CA2054190A1 - Proteins suitable for combating disease and for protecting grazing cattle from ticks - Google Patents

Abstract

O.Z. 0050/41125 Novel proteins and the preparation thereof Abstract A novel protein with the molecular weight of about 15,000 dalton and the N terminus SDYEFPPPKKXRPG is described. The protein is suitable for controlling diseases and for protecting grazing cattle against ticks.

Description

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 ;..... . . : - ' - . ' ' 2cs~o - 2 - O.Z. 0050/41125 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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- 3 - O.Z. 0050/41125 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.

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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~C5~ 0 ~ - 4 - O.Z. 0050/41125 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.

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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ZC5~1~0 ` ~- - 5 - O.Z. OOS0/41125 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.

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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Claims (4)

- 6 - O.Z. 0050/41125 Claims

1. A protein which has the N-terminal amino-acid sequence SDYEFPPPKKXRPG in which X is S or N, and the molecular weight of about 15,000 dalton, and the muteins thereof.

2. A protein as claimed in claim 1 for use for controlling diseases.

3. The use of the protein as claimed in claim 1 for preparing antibodies against tick attack and for immuniz-ing grazing cattle against tick attack.

4. A process for preparing the protein as claimed in claim 1, which comprises a) homogenizing ticks, removing the insoluble com-ponents from the homogenate, precipitating inactive proteins from the supernatant by precipitation with trichloroacetic acid, and subjecting the protein remaining in solution after precipitation with acetone to a chromatography, or b) preparing the protein by known genetic engineering methods.