Chemistry: molecular biology and microbiology – Vector – per se
Patent
1994-05-18
1995-10-03
Wax, Robert A.
Chemistry: molecular biology and microbiology
Vector, per se
435 691, 4352402, 4352523, 514 2, 514 12, 530300, 530324, 536 221, 536 231, 536 235, C12N 1500, C12P 2106, A61K 3800, C07K 100
Patent
active
054551811
DESCRIPTION:
BRIEF SUMMARY
FIELD OF THE INVENTION
The present invention relates to novel thrombin-inhibitory proteins from the terrestrial leech Haemadipsa sylvestris and to processes for the preparation thereof.
BACKGROUND OF THE INVENTION
Thrombin inhibitors are important therapeutic substances used, for example, for the prophylaxis or treatment of thromboses or arterial reocclusions.
EP 142 860 describes the thrombin inhibitor hirudin from the medical leech (Hirudo medicinalis) with its primary structure. Furthermore, the preparation of hirudin by genetic manipulation is disclosed, for example, in EP 168 342.
SUMMARY OF THE INVENTION
We have now found novel thrombin-inhibitory proteins from the terrestrial leech Haemadipsa sylvestris.
The novel proteins have the following physicochemical properties. A molecular weight of 11200.+-.1000 dalton is assigned to them by molecular sieve chromatography. A molecular weight of 5000.+-.1000 dalton is determined in an SDS polyacrylamide gel.
The proteins bind specifically to a thrombin affinity column. They inhibit the biological activity of thrombin in an in vitro enzyme assay.
The following N-terminal amino-acid sequence was determined for the proteins (SEQ ID NO: 3): Ile-Arg-Phe-Gly-Met-Gly-Lys-Val-Pro-Cys-D-A-Gly-Glu-Val-Gly-Tyr-Thr-Cys-As p-Cys-Gly-B-C-Ile-Cys-Leu-Tyr-Gly-Gln-Ser-Cys-Asn-Asp-Gly-Gln-Cys-Ser-Gly-A sp-Pro-Lys-Pro-Ser, where A is Asp or Phe, B is Glu or Trp, C is Lys, Asn or Asp, and D is Pro or Leu.
The novel proteins can be isolated from terrestrial leeches of the genus Haemadipsa. For this, the leeches are expediently homogenized in a buffer at pH 6-9, preferably pH 7-8, in a homogenizer, preferably a mixer. The insoluble constituents are then removed, preferably by centrifugation.
The proteins can be further purified from the resulting solution by chromatographic methods, preferably ion exchange chromatography and/or affinity chromatography. A purification step by thrombin affinity chromatography is particularly preferred.
After affinity chromatography on a thrombin column it is possible to separate various isoproteins with thrombin-inhibitory activity by reversed phase HPLC (see FIG. 1).
The purification of the proteins can be followed by a thrombin activity assay. It is expedient to use for this an optical assay in which a chromogenic substrate, for example Chromozym TH, is converted by thrombin. The fractions containing the novel proteins can be identified on addition to this optical assay by their thrombin-inhibiting action.
Genetic manipulation processes are particularly suitable for preparing the proteins according to the invention.
To do this, a leech 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 probe whose sequence is obtained from the N-terminal amino-acid sequence described above by translation back in accordance with the genetic code. The appropriate gene can be found and isolated by hybridization with this DNA probe.
However, the polymerase chain reaction (PCR) technique can also be employed to prepare the appropriate gene. For example, it is possible with the aid of a primer whose sequence has been obtained by translation back from the N-terminal amino-acid sequence described above, and of a second primer whose sequence is complementary to the 3' end of the cDNA gene fragment, preferably with the sequence poly(dT), to prepare the cDNA gene fragment for the protein according to the invention by the PCR technique. The appropriate gene can also be isolated by setting up a leech expression gene bank and screening it with an antibody directed against the protein according to the invention.
A cDNA which codes for a protein according to the invention is depicted in SEQ ID NO: 22.
Other suitable DNA sequences are those which, although their nucleotide sequence differs from that detailed in SEQ ID NO: 22, do code, as a consequence of the degeneracy of the genetic code, for the polypeptide chain detailed in SEQ ID NO: 22, or parts
REFERENCES:
Primary structures of new `iso-hirudins, Scharf et al. Febs Letters, vol. 255, No. 1, Sep. 1989.
Bialojan Siegfried
Friedrich Thomas
Kroeger Burkhard
Strube Karl-Hermann
BASF - Aktiengesellschaft
Kim Hyosuk
Wax Robert A.
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