Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Hydrolase
Patent
1994-06-30
1998-09-01
Wax, Robert A.
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Hydrolase
4351723, 435219, 536 232, C12N 952, C12N 1558
Patent
active
058010378
DESCRIPTION:
BRIEF SUMMARY
The present invention relates to signal-peptide-free expressed forms of the bacterial plasminogen activator staphylokinase (abbreviated form: SAK), including novel forms of staphylokinase, and also to processes for their production by means of genetic engineering and to their use. The invention thus includes DNA sequences which code for the SAK forms according to the invention, recombinant plasmids on which these DNA sequences are present coupled with highly effective expression signals, selected recombinant host cells which, after incorporation each time of one of these expression plasmids, are capable under cultivation conditions of the biosynthesis of the SAK forms according to the invention, and to part-processes for this cultivation and for the isolation of the staphylokinase forms (target polypeptides) from the culture soups obtained.
In conjunction with this, the invention also comprises the first-time provision of monoclonal anti-staphylokinase antibodies (anti-SAK mABs).
As has been known since the studies conducted by CH. LACK (Nature, 161 (1948), 558-560) and by K. C. ROBBINS et al. (J. Biol. Chem., 242 (1967), 2333-2342) and by K. W. JACKSON et al. (Methods in Enzymology, 80, (1981), 387), staphylokinase, a polypeptide from Staphylococcus aureus strains, is in the position to arrange the transformation of the proenzyme plasminogen localized in human or animal blood plasma into the fibrinolytically active enzyme plasmin, without itself displaying proteolytic activity. The mechanism of the activation is still largely unexplained.
The staphylokinase forms according to the invention are thus suitable as pharmaceutical preparations in human and also optionally in veterinary medicine during the treatment of thromboembolic angioses, while the anti-SAK mABs can be used both during the obtaining of active ingredient and, as is explained in more detail below, as pharmaceutical preparations.
Staphylokinase (molecule length: 136 amino acids) is a member of the group of those substances which can effect an activation of the fibrinolytic system via the transformation of plasminogens to plasmin and the cleavage of the fibrin caused by this. The best-known endogenous activators are the human-tissue plasminogen activator (t-PA) and urokinase. Another plasminogen activator of bacterial origin is streptokinase. The said substances are already used therapeutically for the treatment of patients with thromboembolic angioses. As the incidence of these illnesses is high, there is a considerable demand for substance quantities with a high purity standard.
Differently from the case of, say, streptokinase, no industrial production processes have been developed for SAK with the help of conventional industrial microbiology, although this bacterial plasminogen activator offers for example the advantages of
This situation had its cause primarily in the fact that a large number of toxic accompanying products are formed by the Staphylococcus aureus producer strains.
After the introduction of the recombinant DNA technique, the synthesis of SAK by recombinant host organisms became possible, with the result that a technically efficient generation of pharmaceutical preparations with this polypeptide as active ingredient was realizable.
Two genes coding for staphylokinase, which have been isolated from the genomes of Staphylococcus aureus phages, are described in the state of the art. Used as gene donors were, on the one hand, the S. aureus phage C (T. SAKO et al., Mol. Gen. Genet., 190 (1983), 271-277; T. SAKO/N. TSUCHIDA, Nucl. Acids Res., 11 (1983), 7679-7693; cf. EP 0 077 664) and, on the other hand, the S. Aureus phage 42D (cf. DD 245 444 and also D. BEHNKE/D. GERLACH, Mol. Gen. Genet., 210 (1987), 528-534). The genes were initially primary-cloned each time on the E. coli plasmid pBR332 and identified by means of DNA sequence analysis after various subcloning steps.
The isolation of another staphylokinase gene has recently been described (D. COLLEN et al., Fibrinolysis, 6 (1992), 226-231). This gene, called STAR, has been obta
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Albrecht Sybille
Behnke Detlev
Guhrs Karl-Heinz
Hartmann Manfred
Schlott Bernhard
Bugaisky Gabriele E.
medac Gesellschaft fur klinische spezialpraparate mbH
Wax Robert A.
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