Note: Descriptions are shown in the official language in which they were submitted.
2121134
O.Z. 0050/42778
Nova~ thrombin-inhibitory Protein from ticks
The present invention relates to a novel
thrombin-inhibitory protein from ticks and a process for
preparing it.
Thrombin inhibitors are Lmportant therapeutic
substances used, for example, for the prophylaxis or
treatment of thromboses or arterial reocclusions.
German Offenlegungsschrift DE 39 31 839 describes
a thrombin inhibitor isolated from the argasid tick
Ornithodoros moubata. This protein has a molecular weight
of about l5,000 Dalton, an isoelectric point at pH 4-5
and the N-terminal amino-acid sequence SDYEFPPPKKXRPG.
European Published Applicatioh EP 345 614 des-
cribes the thrombin-inhibitory agent amblyommin which is
isolated ~rom bont ticks. This is a protein with a
molecu}ar weight of 20,000-30,000 Dalton and an isoelec-
` tric po~int of 5.05-5.65.
However, to~date, no protein with a thrombin-
inhibitory~actLon~has~ been found to be suitable as drug
20~ in ~terms`~o~f ~high activity,~;lack of~ antigenicity, long
bi~1 ~ ca1~half-life, and~few side effects, such as risk
of~ hemorrhage~
It is an ob~ect of the present invention to
provide~novel thrombin inhibitors which are suitable as
25~ drùgs on the~basis of the abovementioned properties.
We~have~ found that th1s object is achieved by a
novel~thro:mbin-inhibitory protein isolated from tick
he~ nove1~protein~ has the following physico-
chemical~properties.~ Mo~lecular sieve chromatography
30~ reveals~;that ;it has a molecular weight of 22,000-28,000
Dalton. A molecular weight of 8000 ~ 1500 Dalton is
determ~néd in'!ah 9DS polyacrylam;de gel. ~eterminatiQn of
; the isoelectric point reveals that it is at a pH below
3.~8~. ~
35~ The protein binds specifically to a thrombin
affinity ~ column~.~ It inhibitQ the biological activity of
thrombLn in an ~n vitro eozyDIe assay.
212113~l ~
- 2 - O.Z. 0050/42778
Silver staining of the protein band in a poly-
acrylamide gel is not possible; it is visible only as an
unstained spot on a stained background.
The following N-terminal amino-acid sequence was
determined for the protein (SEQ ID NO~
Leu-Asn-Val-Leu-Cys-Asn-Asn-Pro-His-Thr-Ala-Asp-Cys-Asn-
Asn-Asp-Ala-Gln-val-Asp-Arg-Tyr-phe-Arg-Glu-Gly-Thr-Thr
-~ Cys-Leu-Met-Ser-Pro.
The complete amino acid sequence of the novel
protein is depicted in SEQ ID NO:; 7.
; Other proteins according to the invention are
~; those which differ from the amino-acid sequence depicted
` -in~SEQ ID NO: 7 by replacement of up to l0 amino acids or
'`,f~ deleti~n of individual amino acids.
-~ 15Amino-acid~sequences of these types can easily be
~; prepared, for example, by genetic engineering via an
appropriately mutated~gene.
The~nove1 protein~can~be isolated from ticks of
the genus~Or ithodoro~ To ~do this,~the ticks are homo-
20~ genized, ~expédiently~ in a buffer at pH 6-9, preferably
pH~7-~8,;~with~a ~homogenizer, preferably~ a mixer. The
insQ1ub1e ~cons~titùents~are then~ removed, preferably by
centrifugation. -
~
The~ protein can be further purified from the
25~ ésult1ng~solutlon~by precipitating other proteins fromthe~ so1ution~with~a~precipitant, preferably trichloro-
acetlc~ac~id,~and~subsequently removing them.
Further purification o~f~the protein is possible
by~-~chro~atographic ~m*thods, preferably ion exchange
30~ : chromatography~and/or affinity chromatography. A purifi-
~- -;Gation ~ step by thrombin affinity chromatography is
par~icular1y~lp eferred.i~ t ~
The purification of the protein can be monitored
by~a thrombin~activity assay. It is expedient to use for
35~ ~ this an optical as~ay in which a chromogenic substrate,
for~example ~Chromozym T, is converted by thrombin. The
fraction- containing the novel protein can be identified
l ~
212i134
- 3 - O.Z. 0050/42778
by ~heir thrombin-inhibiting action on addition to this
optical assay.
Genetic engineering methods are particularly
suitable for preparing the protein according to the
~ 5 invention.
-~ To do this, a cDNA gene bank from the tick is
constructed in a convention~l manner. It is possible to
isolate the gene coding for the protein according to the
invention from this gene bank by, for example, preparing
~ 10, a DNA probe whose sequence is obtained from the
`~ N-terminal amino-acid sequence described above by trans-
lation back using the genetic code. The appropriate gene
can~be found and~isolated by hybridization with this DNA
probe.
~ ~ However, it is also possible to employ the
~ polymerase chain reaction-(PCR) technique to prepare the
,~ appropriate gene. For'example, a primer whose sequence
has~been obtained by translation back from the N-terminal
amino-acid~sequence~described~above~,~and a second prLmer
20 ~ who~se~s~equence~ complementary to~the 3' e~d of the cDNA
gene~fr ~ nt,~preferably with the sequence poly(dT), can
~`'"'`~ be~us~ed t~prepare the cDNA gene fragment for the protein
a~cording~to the invention by the~PCR technique. The
' appropriate gene can~al o be isolated by~constructing an
25~ e;xpress~ion-~gene~ bank~from ticks and screening this with
"~ an,~antibody directed against the protein according to the
invention.'
The~gene~for the protein describet in the intro-
duction~has~the~nucleotide sequence depicted in SEQ ID
30~ NO;~6.~
Apart f~rom this gene, other suitable nucleotide
equences 'àrel!~tho~e-lwhiich code for the amino-acid
,
sequence depLcted in SEQ ID NO: 7 but which, a~ a conse-
quence of the degeneracy of the genetic code, have a
~ nucleotide sequence~differing from SEQ ID NO: 6. Genes of
this~type can be prepared by total chemical synthesis or
by mutagene~is of the SEQ ID NO: 6 nucleotide sequence.
2121134
- 4 - O.Z. 0050/42778
- Once the appropriate gene has been isolated, it
can be expressed by genetic engineering methods in
organi~ms, eg. in bacteria, yeasts, eukaryotic cells,
with the aid of an expression vector in a conventional
manner. Preferably used are proka~yotes such as E. coli,
and vector~ with high-level expression, eg. under the
control of the inducible tac promoter as present eg. in
~- the plasmid pMAL-p2 (Protein Fusion and Purification
System, Gene~xpress; New England Biolabs). This results
in periplasmic expression of a fusion protein comprising
the maltose binding protein and the described thrombin
inhibitor. The fusion partner can be deleted enzymatic-
~ ally after purification. The protein can be isolated from
- these recombinant host systems on the basis of the
physicochemical properties described above.
The general procedure for the preparation by
genetic engineering of a novel protein when the partial
, ~
- amino-acid~sequence is known is described in textbooks of
genetic~engineerlng, for example E~.L. Winnacker, Gene und
Klone~, Verlag Chemie, Weinhei , 1984. The experimental
conditions for~ the individual~methods, such as construc-
tion;~of~ a~gene~bank, hybridization and expression of a
gèné~are~ described in J. Sambrook et al. J Molecular
Cloning, Cold~Spring Harbor Laboratory, 1989.
;~ The~ protein ~according to the invention is pre-
ferably used in the form of its pharmaceutically accep-
table ~salts.
The novel protein~ has anticoagulant properties.
It can ~be used~, ~for example, for the prophylaxis of
thromboses or arterial reocalusions, for the treatment of
-~ ~ thromboses, for conserving blood or in extracorporeal
CirCùlation8`. " !~ ! ", ~ ! '
~ The invention is further illustrated by the
~ ~ folloving -ampl-J.
;~ ~
212113~
- 5 - O.Z. 0050/42778
Exa~ple 1
Purification of the thrombin-inhibitory protein from
ticks
A laboratory culture of the ticks (Ornithodoros
s moubata) was maintained at 28~C and 80~ relative
humidity. The ticks were allowed to feed off rabbits at
14-day intervals. Ticks in all stage~ of development were
frozen at -20C.
60 g of ticks were homogenized with 400 ml of
20 mM phosphate buffer, 150 mM NaCl (pH 7.5). The homo-
genate wa~ centrifu~ed at 7000 rpm (Sorvall RC-SB, rotor
~ GS-3) for 15 minutes. The precipitate was discarded, and
-~ 25 ml of 50~ by weight trichloroacetic acid was added to
the supernatant dropwise over a period of 15 minutes.
The mixture was then centrifuged (20 minutes,
7000 rpm) and the supernatant was neutralized with sodium
hydroxide solution.
The~neutralized supernatant was introduced into
the~ dialysi~s~ tubing (exclusion~vo~lume 300 Da) and dia-
~ 20 ~ lyzéd~several times against 10 times~the volume of 20 mM
-~ sodium~phosphate, 150 mM NaCl, pH 8Ø
The dialyzed protein solution was loaded onto a
Q-Sepharose column (Pharmacia) (60 ml/h) equilibrated
with~ 20 mN ~sodium phosphate buffer pH 8.0 (diameter
25 ~ .5~cm,~height 6 cm, volume~11 ml).
The~calumn was~washed with 10 column volumes of
equilibration buffer.
Then~a linea~r gradient from 50 ml of 20 mM sodium
phosphate~tpH~8.03~ to 50 ml of 20 mM sodium phosphate
30 ~ (pH 8.0), 1 M NaCl was applied.
-- Active fractions (measured by thrombin inhibi-
` tions) w~èrè dollected.'~
he combined active fractions from the Q-
Sepharose chromatography were loaded onto a copper
chelate chromatogràphy column (diameter 2.5 cm, height
6 cm, ~volume 30 ml) equilibrated with 20 mM sodium
phosph~te (pH 8.0), l50 m~ NaCl. The column wa~ washet
21211~ l
- 6 - O.Z. 0050/42778
with 10 column volumes of equilibration buffer
(1.5 ml/min) and eluted with a gradient rom 150 ml of
20 mM sodium phosphate (pH 8.0), 150 mM NaCl to 150 ml of
20 mM sodium phosphate (pH 8.0)r 150 mM NaCl, 100 mM
imidazole.
Active fractions tmeasured by thrombin inhibi-
~; tion) were collected.
The combined active fractions were~loaded onto an
affinity column with immobilized thrombin (diameter
1.5 cm, height 6.5 cm, volume 11.5 ml, 60 ml/h). The
colu~n was prepared a~ described in Example 3.
` The column was equilibrated with 20 mM sodium
phosphate pH 7.5. After the protein solution had been
loaded onto the column it was washed with 10 column
volumes of equilibration buffer until the absorption at
280 nm decreased to zero.
It was then washed with 0.5 M NaCl, 20 mM sodium
phosphate buffer pH 7.5. This removed non-specifically
a~sorbed~material.~
20 ~ Protein specifically bound to thrombin was eluted
wi~th~0.1 M~glycine, 0.5 M:~NaCl pH 2~8. The column was
then~ immediately read~usted to pH 7.5 with phosphate
buffer.
The individual fractions ~ere neutralized with
25 ~ O 1 N NaOH~ and t-sted for their inhibitory action on
The fraction~ eluted by glycine/NaCl buffer
pH~2.8 had a thrombin-inhibiting action.
he collected active fractions were, after
neutralization, diluted with water (1:10) and loaded onto
- a Mono-Q column (Pharmacia, volume l ml).
`~ The c~lumn Iwasi equilibrated with 20 mM sodium
phosphate buffer pH 7.S, 150 mN NaCl ~buffer A). It was
washed with buffer A until the absorption decreased to
zero ~10 minute ). The buffer was then changed over the
course of 50 mlnuteQ to 20 mM sodium phosphate, pH 7.5,
80~0 mM NaCl (buffer B) (flow rate 0.5 ml/min).
2121134
- 7 - O.Z. 0050/42778
Thrombin-inhibiting fractions were collected.
The collected fractions were further purified on
an RP 318- ~Biorad) HPLC column. The column was equi-
librated with 0.1% by weight trifluoroacetic acid (TFA)
in distilled water. The combined active fractions were
loaded onto the column which was then eluted with a
gradient to 0.1% TFA, 100% acetonitrile and a flow rate
of 1 ml/min over the course of 1 hour. The absorption was
determined at 280 nm, and 0.5 ml fractions were collec-
ted. The collected fractions were concentrated to dryness
and taken up in phosphate-buffered saline ~PBS) (0.8 g/l
` NaCl; 0.2 g/l HCl; 0.144 g/l sodium phosphate; 0.2 g/l
calcium phosphate, pH 7.5) and the inhibitory activity
was determined.
The protein was determined by the method of
Bradford (Anal. Biochem., 72, (1976) 248-254) using
bovine serum albumin (Boehringer Mannheim) as standard
pro~ein.
, -:
- Example 2
20~ ~ Determination of the inhibition of thrombin by the
;inhibitor
Thrombin (Boehringer ~annheim) wa~ dissolved to
a final concentration of 25 mU/ml in phosphate buffered
saline.
~ - ~Chromozym TH (Boehringer Mannheim) was dissolved
in~20 ml of H2O per vial.
50 ~1 of thrombin solution and 100 ~1 of
Chromozym~and 25 ~1 of sample or buffer were placed in
the~wèlls of a microtiter plate. The absorption at 405 nm
~- 30 was measured at 37C immediately thereafter at time 0 and
`after 3l~ minut!es~
When the sample was deeply colored another
control without thrombin was treated as described abo~e.
The activity of thrombin liberates a dye which
35 - absorbs at 405 nm from the chromogenic substrate. Inhibi-
tion of the thrombin by an inhibitor is evident from a
21211:~
- - 8 - O.Z. 0050l42778
sma~ler increase in absorption a~ 405 n~ and was quan-
tified using a calibration plot.
Example 3
Preparation of an affinity column with thrombin as ligand
a) Coupling:
2 g of CNBr-activated Sepharose (Pharmacia) were
washed with 200 ml of 1 mM HCl on a suction funnel.
The gel was taken up in 100 mM NaHCO3, 500 mM NaCl
pH 8.3 and Lmmediately mixed with lO,000 units of
~ 10 thrombin (Sigma) in 100 mM NaHCO3, 500 mM NaCl,
- ~ pH 8.3.
~ The solution was gently shaken at 4C for 24 hours.
.
; b) Blocking: ,
The gel material was aIlowed to settle and then
15;~ washed with 100 mN NaHCO3, 500 mM NaCl, pH 8.3. The
;Sepharose was then~ incubated with 100 mM NaHCO3,
5DO~mM NaCl, I M thanolamlne pH 8.3 for 2 hours.
c) Preparation:
, ., ~ :
To remove unbound ~thrombin, before use the gel
~ material is washed once more in the column with 20
column volumes~: of PBS pH 7 . 4 .
.- Example 4
Determination of the molecular weight by,molecular s!ieve,
, chromatography
Material purified by Mono-Q chromatography was
passed at a flow rate of 1 ml~min in 20 mM sodium phos-
phate, 150 mM NaCl, pH 7.5 through a Spherogel- TSg 3000
SW molecular sieve column (Pharmacia, diameter 7.5 mm,
212113~
- - 9 - O.Z. 0050/42778
hei~ht 60 cm).
The reference proteins were treated to the same
procedur~. lserum albumin MW 67,000 Da, ovalbumin
MW 45,000 Da, chymotrypsinogen A MW 25,000 Da).
The logarithm of the molecular weight of the
reference proteins was plotted against their elution
time.
The thrombin inhibition by the eluted fractions
of the sample was determined.
The logarithm of the molecular weight of the
-~ ~ inhibitor was obtained from the intercept of the elution
~`~ time on the calibration line.
The ~ molecular weight was found to be
22,000-28,000 Dalton.
,,
-~ 15 ExampIe S
- Detér~ination of the molecular weight by tricine SDS
polyacrylamide gel electrophoresi~
(Reference~ Analytical;Bioc~hemistry, I66, (1987)
;368-379~Tricine-Sodium Dodecyl Sulfate-Polyacrylamide Gel
20~ Elec~rophores~is~for~ the~ Separation~of Proteins in the
rangè~from l-1000 kDa~, Schagger, H. and von Jagow, G.)
Thè~gel electrophoresis wa~carried~out as stated
in~ehe~referenGe~at 20 mA and 1400 V, 30 watt.
Tho~molecular wei~ht determined by thi~ method
25~ was~8000 +~1500~D4lton~
The referencè proteins were intact myoglobin
17~.2~kDa~,~myoglobin:~I+II 14.6 kDa, myoglobin I 8.2 kDa,
yoglobin~ 6.4~kDa,~yo~globin III 2.6 kDa and myoglobin
Example 6
Determ~hation!l~f the}seiquence of~the inhibitor
Reduction and carboxymethylation
2.8~ml~of protein solution (0.02g mg/ml) were
mixed with 0.~28 ml of buffer (1 M tris/HCl, O.S M guani-
35;~ dine hydrochloride,~ pH 8.6). Then 0.116 ml of dithio-
threitol ~DTT,~ lO m~g/ml) was added and the mixture was
" 212113~
- 10 - O.Z. 0050J42778
inc~ated at 37C for 10 ~minutes. After addition of
O.185 ml of iodoacetamide (10 mg~ml) the mixture was
incubated at 37C for 90 minu~es. The reaction was
stopped with ~.073 ml of DTT as above.
~ 5 The protein was purified by renewed reversed
- phase HPLC on RP 318-. The mixture was adjusted to a
-~ final concentration of 0.1~ by weight trifluoroacetic
acid (TFA) and separated in an HPLC system from Hewlett
Packard (HP 1090 Liquid Chromatograph). The column was
washed with solvent A (0.1% TFA, 100~ H2O) for 5 minutes.
Then the proportion of solvent B (90% acetonitrile, 10%
H2O, 0.1~ by weight TFA) was increased to 50~ over the
course of 120 minutes. The absorption of the eluate at
214 and 280 nm was measured. Absorbing fractions were
collected. The protein was identified by SDS gel electro-
phoresis and subjected to sequence analysis in an applied
biosystems 477 A protein sequencer in accordance with the
~,.
manuf~acturer's instructions.
T~he ~following sequence was obtained (SEQ ID
NO~
Leu-Asn-Val-Leu-Cys-Asn-Asn-Pro-His-Thr-Ala-Asp-Cys-Asn
-Asn-Asp-Ala-Gln-Val-Asp-Arg-Tyr-Phe-Arg-Glu-Gly-Thr-Cys-
Leu-Met-Ser-Pro.
ampl-~7~
25 ~ Determination of the isoelectric point by isoelectric
focusing
The determination was carried out with an LKB
Mult~iphor~2117 ~Horizontal Qystem) and an LXB 2103 power
supply~.~Precast gels were employed (Pharmacia Ampholine
PAGplate pH 3.5-9.5). The standard proteins employed were
amylogluco~idase,l pH~315; soybean tryp$in inhibitor,~
pH 4.55; ~-lactoglobulin A, pH 5.2; bovine carbonic
anhydra-e, pH 5.857 human carbonic anhydra~e, pH 6.55;
hor e myoglobin, pH 6.85 and 7.35; lentil lectin,
~: 35 pN~8.15, 8:45, 8.65 and tryp~lnogen, p~ 9.3.
212113~
- 11 - O.z. 0050/42778
Focusing conditions: 1500 volt, 30 watt.
Buffer: Anode lM phosphoric acid
Cathode lM sodium hydroxide solution
The plates were prefocused for 30 minutes to
produce a pH gradient. The samples were loaded onto
filter disks which lay on the gel. Focusing was continued
for 30 minutes, the filter disks were removed and, after
a further 30 minutes, the focusing was stopped. The gels
were Lmmediately cut into 2 mm slices and transferred
into distilled water. The protein eluted out of the gel
slices overnight. The location of the thrombin inhibitor
was determined by a thrombin inhibition assay. The pH can
also be determined directly using a pH electrode. The
reference substance hirudin had an isoelectric point of
pH 3.5 and below. The novel inhibitor had an isoelectric
point below pH 3.8. ~
Example 8
Prepa~ration of a DNA sequence which codes for a thrombin-
inhibitory protein.
;~; 20 a); ~Isolation of RNA and~preparation of a cDNA bank
Total ~NA was obtained from whole anLmals of the
spec~e~ Ornithodoro~ moubata by disruption in gua~idinium
thiocyanate. This wàs done using the materials and
according~ to the instruction in the RNA isolation kit
from Stratagene, La Jolla, CA, USA (Catalog No.: 200345).
The polya~denylated mes~enger RNA was selected
~ from~the total RNA~by oligo(dT) affinity separation. This
- ~ ~ process was carried out with materials and according to
the instructions in the PolyATtract mRNA isolation system
from P~romega, Madlison', WI, USA (Catalog No.: ZS200).
cDNA was synthesized from polyadenylated mes-
-~ senger RN~ using the materials and according to the
-~ instructions in the ZAP-cDNA synthesis kit from
Stratagene, La Jolla, CA, USA (catalog No.: 200400) and
-; 35 was then cloned into the Eco RV restriction site of the
,,,,, ~ ~ .
,~ ~
2~2113~
- 12 - O.Z. 0050/42778
plas~id pBluescript II SK from Stratagene, La Jolla, USA
(Catalog No.: 212205).
b) Preparation of oligonucleotide probe~ for the PCR
Peptides from the protein sequence described in
Example 6 were used as basis for the cloning of cDNA
fragments by the polymerase chain reaction (PCR, see
Molecular Cloning, 2nd edition (1989), Sambrook, J. et
al., CS~ Press, page 14.1 et seq.).
On the basi~ of the genetic code it is possible
to deduce the nucleic acid sequence (SEQ ID NO: 2):
5'-TGY AAY AAY CCN CAY ACN GC-3'
from the peptide sequence I):
NH~-Cys-Asn-Asn-Pro-His-Thr-Ala (Pos 5-11)
and the nucleic acid sequence (SEQ ID NO: 3):
5'-CCN CAY ACN GCN GAY TGY AA TG-3'
from the peptide sequence II):
~H2-Pro-His-Thr-Ala-Asp-Cys-Asn (Pos 8-14),
each of the coding DNA strand. Because of the known
degeneracy of the genetic code, a plurality of nucleo-
tides ~N: A, C, G, T; Y: C, T;) can ~e used at some
positions. The complexity of the mixture is therefore
256-fold for SEQ ID NO: 2 and 512-fold for SEQ ID NO: 3.
The said sequences were synthesized as oligonucleotides.
The syntheses were carried out with an Applied
Biosystems type 360A DNA synthesizer. The oligonucleo-
tides were purified, after removal of the protective
groups, by gel electrophoresis on a polyacrylamide~urea
gel.
- c) PCRs and cloning of a part cDNA sequence
The polymerase chain reaction was carried out in
accordance with known protocols ~see Molecular Cloning,
2nd edition (1989), Sambroiok, J. et al., qSH Press, ~age
14.1 et seq.). A Perkin-Elmer DNA thermal cycler was used
for this. The principle of ~internal~ primers described
by Frohmann, M.A. et al. (Proc. Natl. Acad. Sci. USA 85,
(1988) 8998-9002, as modiied by Fritz, J.D. et al.
(Nucl. Acids Re~. 19, (1991~ 3747) was employed in this
212113~
- 13 - O.z. 0050/42778
case_
Specifically, the cDNA from a) was amplified with
20 pmol each of the oligonucleotides SEQ ID NO: 2 and the
synthetic T7 prLmer (deri~ed from pBluescrip~ II SK). ~he
conditions for this were 95C for l', 72C ~or 3' for
35 cycles.
The PCR products were fractionated by electropho-
resis on a 1.2% LMP agarose TBE gel.
Five slices were cut out of the gel over the
entire length of the "streak" and were melted as separate
fractions with DNA fragments of increasing molecular
mass.
Aliquots of these fractions we~e then used
separately in a second PCR with 20 pmol of each of the
oligonucleotides ii) and the synthetic M13-20 primer
(derived from pBluescript II SK).
The agarose content never exceeded 1/10 of the
volume of the PCR mixture. Reaction conditions: 95C for
1', 50C for 2', 72C for 3' for 35 cycles.
Fractionation by gel electrophoresis of the
amplification products of these fraction~ clearly showed
a reduction in the complex spectrum of products from the
irst PCR as far as a defined band after the second PCR.
The PCR pro~uct~ selected in this wa~- ~ere eluted
by standard methods. 5ubcloning into the EcoRV cleavage
- site of the ~ector pBlue~criptR5 and multiplication of
the plasmid in E. coli DH5alpha were followed by sequence
analysis of a clone which showed an open reading frame of
~1 amino acids (SEQ ID NO: 6) which agreed wilh the pre-
dicted protein sequence ~SEQ ID NO~
The DN~ and the amino acid sequence derived
therefrom are depicted in SEQ ID NO: 6.
d) Heterologous expression
The following oligonucleotides were synthesized
for the heterologous expression of the sequence of 91
amino acids in E. coli:
Nucleic acid sequence SEQ ID NO: 4
21 211~4
- 14 - O.Z. 0050/42778
and
nucleic acid sequence SEQ ID NO: 5
SEQ ID NO: 4 is derived from SEQ ID NO: 1; it
corresponds to the sequence for the amino acid~ in
positions 1 - 14; SEQ ID NO: 5 is deri~ed from SEQ ID
NO: 6 and correspond~ to the sequence of the complement-
ary strand for amino acids 87 - 91 and the stop codon.
The additional 15 nucleotides at the 5' end of SEQ ID
NO; 5 are intended to reconstitute a Sal I cleavage site
for cloning purposes.
A PCR with these two oligonucleotides and the DNA
cloned in c) as template led to the isolation of a cDNA
:~ sequence which contains the complete coding region of the
- abovementioned thrombin inhibitor (SEQ ID NO: 6). The
reaction conditions were 10 ~l of template, 20 pmol each
of oligonucleotides SEQ ID NO: 4 and SEQ ID NO: 5; 95C
for 1', 55C for 2', 55C for 2', 72C for 3' for
3S cycles.
The PCR product was then cut with Sal I, analyzed
20~ by~gel e~leotrophore8is and eluted by stand rd m~thods.
For heterologous expression in E. coli, the PCR
fragment~was subcloned into the inducible vector which is
expres~sed in the periplasm, pNal-p2 from New England
Biolabs, Beverly, M~, USA (Catalog No. 800-65 S).
To do this, pMal-p2 was digested w.Lth Xmn I and
Sal I, fractionated by gel electrophoresis, and the
;vector band was eluted by~standard method~ and ligated to
the PCR fra~ment.
In this wày the sequence of the novel thrombin
~ 30~ inhibitor tSEQ~ID NO: 6) was fused directly to the 3' end
: of the bacterial gene for the maltose binding protein
(mai E).
~: The constr~lct was tran~formed by standard methods
into the E. coli strain TBl (New England Biolab~).
Growth of transformed cell~, induction with IPTG
and workup of the periplasmic fraction, and liberation of
the thrombin inhibitor by cleavage with factor Xa were
,. . - ~
.~
. .
.
,. ~
21211~4
- 15 - O.Z. 0~50~2778
carr-ied out exactly as stated by the manufacturer. The
individual steps were analyzed by SDS gel :
electrophoresis.
The activity of the recombinant thrombin inhibi-
tor in the thrombin inhibition assay was entirely com-
parable to the natural inhibitor from Ornithodoros -
moubata. i
i ~ .
'
::,.- -: : : :
: : :
- . ` . : ` ' ! I i , i i ~ ! ,,
'~. ., ~,' ':
'~ ~;,,
~ :,
" .
~' ~
` ~ . '.
~ .
2 11~ 1 1 3 4 o. z . 0050/42778
. SEQUENCE LISTING
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(H) TE~EFAX: 0621/6043123
(I) TELEX: 1762175170
(ii) TITLE OF THE INVENTION: Novel thrombin-inhibi-
tory protein from ticks `~-
~iii) NUMBER OF SEQUENCES: 7
( iv) COMPUTER READABLE FORM: ~`
(A) MEDIUM TYPE: Floppy disk . ~-
. (B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
D) SOFTWARE: PatentIn Release #1.0, Version #1.25 (EPO)
-~ : (v] CURRENT APPLICATION DATA: . .
APPLICATION NUMBER:
l2) INFO~MATION FOR SEQ ID NO:l: -~
~ (i) SEQUENCE CHARACTERISTICS~
;~ (A) LENGTH: 33 amino acids .
B) TYPE: amino acid :
: (C) STRANDEDNESS: single
~; (D) TOPOLOGY: linear ~:
i) MOLECULE TYPE: protein
(iiil HYPOTHETICAL: NO
(iv) ANTI-SENS~E: NO
(v) FRAGMENT TYPE: N-terminal
. . ~
(vi) ORIGINAL~SOURCE: ~-
A) ORGANISM: Ornithodoros moubata
( D ) DEVELOPMENTAL STAGE: adult
,, (F) TIS$UE TYPE;~,whole animals
(ix) FEATURE:
- (A) NAMEtKEY: Peptide
B) LOCATION: 1..33
~ ~ ~xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:
- Leu Asn Val Leu Cys Asn Asn Pro His Thr Ala Asp Cys Asn Asn Asp
- 1 5 10 15
Ala Gln Val Asp Arg Tyr Phe Arg Glu Gly Thr Thr Cys Leu Met Ser
::
'
.
2121134 ;~
: 17 ~ ~
. . ~.
" .:,,.;
Pro `;~`
(2) INFO~MATION FOR SEQ ID NO:2: . .
(i) SEQUENCE CHARACTERISTICS:
(A~ LENGTH: 20 ~ase pairs ~ ~ :
(B) TYPE: nucleic acid ~.
(C) STRANDEDNESS: single -~.
(D) TOPOLOGY: linear .~
(ii) MOLECULE TYPE: DNA (genomic) .`
(iii) HYPOTHETICAL: YES . ~.
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2~
TGYAAYAAYC CNCAYACNGC 20
(2) INFORMATION FOR SEQ ID NO:3
(i) SEQUENC:E CHARACTERISTICS: :-~
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid .
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear ;~
(ii) MOLECULE TYPE: DNA (genomic)
liii) HYPOTHETICAL: YES
(i~j ANTI-SENSE: NO `:
:: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: ~,
: CCNCAYACNG~CNGAYTGYAA 20
(2) INFORMATION FOR SEQ ID NO:4: .
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 42 base pairs .
(B? TYPE: nucleic acid
:~ (C)~STRANDEDNE$S: single
(D) TOPOLOGY: linear
(i.i) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL:: NO
: (iv) ANTI-SENSE: NO
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:
TTGAATGTGT TGTCCAATAA TCCGCATACG GCCGATTGCA AC 42
(2),INFORMATION FOR SEQ ID N,0-5i;~
(i) SEQUENCE CHARACTERISTICS:
~(A) LENGTH: 33 base pairs
:~ ~ (B) TYPE: nucleic acid
~: (C) STRANDEDNESS: single ;~
. (D) TOPOLOGY: linear :~
: (ii) MOLECULE TYPE: DNA (genomic) :~:
. (iii) HYPOTHETICAL: NO
, .
212113~
18
(iv) ANTI-SENSE: NO ~.
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: .~.
GGGGGGGTCG ACGTGCTAGT ACCTTA~.;TG TTT 33 :-.
(2) I~FORMATION FOR SEQ ID NO:6: ::~
(i) SEQUENCE C~ARACTERISTICS:
~A) LENGTH: 291 base pairs
(B) TYPE: nucleic acid
(CJ STRAN~EDNESS: single
(D) TOPOLOGY: linear
(il) MOLECULE TYPE: cDNA to mRNA
(iii) HYPOTHETICAL: NO ;~
- (iv) ANTI-SENSE: NO ~-~
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Ornithodoros moubata ~.
(D) DEVELOPMENTAL STAGE: adult ~^
(F) TISSUE TYPE: whole animals
(i~) FEATURE: . ~.
~(A) NAME/KEY: CDS
(B) LOCATION: 1..277
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: :
TTG AAT GT~ TTG TCC AAT AAT CCG CAT ACG GCC GAT TGC AAC AAT GAT 48
Leu Asn Val Leu Ser Asn Asn Pro His Thr Ala Asp Cys Asn Asn Asp
1 :5 10 : 15 ~:`--.
GCA CAG GTT GAC AGA TAT TTT AGG GAG GGG ACA ACG TGC CTA ATG TCC 96
Ala Gln Val Asp Arg Tyr Phe Arg Glu Gly Thr Thr Cys Leu Met Ser
20 25 30
CCA~GCA TGC~ACG AGC GAA GGA TAC GCC TCT CAG CAC GAA TGT CTC AGG 144 .~
Pro Ala Cys Thr Ser Glu Gly Tyr Ala Ser Gln His Glu Cys Leu Arg ~:
35 40 45
CCT GCT TTG TTG GCG GGG AAG ACC ACA GCA GTG AAA TGC ACA GCT CAT 192 ~:
Pro Ala Leu Leu Ala Gly Lys Thr Thr Ala Val Lys Cys Thr Ala His -
50 : 55 : 60 ~:~
GCC TTG GTG ACC CGC CCA CTT CCT GCG CGG AAG GCA CGG ACA TCA CCT 240
Rla Leu Val Thr Arg Pro Leu Pro Ala Arg Lys Ala Arg Thr Ser Pro
65 70 75 80 ~.
ACT ACG ATT CTG ATA GCA A~A CAT,GTA AGG TAC TAG .C AGGTCGACCC ~ 287
Thr Thr Ile Leu Ile Ala Lys His Val Arg Tyr * :~
CCCC . 291
(2) INFORMATION FOR SEQ ID NO:7:
' ~.
~;
212113A
19 !.
(i) SEQUÉNCE CHARACTERISTICS~
(A~ LENGTH: 92 amino acids
(B) TYPE: amino acid .~
(D) TOPOLOGY: linear .;i
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ I~ NO:7:
Leu Asn Val Leu Ser Asn Asn Pro His Thr Ala Asp Cys Asn Asn Asp ~;
1 5 10 15 :~
Ala Gln Val Asp Arg Tyr Phe Arg Glu Gly Thr Thr Cys Leu:Met Ser
20 . 25 30 ~;
Pro Ala Cys Thr Ser Glu Gly Tyr Ala Ser Gln His Glu Cys Leu Arg ,-.
Pro Ala Leu Leu Ala Gly Lys Thr Thr Ala Val Lys Cys Thr Ala His .
: 50 55 60
Ala Leu Val Thr Arg Pro Leu Pro Ala Arg Lys Ala Arg Thr Ser Pro
65 70 75 80
Thr Thr Ile Leu Ile Ala Lys His Val Arg Tyr *
--
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