture medium by chromatography, eg. affinity chromato
graphy on arginine-Sepharose~, Matrex-RedA-Sepharose ,
heparin-Sepharose or ion exchange materials in a conven-
tional manner (Lit.: Guide to Protein Purification,
Murray P. Deutscher [ed), Academic Press 1990).
The purification of the fibrinogenases can
likewise be purified [sic) directly from the venom of A.
rhodostoma by a combination of suitable chromatographic
methods, preferably using Matrex-redA-Sepharose , heparin
Sepharose~, arginine-Sepharose , conA-Sepharose , Q
Sepharose , S-Sepharose and chromatofocussing as
described in Example 5. Particularly suitable for the
final purification are HPLC methods.
The present invention also relates to drugs which
contain the proteins prepared according to the invention,
where appropriate in a pharmaceutically tolerated carrier
or excipient. The drugs can also contain combinations of
the proteins prepared according to the invention with
other pharmacologically active substances such as
thrombolytics (tPA, streptokinase), hirudin or throm-
boxane receptor antagonists.
Further embodiments of the invention are des-
cribed in detail in the Examples.
For methods of genetic manipulation, reference
may be made to, for example, the handbook by Maniatis et
al., Molecular Cloning, Cold Spring Harbor Laboratory,
1982 or DNA cloning Vol. I - III, IRI [sic] Press 1985 -
87, edited by D.M. Glover.
The polypeptides according to the invention are
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suitable for the treatment of glomerulonephritis, myo-
cardial infarct, non-ischemic stroke, disturbances of
peripheral arterial blood flow (especially athero-
sclerosis obliterans, thrombangitis obliterans, diabetic
microangiopathy and Raynaud's disease), unstable angina
pectoris, deep vein thrombosis and other thromboses,
rethrombosis after thrombolysis or vascular surgery, such
as angioplasty, and for preventing thromboses in extra-
corporeal circulations.
EXAMPLE 1
Isolation of a fibrinogenase cDNA clone from the Malayan
pit viper (Agkistrodon rhodostoma)
1 g of venom gland tissue from a 5-year old snake
of the genus [sic] Agkistrodon rhodostoma was disrupted
in 6 M guanidinium thiocyanate, 5 mM sodium citrate (pH
7.0), 0.1 M 2-mercaptoethanol, 0.5~ sarkosyl in an ULTRA-
TURRAX~. Large cell detritus was removed by centrifugation
at 3000 rpm. The RNA was removed by centrifugation
through a 5.7 M CsCl cushion at 45,000 rpm overnight. The
polyA+-containing RNA fraction was then isolated by
affinity chromatography on oligo(dT)-cellulose.
The polyA+ RNA was converted into single-stranded
cDNA using AMV reverse transcriptase and oligo(dT)12-18
as primer. The second strand was synthesized using E.coli
DNA polymerase I. An EcoRI adaptor of the sequence 5'AATT
CCATGG ATG CATGC 3' was attached to the double-stranded
cDNA using T4-DNA ligase. The commercial phage vector
a gt 10 (Fig. la, lb) was linearized with the restriction '''~f
enzyme EcoRI. The two DNAs were ligated together and
packaged with the commercial packaging extract to give
infectious phages. The recombinant phages were plated out
with E.coli C 600 Hfl on NZYDT plates and incubated at
37°C overnight. The resulting cDNA library contained 2x106
independent clones. Amplification of the cDNA library by
conventional methods was followed by plating out of
500,000 phages with C 600 Hfl cells. The phages were
transferred to nitrocellulose filters, lyzed with 0.5 N
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NaOH/1.5 M NaCl, and the denatured DNA was firmly bound
to the filter by baking at 80°C for 2 hours. The filters
were prehybridized in 6 x SET buffer (1 x SET = 0.15 M
NaCl, 15 mM tris/HC1, pH 7.4, 1 mM EDTA), 0.1% SDS and
5 x Denhardt's solution (100 x Denhardt = 1 g of Ficoll,
1 g of polyvinylpyrrolidone, 1 g of 8SA per 50 ml) at
68°C for 4 h.
Hybridization was carried out with a nick-trans-
lated cDNA (Fig. 2) which codes for ancrod protein.
The filters were incubated in a solution which
contained 2 x SET, 0.1% SDS, 30% formamide, 5 x
Denhardt's and 10% dextran sulfate at 42°C overnight
while shaking gently. They were then washed several times
with 2 x SET/0.1% SDS at 42°C, dried and exposed to an X-
ray film. Clones which gave a radioactive response in the
screening were isolated and cultured further in order to
obtain the corresponding phage DNA.
Phage DNA was prepared by incubating the purified
phages with protenase [sic] K (ad 60 ~g/ml) at 55°C for
1 h and subsequent phenol/chloroform extraction. Addition
of 3 volumes of ethanol (-20°C) resulted in precipitation
of the phage DNA, which was transferred with a sterile
injection needle into 70% ethanol, washed and briefly
sedimented. The pellet was briefly dried in air and then
suspended in TE buffer.
The purified phage DNA was transferred to nitro-
cellulose filters, renatured, reneutralized, baked and
prehybridized as described above. Hybridization was then
carried out under stringent conditions, using a radio-
labeled oligonucleotide probe which was homologous to the
ancrod-encoded [sic] cDNA:
5' GTC TAC GAT TAT CGT GAC TGG GTC AA 3'
The filters were incubated in a solution which
contained 2 x SET, 0.1% SDS, 30% formamide, 5 x
Denhardt's and 10% dextran sulfate at 42°C overnight
while shaking gently. They were then washed several times
in 2 x SET/0.1% SDS at 60°C, dried and exposed to an
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X-ray film.
The DNA which did not hybridize under these
conditions was subconed [sic] in the single-stranded
phage MB for further analysis.
EXAMPLE 2
Preparation of single-stranded DNA which codes for ancrod
The starting point were [ sic ] the phage DNA which
did not hybridize with the ancrod-specific
oligonucleotide as described in Example 1. They were each
separately cut preparatively with the restriction enzyme
Eco RI. The Eco RI fragments which contained the cDNA
inserts were eluted from the gel by electrophoresis. 30
ng of each of these fragments were ligated at 4°C for 12
h with 100 ng of the commercial cloning vector M13mp18 or
M13mp19 (Fig. 3) which had been cut with Eco RI. The
volume of the ligation mixture was 10 ~1. Ligation was
stopped by heating at 80°C for 5 min.
1/10 of the volume of each ligation mixture was
employed to transform 100 ~l of competent SR 101 cells.
After the transformation was complete, 60 ~1 of 0.2 M
IPTG solution and 120 ~sl of XGal (20 mg/ml) were added to
the transformation mixture. The resulting mixture was
plated out in NZYDT top agar on NZYDT agar plates con-
taining 200 ~1 of SR 101 cells (ODsoo-1)~ The NZYDT medium
is commercially available (GIBCO-BRL). Clones which
contained cDNA inserts were identifiable because the
plaques were not stained blue. DNA sequence analysis
(Sanger et al., Proc. Natl. Acad. Sci. USA 74, (1977)
5463-67) was used to elucidate the sequence of this cDNA
insert (sequence listing, Nos. 6 to 9).
EXAMPLE 3
Construction of vectors for the expression of ancrod in
eukaryotic cells
SV40 DNA was cut with the restriction enzymes
BamHI and BclI, and the 0.24 kb fragment was prepared by
gel electrophoresis (Fig. 4). The ends were filled in '~---'
with the Klenow fragment in the presence of the four
_ ~~~33~'~~
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deoxynucleotide triphosphates dATP, dCTP, dGTP and dTTP.
XhoI linkers were then ligated on.
In parallel the commercial vector pUCl8 was
linearized with SmaI. XhoI linkers were then likewise
attached. The DNA of this vector (puCl8Xho) was linear
ized with Xhol, treated with alkaline phosphatase and
ligated to the 0.24 kb XholI [sic] SV40 fragment (see
above). The result was pSVpA.
pSVpA DNA was cleaved preparatively with XhoI and
incubated with Klenow polymerase in the presence of the
four dNTPs as above. The 0.24 kb fragment was isolated
from the gel.
At the same time, the eukaryotic expression vec
tor CL28XhoBPV, produced by ligation of CL28x and pB2-2
(Reddy et al. DNA 6, (1987) 461-72) was partially cut
with the restriction enzyme XbaI, ie. the incubation time
was restricted so as to result in molecules cleaved at
only one of the two XbaI recognition sequences, ie.
linearized (Fig. 5) . The mixture was then reacted with
Klenow polymerase and dNTPs as described. The linear
molecules were subsequently isolated by gel electro-
phoresis.
The linear pCL28XhoBPV fragments were then
ligated with the pretreated 0.24 kb SV40 fragment. After
transformation and screening of minilysates, a clone
which carried the SV40 fragment in the former XbaI site
located about 0.15 kB [sic] 3' of the XhoI site was
isolated; this DNA (pCL28XhoBPV-SVpolyA) carried the SV40
transcription stop signals of the early genes.
Plasmid DNA from pCL28XhoBPV-SVpolyA was linear-
ized with Xhol and treated with alkaline phosphatase. At
the same time, the cDNA inserts which did not hybridize
with the Ancrod-specific oligonucleotide as described in
Example 2 were provided with Xho linkers using T4 ligase.
The two fragments were connected together using T4
ligase. After transformation and analysis of minilysates,
a clone which contained the cDNA inserts singly and in
~~8'~~g~b
O.Z. 0050/41781
the correct orientation was isolated: pCL28BPV-fibro-
genase [sic] I-IV.
EXAMPLE 4
Transfection and establishment of cell lines
c127I cells (J. Virol. 26 (1978) 292; ATCC
catalog of cell lines and hybridomas 5th edition, 1985,
p.142) were transfected with BPV expression plasmids
using the calcium phosphate coprecipitation method
(Virology 52 (1973), 456, DNA cloning; Volume II, ed.
D.M. Glover IRL Press, (1985) 143ff and 213).
DMEM (Dulbeccos~s Modified Eagles Medium) + 10~
FCS (fetal calf serum) in 60 mm Petri dishes was inocu-
lated with 5 x 105 C127I cells. The next day the medium
was changed to MEM (Modified Eagles Medium) containing
25 nM Hepes + 10$ FCS. A Ca phosphate coprecipitate was
formed with 10-5 g of CsCl-purified plasmid DNA and was
cautiously placed on the C172I cells. The cells were
incubated at 37°C, 7~ C02 for 4 h. A subsequent glycerol
shock treatment considerably increased the efficiency of
transfection. For this, 4 h after addition of the preci-
pitate the medium was aspirated off from the cells. The
cells were incubated with 2 ml each [sic] of 15~
glycerol/HBS (DNA cloning Vol. II, page 152) in a 60 mm
Petri dish at room temperature for 3 min. The
glycerol/HBS solution was aspirated off and the cell lawn
was washed with 3 ml of DMEM + 10~ FCS. The cells were
incubated with DMEM + 10 ~ FCS at 3 7 ° C , 7 ~ COZ . The DMEM +
10% FCS was aspirated off and replaced by fresh three
times a week. After 2 - 3 weeks, the transfected cells
which contain the BPV genome were evident as collections
of transformed cells, called foci.
After the foci had been subcloned, the medium
supernatants from the individual subclones were tested
for fibrinogen-cleaving activity by conventional methods.
For production, after the cell lines had reached
confluence they were maintained in serum-free DMEM. The
novel fibrinogenases can be purified by conventional
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methods from the serum-free cell culture supernatant
obtained in this way and used for pharmacological and
chemical analyses.
EXAMPLE 5
Isolation and purification of a fibrinogen-cleaving
enzyme (sequence listing, No. 5) from the venom of
Agkistrodon rhodostoma
550 mg of crude venom (dry substance) from
Agkistrodon rhodostoma were taken up in 20 ml of 20 mM
Na2HP0,,, 0.01 Tweeri 80, 500 mM NaCl, pH 7.0 {= buffer
A).
a) Chromatography on Matrex Red A-Sepharose
A chromatography column {diameter 2.5 cm, length
5.1 cm) was packed with 25 ml of Matrex red
A-Sepharose (from Amicon). The column was equilibrated
with 100 ml of buffer A and then loaded with the
dissolved crude venom. The column was washed with
45 ml of buffer A (flow rate 120 ml/h) and then eluted
with 85 ml of buffer B, which was composed of 20 mM
Na2HP04, 2 M NaCl, 0.01 Tween, pH 7Ø The W-active
fraction (280 nm) was collected.
The eluate was dialyzed twice against 2.5 1 of 20 mM
NaZHP04, 0.01$ Tweeri 80, pH 7.0 (= buffer C) in a
dialysis tube (Visking size 8.32/32) for 2 h each
time. The conductivity of the dialyzed tubes [sic] was
about 2.2 mS/cm (4°C).
b) Chromatography on arginine-Sepharose
A chromatography column (diameter 2.5 cm, length
10 cm) was packed with arginine-Sepharose (from
Pharmacia) and equilibrated with 200 ml of buffer C.
The dialyzed eluate (vol. about 140 ml) from the
Matrex red A-Sepharose was loaded on the column with
a flow rate of 120 ml/h.
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The flow-through from the column (about 180 ml) was
collected and processed further. Still bound to the
column was, inter alia, ancrod which can be obtained
by elution with arginine salts.
c) Chromatofocussing
A chromatography column (diameter 0.5 cm, length 5 cm)
was packed with 1 ml of PBE~ 94 gel material (from
Pharmacia). The column was equilibrated with 5 column
volumes of 20 mM tris/HC1, 0.01 Tweeri 80, pH 8.0
(= buffer D).
The column was loaded with 20 ml of the flow-through
from the arginine-Sepharose .
The chromatography was carried out with a linear
gradient from buffer D to 20 mM acetic acid/HC1, 0.01
Tweeri 80, pH 2.0 (= buffer E) in 25 min with a flow
rate of 1 ml/min. After this time, the column was
eluted with buffer E for a further 13 min. The W-
active fraction (280 nm) eluted during this was
collected. About 1 ml of a protein solution which
contained, according to protein determination (method:
Anal. Biochem. 153, 267-271), about 0.04 mg/ml was
obtained.
d) Characterization of the purified fibrinogenase V
dl) SDS gel electrophoresis
Comparing with standard proteins, the protein
solution showed a main band {about 70 to 90$) at
42000 Dalton.
d2) N-terminal sequencing
The N-terminal sequence of the purified protein
solution was determined (see sequence listing,
sequence No. 5).
_ ~os~~~~
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d3) Fibrinogenase assay
Fibrinogenase activities were determined by
converting fibrinogen with the enzyme to be
assayed into deAA fibrinogen.
This reaction was associated with an increase in
turbidity which was followed by photometry (DD
[sic] 340 nm).
The activity was quantified by calibration with
an ancrod standard (Arwiri) of 3000 U/mg.
The fibrinogenase activity of the purified enzyme
was about 500 U/mg.
2~8~~~
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Sequence listing
Sequence No. 1: 234 amino-acid sequence
Val Ile Gly Gly Asp Glu Cys Asn Ile Asn Glu His Arg Phe Leu Val
10 15
Ala Leu Tyr Asp Ser Thr Thr Arg Asn Phe Leu Cys Gly Gly Val Leu
20 25 30
Ile His Pro Glu Trp Val Ile Thr Ala Lys His Cys Asn Lys Lys Ser
35 40 45
Met Val Leu Tyr Leu Gly Lys His Lys Gln Ser Val Lys Phe Asp Asp
50 55 60
Glu Gln Glu Arg Phe Pro Lys Glu Lys His Phe Ile Arg Cys Asn Lys
65 70 75 80
Pro Arg Thr Arg Trp Gly Glu Asp Ile Met Leu Ile Arg Leu Asn Lys
85 90 95
Pro Val Xaa Asn Ser Glu His Ile Ala Pro Leu Ser Leu Pro Ser Gly
100 105 110
Pro Pro Ile Val Gly Ser Val Cys Arg Val Met Gly Trp Gly Ser Ile
115 120 125
Asn Lys Tyr Ile Asp Val Leu Pro Asp Glu Pro Arg Cys Ala Asn Ile
130 135 140
Asn Leu Tyr Xaa Tyr Thr Val Cys Arg Gly Val Phe Pro Arg Ile Gly
145 150 155 160
Lys Lys Ser Lys Ile Leu Cys Ala Gly Asp Leu Gln Gly Arg Leu Asp
165 170 175
Ser Cys His Cys Asp Ser Gly Gly Pro Leu Ile Cys Ser Glu Glu Phe
180 185 190
His Gly Ile Val Tyr Arg Gly Pro Asn Pro Cys Ala Gln Pro Asp Lys
195 200 205
Pro Ala Leu Tyr Thr Asn Ile Phe Asp His Leu His Trp Ile Leu Ser
210 215 220
Ile Val Ala Gly Xaa Ala Thr Cys Tyr Pro
225 230
2~~~~~
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Sequence listing
Sequence No. 2: 236 amino-acid sequence
Val Val Gly Gly Asp Glu Cys Asn Ile Asn Glu His Arg Phe Leu Ala
10 15
Leu Val Tyr Ile Thr Ser Gly Phe Leu Cys Gly Gly Thr Leu Xab His
20 25 30
Pro Glu Trp Val Val Ser Ala Ala His Cys Ala Arg Gly Glu Ile Glu
35 40 45
Val Phe Phe Gly Val His Ser Leu Lys Asp Ile Arg Thr Asn Lys Asp
50 55 60
Val Gln Lys Arg Val Ala Lys Glu Met Phe Phe Cys Leu Ser Ser Lys
65 70 75 g0
Xaa Tyr Thr Lys Trp Asp Lys Asp Ile Met Leu Ile Lys Leu Asp Ser
85 90 95
Pro Val Xaa Asn Ser Thr His Ile Ala Pro Ile Ser Leu Pro Ser Ser
100 105 110
Pro Pro Ser Val Gly Ser Val Cys Arg Val Met Gly Trp Gly Val Thr
115 120 125
Thr Ser Pro Xaa Gly Thr Xab Pro Ser Val Pro His Cys Ala Asn Ile
130 135 140
Asn Ile Leu Asp Tyr Xab Val Cys Arg Ala Ala Arg Pro Lys Leu Pro
145 150 155 160
Ala Lys Ser Arg Thr Leu Cys Ala Gly Ile Leu Glu Gly Gly Lys Ser
165 170 175
Ala Cys Asp Gly Asp Ser Gly Gly Pro Leu Asn Cys Asn Gly Glu Ile
180 185 190
Gln Gly Ile Val Ser Trp Gly Gly Asn Ile Cys Ala Gln Pro Arg Lys
195 200 205
Pro Ala His Tyr Xab Lys Val Ala Asp Tyr Thr Asp Trp Ile Lys Ser
210 215 220
Ile Ile Ala Gly Xaa Thr Thr Ala Thr Cys Pro Pro
225 230 235
~~i832~~
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Sequence listing
Sequence No. 3: 236 amino-acid sequence
Val Ile Gly Gly Ala Glu Cys Asn Val Asn Glu His Arg Phe Leu Val
10 15
Ala Leu Tyr Asp Xaa Leu Thr Gly Thr Leu Gln Cys Gly Gly Thr Leu
20 25 30
Ile His Pro Glu Trp Val Leu Thr Ala Ala His Cys Asp Arg Lys Ser
35 40 45
Met Val Ile Tyr Leu Gly Met His Xaa Lys Ser Val Asn Asn Asp Asp
50 55 60
Gln Gln Arg Arg Ser Ala Lys Glu Lys Tyr Phe Phe Ser Cys Ser Lys
65 70 75 80
Ser Ile Ala Ala Trp Glu Lys Asp Ile Met Leu Ile Arg Leu Asp Ser
85 90 95
Pro Val Xaa Asn Ser Thr His Ile Ala Pro Leu Ser Leu Pro Ser Arg
100 105 110
Pro Pro Thr Val Gly Ser Val Cys Arg Val Met Gly Trp Gly Ala Ile
115 120 125
Thr Ser Pro Lys Glu Thr Tyr Pro Glu Val Pro His Cys Thr Asp Ile
130 135 140
Asn Leu Leu Xaa Tyr Ser Glu Cys His Gly Asp Phe Pro Arg Leu Arg
145 150 155 160
Ala Thr Ser Arg Ile Leu Cys Ala Gly Val Leu Gln Gly Gly Ile Asp
165 170 175
Thr Cys Asn His Asp Ser Gly Gly Pro Leu Ile Cys Asp Glu Gln Phe
180 185 190
Gln Gly Ile Val Ser Trp Gly Pro Tyr Pro Cys Ala Gln Pro Arg Asn
195 200 205
Ala Ala Ile Tyr Thr Lys Val Phe Asn Tyr Leu Val Trp Val Trp Ser
210 215 220
Thr Ile Ala Gly Xaa Thr Thr Val Thr Cys Pro Pro
225 230 235
w
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Sequence listing
Sequence No. 4: 234 amino-acid sequence
Val Val Asn
Gly Ile
Gly Asn
Asn Glu
Glu His
Cys Arg
Phe
Leu
V
l
5 a
10 15
Ala Ile Phe Xab Phe Val Cys Ala Gly Thr Leu Ile His
Ser
Thr
Gly
20 25 30
Pro Glu Trp Val Val Thr Ala His Cys Glu Ser Thr Asp Leu Lys
Ala
35 40 45
Met Lys Phe Gly Met His Ser Lys Lys Val Gln Asn Glu Asp Glu Gln
50 55 60
Thr Arg Asn Ala Lys Glu Lys Phe Ile Cys Pro Asn Lys Lys Asn Asp
65 70 75 80
Glu Val Leu Asp Lys Asp Ile Met Leu Ile Lys Leu Asn His Pro Val
85 90 95
Ser Asn Ser Glu His Ile Ala Pro Leu Ser Leu Pro Ser Ser Pro Pro
100 105 110
Ser Val Gly Ser Phe Cys His Ile Met Gly Trp Gly Ser Ile Thr Pro
115 120 125
Val Lys Val Thr Phe Pro Asp Val Pro His Cys Ala Asn Ile Asn Leu
130 13 5 140
Leu Glu Glu Ala Glu Cys His Ala Gly Tyr Pro Glu Val Leu Ala Glu
145 150 155 160
Tyr Arg Thr Leu Cys Ala Gly Ile Val Gln Gly Gly Lys Asp Thr Cys
165 170 175
Met Tyr Asp Ser Gly Gly Pro Leu Ile Cys Asn Glu Gln Val Gln Gly
180 185 190
Ile Val Ser Tyr Gly Ala His Pro Cys Gly Gln Pro Leu Lys Pro Gly
200 , 205
Ile Tyr Thr Arg Leu His Asp Tyr Asn Asp Trp Ile Asn Ser Ile Met
210 215 220
Ala Gly Asn Thr Ala Val Thr Pro Pro
Cys
225 230
2~~3~~~
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Sequence listing
Sequence No. 5: 25 amino-acid sequence
Val Ile Gly Gly Asp Glu Cys Asn Ile Asn Glu His Pro Phe Leu Val
10 15
Ala Val Tyr Glu Glu Thr Ala Gly Ala
20 25
~0~32~~
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Sequence listing
Sequence No. 6: 1096 nucleotide sequence corresponding
to amino-acid sequence No. 1
Strandedness : double-stranded
Topology: linear
Molecule type . cDNA to mRNA
Original source: Agkistrodon rhodostoma
The region coding for the protein of sequence No. 1
starts at base 144 and terminates at base 841.
GAATTCCATGGATGCATGCG TTTGGGACTG GGATCTTACA GGCAAAGAGC TTTCTGTGCA 60
GAGTTGAAGCTATGGTGCTG ATCAGAGTGC TAGCAAACCT TGTGATACTA CAGCTTTCTT 120
ACGCACAAAAGTCTTCTGAA CTGGTCATTG GAGGTGATGA ATGTAACATA AATGAACATC 180
GTTTCCTTGTAGCCTTGTAT GACAGTACGA CTCGGAATTT TCTCTGTGGT GGGGTTTTGA 240
TCCATCCGGAATGGGTGATC ACTGCTAAAC ACTGCAACAA GAAAAGTATG GTCCTATACC 300
TTGGTAAGCATAAACAAAGT GTAAAATTTG ACGATGAGCA GGAAAGATTC CCAAAGGAGA 360
AGCACTTTATTCGCTGTAAC AAACCCCGTA CCAGATGGGG CGAGGACATC ATGTTGATCA 420
GGCTGAACAAACCTGTTAAC AACAGTGAAC ACATCGCTCC TCTCAGCTTG CCTTCCGGCC 480
CTCCCATTGTGGGCTCAGTT TGCCGTGTTA TGGGATGGGG CTCAATCAAT AAATATATAG 540
ACGTTTTGCCCGATGAACCT CGTTGTGCTA ATATTAACCT GTACAATTAC ACGGTGTGTC 600
GTGGAGTTTTTCCAAGGATA GGAAAGAAAA GCAAAATATT GTGTGCAGGT GACCTGCAAG 660
GACGCCTAGATTCATGTCAC TGTGACTCTG GGGGACCTCT CATTTGTAGT GAAGAATTCC 720
ATGGCATTGTATATCGGGGA CCCAATCCTT GTGCCCAACC AGATAAGCCT GCCCTCTACA 780
CCAACATCTTCGATCATCTT CACTGGATCC TTAGCATTGT GGCAGGAAAT GCAACTTGCT 840
ATCCATAAAACCTTTTGAAA TAGTTAAGTG GAGAAAATGT AACATATTAG TAAATCTCTT 900
CTATATCCTTGCATTGGAAC ATATTCCCAG GCTGTAAGCT TTTTAGACTC AAATAGGACT 960
ACCTTTGGAGTAAGAAGTGC TCAAAATAGT GCTGCAGGGA TCATGTCCCA TTTAATTTCA 1020
GTTTAAAACAGTCTCCATAG ATTGGAGGCC TGTTTAGGGT TAGGTGCAAA TTTCTGACTC 1080
TAAATGGACCATTCCC
._ ~~~~~s~
- 19 - O.Z. /41781
Sequence listing
Sequence No. 7: 1333 nucleotide sequence corresponding
to amino-acid sequence No. 2
Strandedness: double-stranded
Topology: linear
Molecule type: cDNA to mRNA
Original source: Agkistrodon rhodostoma
The region coding for the protein of sequence No. 2
starts at base 231 and terminates at base 935.
ANCCCCCTTT NNNGGNGGGG GGGGNCCAGA AGTTNCCCAG ATTNCTTGGC CACCCCGGTT 60
GCTTAATTTG ATCAAATAAA GTGCTGCTTG ATCCAAGAAA TTCTCCGCTT GGGTTATCTG 120
ATTAGGCAAA CAGCTTGCCA CGCAGAGTTG AAGCTATGGT GCTGATCAGA GTGCTAGCAA 180
ACCTTCTGAT ACTACAACTT TCTNACGCAC AAAAGTCATC TGAACNGGNC GTTGGAGGTG 240
ATGAATGTAA CATAAATGAA CATCGTTTCC TTGCACTCGT GTATATCACT AGTGGTTTTC 300
TCTGCGGTGG GACTTTGANC CACCCGGAAT GGGTGGTCAG TGCTGCACAT TGCGCTAGGG 360
GAGAAATAGA GGTATTCTTT GGTGTGCATA GCCTAAAGGA TATACGGACA AATAAGGATG 420
TGCAGAAAAG AGTCGCAAAG GAGATGTTCT TTTGCCTCAG TAGCAAAAAC TATACCAAAT 480
GGGACAAGGA CATCATGTTA ATCAAGCTGG ACAGTCCTGT TAACAACAGT ACTCACATCG 540
CGCCTATCAG CTTGCCTTCC AGCCCTCCCA GTGTGGGCTC AGTTTGCCGT GTTATGGGAT 600
GGGGCGTAAC CACATCTCCT AATGGGACTA TNCCCAGTGT NCCTCACTGT GCTAACATTA 660
ACATACTCGA TTATNCGGTG TGTCGAGCAG CTAGGCCAAA GTTGCCGGCG AAAAGCAGAA 720
CATTATGTGC TGGTATCCTG GAAGGAGGCA AAAGTGCATG TGACGGTGAC TCTGGGGGAC 780
CCCTCAACTG TAATGGAGAA ATCCAGGGCA TTGTATCTTG GGGGGGTAAT ATTTGTGCTC 840
AACCGCGTAA GCCTGCCCAC TACNCCAAGG TCGCCGATTA TACTGATTGG ATTAAGAGCA 900
TTATTGCAGG AAATACAACT GCAACTTGCC CCCCGTGAAA ATTTTTGAAA AACTTAAGAG 960
GAGAAAATAC ATCTCTTCTA TATCCCTAGC CATATTCAAT TACATTGGAA TATATTCCCA 1020
AGTTAACTCT ACATCAACAA AAAATCCTAC NAAACAACAA CAGAGAAGGA GCAGATAAAA 1080
GAGATAAATG GTACAAAATT GAGAATCAAG ACTTAAAGAT GGAACTTAAG AAAACAAGGA 1140
ACCATGATTT AATCCTTGTG GGGGGGGAAA TCACAAGAAT TGGAAAAAAA CAACTTATCC 1200
CTTAGACAGC AAACTAAATC TGAGGACAAG AAAACAGATT GGATAAAATG GACTGTAGAA 1260
ATGTCAGGAA CATCGGAGAG AAAGGAAATA ATAAGAGAAG CAAAAAAAAA AAAAGCATGC 1320
ATCCATGGAA TTC
~~s~~s~
- 20 - O.Z. 0050/41781
Sequence listing
Sequence No. 8: 988 nucleotide sequence corresponding
to amino-acid sequence No. 3
Strandedness: double-stranded
Topology: linear
Molecule type: cDNA to mRNA
Original source: Agkistrodon rhodostoma
The region coding for the protein of sequence No. 3
starts at base 197 and terminates at base 904.
AACAATAAAGNCTGCNTGAN CAAGAAGCNN CTGCTTAGCT TATCTGATAA GATTGACATG 60
TATCTCAAGCTTAAGTTGGG ACTGGGATCT TACAGCAAAG AGCTTTCCAC GCAGAGTTGA 120
AGCTATGGTGCTGATCAGAG TGCTAGCAAA CCTTCTGATA CTACAGCTTT CTTACGCACA 180
AAAGTCTTCTGAACTGGTCA TTGGAGGTGC TGAATGTAAC GTAAATGAAC ATCGTTTCCT 240
TGTAGCCTTGTATGACAATT TGACTGGGAC TTTGCAGTGT GGTGGGACTT TGATCCACCC 300
GGAATGGGTGCTCACTGCTG CGCACTGCGA CAGGAAAAGT ATGGTCATAT ACCTTGGTAT 360
GCATAACAAAAGTGTAAACA ATGACGATCA GCAGAGAAGA TCCGCAAAGG AGAAGTACTT 420
TTTTAGCTGTAGCAAAAGCA TTGCCGCATG GGAAAAGGAC ATCATGTTGA TCAGGCTGGA 480
CAGTCCTGTTAACAACAGTA CACACATCGC CCCTCTCAGC TTGCCTTCCA GACCTCCCAC 540
TGTGGGCTCAGTTTGCCGTG TTATGGGATG GGGCGCAATC ACATCTCCTA AAGAGACTTA 600
TCCTGAGGTCCCTCATTGTA CTGACATTAA CCTGTTAAAT TATTCGGAGT GTCATGGAGA 660
TTTCCCACGGTTGCGGGCGA CAAGCAGAAT ATTGTGTGCA GGTGTCCTGC AAGGAGGCAT 720
AGATACATGTAATCATGACT CTGGGGGACC TCTCATCTGT GATGAACAAT TCCAGGGCAT 780
TGTATCTTGGGGACCCTATC CTTGTGCCCA ACCGCGTAAC GCTGCCATCT ACACCAAAGT 840
CTTCAATTATCTTGTCTGGG TCTGGAGCAC TATTGCAGGA AATACAACTG TGACTTGCCC 900
CCCATGAAAACATTTTTATT TCCACAAAGG AGTTTCCAAA GGAATTAAAA CTAAATAATG 960
TGGTAAAAAAAAAAAAAAAA A~~~AAAAA
._.. - 21 - 0. Z . 00~
Sequence listing
Sequence No. 9: 957 nucleotide sequence corresponding
to amino-acid sequence No. 4
Strandedness: double-stranded
Topology: linear
Molecule type: cDNA to mRNA
Original source: Agkistrodon rhodostoma
The region coding for the protein of sequence No. 4
starts at base 210 and terminates at base 911.
CTNAATTNNA CAAAAAAAGT GCTGCTTGGT CAAGAGGTNC TCCGCTTCGG TTATCTGATT
60
AGATTGATAC GTATCTCAAG TATAAGTTTG GGACTGGGAT CTTACAGGAA AACAGCTTTC
120
CGTGCAGAGT TGAAGTTATG GTACTGATCA GAGTGCTAGC AAACCTTCTG ATACTACAGC
180
TTTCTTACGC ACAAAAGTCA TCTGAACTGG TCGTTGGAGG TAATGAATGT AACATAAATG 240
AACATCGTTT CCTTGTAGCC ATCTTTAACT CTACTGGGTT TGTCTGCGCT GGGACTTTGA 300
TCCACCCAGA ATGGGTGGTC ACTGCTGCAC ACTGCGAGAG TACGGATCTC AAGATGAAGT 360
TTGGTATGCA TAGCAAAAAG GTACAAAATG AGGATGAGCA GACAAGAAAC GCAAAGGAAA 420
AGTTCATTTG TCCCAATAAG AAAAACGATG AAGTACTGGA CAAGGACATT ATGTTGATCA 480
AGCTGAACCA TCCTGTTAGC AATAGTGAAC ACATCGCGCC TCTCAGCTTG CCTTCCAGCC 540
CTCCCAGTGT GGGCTCATTT TGCCATATTA TGGGATGGGG CTCAATCACA CCTGTTAAAG 600
TGACTTTCCC CGATGTCCCT CATTGTGCTA ACATTAACCT ACTCGATGAT GCAGAGTGTC 660
ATGCAGGTTA CCCTGAGGTG CTGGCAGAAT ACAGAACATT GTGTGCAGGT ATCGTGCAAG 720
GAGGCAAAGA TACATGTATG TATGACTCTG GAGGACCTCT CATCTGTAAT GAACAAGTCC 780
AGGGCATTGT ATCTTATGGG GCGCATCCTT GTGGCCAACC TCTTAAGCCT GGTATCTACA 840
CCAGGCTCCA TGATTATAAT GACTGGATCA ACAGCATTAT GGCAGGAAAT ACAGCTGTGA 900
CTTGCCCCCC GTGAAAACTT TAGTATCAGA AGGTTTGCTG CATGCATCCA TGAATTC
