Note: Descriptions are shown in the official language in which they were submitted.
ZC5~90
O.Z. 0050/41125
Novel proteins and the preparation thereof
Description
The present invention relates to novel proteins
and the preparation thereof.
It is already known that leeches produce a
substance, hirudin, which inhibits blood clotting ~cf.
The Nerck Index, 10th edition, No. 4613). Hirudin is a
polypeptide with the molecular weight of about 6,500.
Substances which inhibit blood clotting are also
produced by other blood-sucking parasites, eg. ticks.
However, these substances have to date neither been
obtained in pure form nor prepared synthetically
(Naturwissenschaften 11, 30 (1961).
A novel protein has now been isolated from ticks.
The invention relates to a novel protein which
has a molecular weight of about 15,000 dalton and has at
the amino terminus the amino-acid sequence
SDYEFPPPKKXRPG
in which X i8 S or N, and to the muteins thereof.
By muteins are meant proteins which are produced
from the novel protein by exchange, deletion and/or
addition of amino acids or peptides in the protein chain
without thereby essentially altering the action of the
novel protein.
The novel protein can be isolated from ticks. For
this, the ticks are taken up in a buffer at pH 6 to 9,
preferably 7.5. After homogenization of the mixture with
a homogenizer, the insoluble components are removed by
centrifugation. Inactive protein is precipitated from the
solution obtained in this way by addition of trichloro-
acetic acid to a final concentration of 2.5 ~, and is
removed by centrifugation. Ether-soluble components are
removed from the solution obtained in this way by
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extraction, and subsequently the protein is precipitated
with cold acetone. The novel protein can be isolated from
the precipitated product by ion exchange and pH gradient
chromatography.
The protein described herein is present in the
ticks in concentration between 1 - 100 ~g/kg. It is
possible to employ known genetic engineering methods (cf.
Maniatis, T. et al.: Molecular Clonings A Laboratory
Manual, Cold Spring Harbor Pre~s, N.Y., 1982) in order to
make the protein available in larger amounts for pharma-
ceutical purposes. For this purpose, the genetic informa-
tion for the novel protein must first be identified, and
the corresponding nucleic acid isolated. For this, the
pure protein i8 reduced with dithiothreitol, then iodo-
acetamide is added to derivatize the free SH groups and
subsequently the protein treated in this way is cleaved
with cyanogen bromide and trypsin into small peptides.
The peptides are fractionated by reversed phase chromato-
graphy. N-terminal sequencing of one of these purified
peptides revealed the sequence SDYEFPPPRKSRPG.
The available peptide sequences now permit, by
the synthesis of corresponding oligonucleotides, an
unambiguous identification of the gene from the genome or
from appropriate c-DNA banks by sequence-specific filter
hybridization.
The genetic information, obtained in this way,
for the protein can then be expressed in various host
cells such as eukaryotic cells, yéasts, 9acillus subtilis
or E. coli by known methods, and the protein can be
obtained in this way. Moreover, in the eukaryotic cells
the protein is produced in glycosylated form.
The muteins which are derived from the novel
proteins by exchange, deletion or additional amino acids
or peptides are preferably prepared by genetic engineer-
ing methods.
The novel protein has anticoagulant properties
and can be employed for preventing and treating vascular
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disorders such a~ myocardial infarct, pulmonary embolism,
arterial embolism and phlebothrombosis. Further suitable
as pre~ervative for blood. The low toxicity of the novel
protein i9 advantageous for these uses.
The novel protein is furthermore suitable for
immunizing grazing cattle against tick-specific proteins.
Thus, it i~ possible for neutralizing antibodies to
interfere with feeding by the ticks. This induces the
ticks to leave the host and thus die.
EXAMPLE 1 TO 3
EXA~PLE 1
Purification of the thrombin inhibitor
A culture of argasid ticks (Ornithodorus moubata)
kept in a laboratory at 28C and 80 ~ relative humidity
was fed at 14-day intervals by allowing them to bite
rabbits. Ticks in all stages of development, before or
after feeding, were frozen at -20C and employed in the
subsequent wo~king up. 10 g of ticks were taken up for
this in 20 ml of phosphate-buffered saline (20 mM sodium
phosphate, 150 mN NaCl, 1 mM EDTA, pH 7.5 and homogenized
at 20C. The insoluble components were removed by
centrifugation.
Trichloroacetic acid (TCA) was added to the clear
red supernatant until the final concentration was 2.5 %
TCA. A voluminous red precipitate formed and was removed
by centrifugation. The solution was then extracted by
shaking 3 times with 1/10 of its volume of ether. The
aqueous solution was neutralized with NaOH.
To this was added 4 times the volume of cold
(-78-C) acetone. The solution stood in the cold on dry
ice during this. A flocculent whlte precipitate formed
during the course of up to 24 h. This was removed by
centrifugation and dried in a desiccator. The yield was
1 ~ of the weight of the ticks employed. In the SDS gel
electrophoresis this product still showed 10 to 20 pro-
tein bands in the low molecular weight range up to 40 kDa
after staining with Coomassie brilliant blue. The
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isoQlectric point of these proteins was between pH 4 and
pH 7.
This protein mixture (200 mg) was now taken up in
phosphate-buffer saline, pH 5, to a protein concentration
of 20 mg/ml and loaded onto a Q-Sepharose column
(Pharmacia Fine Chemicals, fast-flow). The volume of the
column was 10 ml. The column was equilibrated with the
same buffer in which the sample was also taken up. After
the column had been washed with three column volumes of
equilibration buffer the column was eluted with a pH
gradient. For this, 10 column volumes of the washing
buffer (pH 5.0) were mixed slowly, via a gradient mixer,
with lO column volumes of washing buffer (pH l to 3). The
thrombin inhibitor eluted at pH 4.5 under these condi-
tions. The protein was obtained in pure and active form
in this way. It showed a molecular weight of 15,000 +
1,000 in the gel electrophoresis. The amino-terminal
sequence of this protein i8S SDYEFPPPKKSRPG. Its isoelec-
tric point is at pH 4 to 5.
EXAMPLE 2
Separation of active from inactive thrombin inhibitor
molecules.
The protein which was homogeneous according to
molecular weight, obtained as in Example l and identified
by thrombin inhibitory action was precipitated as de-
scribed in Example l by a 4-fold excess of cold acetone
and dried. The residue was then taken up in phosphate-
buffered saline, pH 7.5 (protein concentration 20 mg/ml)
and loaded (about 0.1 mg) onto a mono-Q column (column
volume 1 ml) equilibrated with the same buffer. The
column was washed at a flow rate of 0.5 ml/min and then
eluted with a gradient from 150 mN NaCl to 350 mN NaCl
(2.5 ml of each) and then from 350 mM NaCl to 450 mN NaCl
(2.5 ml of each), followed by from 450 mN NaCl to 550 mN
NaCl (10 ml of each) and finally to 800 mN NaCl. The
protein appeared between 350 mM NaCl and 550 mN NaCl.
However, activity (measured as thrombin inhibitory
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action) was associated only with an absorption maximum at
450 mM NaCl. Sequence analysis of all the eluted proteins
revealed, however, that all have the ~ame amino-terminal
~equence and thus are identical with respect to their
S primary structure.
EXAMPLE 3
Renaturation of the inactive thrombin inhibitor molecules
The inactive thrombin inhibitor fraction obtained
in 2 was precipitated as described in Example 1 and taken
up in a renaturation buffer which contained 8 M urea and
200 mM dithiothreitol (DTT). After 1 h at 37C, the
protein was dialyzed against 100 times the volume of
phosphate-buffered saline (exclusion volume of the
dialysis tube 10 kDa). After dialysis at 4C for 2 h, the
protein was active and showed on the mono-Q column after
elution by a salt gradient the same behavior as the
active fraction in Example 2.
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