Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Peptide containing doai
Patent
1994-03-18
1996-12-17
Wax, Robert A.
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Peptide containing doai
530350, 435214, A61K 3855, C07K 1481, C12N 974
Patent
active
055853501
DESCRIPTION:
BRIEF SUMMARY
The present invention relates to a novel thrombin-inhibitory protein from ticks, and to processes for its preparation.
Thrombin inhibitors are important therapeutic substances used, for example, for the prophylaxis or treatment of thromboses or arterial reocclusions.
DE-A 39 31 839 describes a thrombin inhibitor which was isolated from the argasid tick Ornithodoros moubata. This protein has a molecular weight of about 15000 dalton, an isoelectric point at pH 4-5 and the N-terminal amino acid sequence SDYEFPPPKKXRPG (SEQ. I.D. NO.: 1)
EP-A 345 614 describes the thrombin inhibitor amblyommin which is isolated from ixodid ticks. This is a protein with a molecular weight of 20,000-30,000 dalton and an isoelectric point at from 5.05 to 5.65.
However, to date no protein with a thrombin-inhibitory effect which is suitable as a drug in terms of high activity, lack of antigenicity, long biological half-life and few side effects such as bleeding tendency has been found.
It is an object of the present invention to provide novel thrombin inhibitors which are suitable as drugs in terms of the abovementioned properties.
We have found that this object is achieved by a novel thrombin-inhibitory protein isolated from ticks.
The novel protein has the following physico-chemical properties: a molecular weight of 26000-29000 dalton was assigned to it by molecular sieve chromatography. A molecular weight of 11000.+-.1500 dalton was determined by electrophoresis in a tricine SDS polyacrylamide gel. A molecular weight of 25700.+-.3000 dalton was determined by L aemmli electrophoresis in an SDS polyacrylamide gel. Determination of the isoelectric point showed that it is at pH 4-5.
The protein binds specifically to a thrombin affinity column. It inhibits the biological activity of thrombin in an in vitro enzyme assay.
The protein band in a polyacrylamide gel cannot be stained with silver; it is visible only as an unstained spot on a stained background.
The amino terminus of the protein is blocked. The following partial amino-acid sequences were determined for the protein:
______________________________________ Sequence I:
Val--Ala--Lys--Phe--Pro--Ala--(Ala)--Asn--
Ser--Gly--Ser--Glu--Thr--Gly (SEQ ID NO: 2)
Sequence II:
His--Ala--(Cys)--Phe--Glu (SEQ ID NO: 3)
Sequence III:
90% Arg--Val--Ser--Asp--Phe--Glu
(SEQ ID NO: 4)
10% Phe--Ala--(Glu/His)--Lys (SEQ ID NO: 5)
Sequence IV:
70% Phe--Val--Tyr--Thr--Ile--Glu
(SEQ ID NO: 6)
30% Ala--Phe--Gln--Gly (SEQ ID NO: 7)
______________________________________
The identification of the amino acids in parentheses is not entirely certain. Sequences III and IV are in each case mixtures of two sequences which occurred in the stated intensities.
The novel protein can be isolated from ticks of the genus Ornithodoros. To do this, the ticks are homogenized, expediently in a buffer of pH 6-9, preferably pH 7-8, using a homogenizer, preferably a mixer. The insoluble constituents are then removed, preferably by centrifugation.
The protein can be further purified from the resulting solution by adding a precipitant, preferably trichloroacetic acid, to precipitate other proteins from the solution, which are subsequently removed.
Further purification of the protein is possible by chromatographic methods, preferably ion exchange chromatography and/or affinity chromatography. Purification by thrombin affinity chromatography is particularly preferred.
The purification of the protein can be followed by a thrombin activity assay, which is expediently an optical assay in which a chromogenic substrate, for example Chromozym T, is converted by thrombin. The fractions containing the novel protein can be identified by their thrombin-inhibiting action when added to the optical assay.
Methods of genetic manipulation are particularly suitable for preparing the protein according to the invention.
To do this, a tick cDNA gene bank is set up in a conventional way. The gene coding for the protein according to the invention can be isolated from this gene bank by, for example, preparing a DNA pro
REFERENCES:
Characterization of Recombinant Tick Anticoagulant Peptide, Journal of Biological Cehmistry, vol. 265, No. 29, Oct. 15, 1990, Neeper et al.
Bialojan Siegfried
Friedrich Thomas
Koerwer Wolfgang
Kroeger Burkhard
BASF - Aktiengesellschaft
Lau Kawai
Wax Robert A.
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