Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Peptide containing doai
Patent
1994-04-26
1996-06-04
Patterson, Jr., Charles L.
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Peptide containing doai
530350, 530856, 514822, 536 235, C07K 1481, A61K 3857, C12N 1515
Patent
active
055232876
DESCRIPTION:
BRIEF SUMMARY
The present invention relates to a novel thrombin-inhibitory protein from assassin bugs and to a process for preparing it.
Thrombin inhibitors are important 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 which has been isolated from the argasid tick Ornithodoros moubata. This protein has a molecular weight of about 15,000 dalton, an isoelectric point at pH 4-5 and the N-terminal amino-acid sequence SDYEFPPPKKXRPG.
European Published Application EP 345 614 describes the thrombin inhibitor amblyommin which is isolated from bont ticks. This is a protein with a molecular weight of 20,000-30,000 dalton and an isoelectric point at pH 5.05-5.65.
However, to date no protein with thrombin-inhibitory action has yet been found to be suitable and advantageous as a drug in terms of high activity, lack of antigenicity, long biological half-life, and few side effects such as risk of hemorrhage.
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 assassin bugs.
The novel protein has the following physicochemical properties. Molecular sieve chromatography shows that it has a molecular weight of 20,000-24,000 dalton. A molecular weight of 12,000.+-.2000 dalton is determined in an SDS polyacrylamide gel. The determination of the isoelectric point shows that it is at pH 3.7-4.7.
The protein binds specifically to a thrombin affinity column. It inhibits the biological activity of thrombin in an in vitro enzyme assay.
The following N-terminal amino-acid sequence was determined for the protein (SEQ ID NO: 1): Glu-Gly-Gly-Glu-Pro-Cys-Ala-Cys-Pro-His-Ala-Leu-His-Arg-Val-Cys-Gly-Ser-As p
The protein according to the invention contains a sequence of 103 amino acids which is shown in sequence listing SEQ ID NO: 3.
The DNA coding for this sequence of 103 amino acids is likewise detailed in sequence listing SEQ ID NO: 2.
The protein according to the invention may additionally contain further amino acids at the C terminus. It is likewise possible for the protein to contain at the N terminus other amino-acid sequences such as natural or heterologous leader sequences or additional amino acids such as methionine.
It is possible to predict from the amino-acid sequence indicated in sequence listing SEQ ID NO: 3 that there are two domains in this molecule. The first domain is limited essentially by the cysteine residues at positions 6 and 48, and the second domain by the cysteine residues at positions 57 and 101.
The invention also relates to DNA sequences which code for proteins having a thrombin-inhibitory action and which are selected from the group formed by 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12 and SEQ ID NO: 14, and sequences a).
These DNA sequences are for thrombin inhibitors having up to 6 domains.
The invention also relates to smaller proteins which contain only one of these two domains. Proteins according to the invention also include those which contain a plurality, preferably up to 10, of such domains.
In this connection, the minimum requirements for a single active domain are the amino acids (from SEQ ID NO: 7) from position 6 to 48, 57 to 101 or 119 to 160 or the amino acids from SEQ ID NO: 17 from position 6 to 48, 57 to 101, 120 to 161, 190 to 232, 241 to 285 or 303 to 344.
N-Terminal and C-terminal extensions do not interfere with the thrombin-inhibiting activity. Extensions of these types may contain sequences such as, for example, the amino acids 1 to 5, 49 to 56, 102 to 118, 161 to 170 from SEQ ID NO: 7 or 1 to 5, 49 to 56, 102 to 119, 162 to 189, 233 to 240, 286 to 302 or 345 to 354 from SEQ ID NO: 17.
These amino-acid sequences, which are distinguished by high flexibility and hydrophilicity, can be employed as spacers between the individual domains. Multidomain proteins of t
REFERENCES:
patent: 5093322 (1992-03-01), Bonin et al.
Bialojan Siegfried
Friedrich Thomas
Kroeger Burkhard
Kuenast Christoph
BASF - Aktiengesellschaft
Patterson Jr. Charles L.
Prouty Rebecca
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