Streptokinase mutants

Details Streptokinase mutants Streptokinase mutants

1999-08-13

2001-10-30

Prouty, Rebecca E. (Department: 1652)

Chemistry: molecular biology and microbiology

Enzyme , proenzyme; compositions thereof; process for...

Hydrolase

C435S069100, C435S320100, C435S252330, C424S192100

Reexamination Certificate

active

06309873

ABSTRACT:

The present invention is related to the field of biotechnology and genetic engineering techniques, particularly to a method for obtaining mutants from streptokinase, to the molecules obtained from this method, as well as the expression vectors and microorganisms for recombinant obtaining.
The streptokinase is a polypeptide of 414 amino acids residues. This is an extracellular protein produced by various strains of beta haemolytic streptococci, with molecular weight about 47.000 dalton and is a potent activator of the fibrinolytic enzyme system in humans (Tillet, W. S. and Garner, R. L. (1933) Exp. Med. 58, 485-502; Tillet, W. S.; Edwards, E. D. and Garner, R. L (1934) J. Clin. Invest 13, 47-78).
Unlike other plasminogen activators, streptokinase does not possess the intrinsic protease activity necessary to activate plasminogen to plasmin. Streptokinase activates plasminogen by the formation of 1:1 molar complex of streptokinase-plasminogen, which serves as the activator of free plasminogen to form plasmin (Schick, L. A. and Castellino, F. J. (1974) Biochem. Biophys. Res. Commun. 57, 47-54).
Streptokinase, urokinase and tissue-type plasminogen activator are at present used as thrombolytic agents in the treatment of disorders which collectively represent one of the greatest causes of death in the world, such as myocardial infarct, pulmonary thromboembolism, surgical complications and other cases of thrombosis.
The streptokinase is a bacterial protein and therefore, antigenic in humans. Antibodies to streptokinase are found in most individuals as a result of recurrent streptococcal infection (Tillet, W. S. and Garner, R. L. (1934) J. Clin. Invest. 13, 47-78). These antibodies are harmful for the use of streptokinase as thrombolytic, because high antibodies titers might neutralize streptokinase activity preventing effective thrombolysis (Urdahl, K. B.; Mathews, J. D.; Currie, B. (1996) Australian and New Zealand J. Med. 26, 49-53; Spottl, F. and Kaiser, R. (1974) Thromb. Diath. Haemorrh.32, 608). Patients are also immunized with streptokinase as a result of thrombolytic therapy and anti-streptokinase antibody titers exponentially rise post-treatment. These high anti-streptokinase antibody titers could neutralize a standard dose of streptokinase if it is administered a second time in therapy (Rao, A. K.; Pratt, C.; Berke, A.; Jaffe, A.; Ockene, L.; Schreiber, T. L.; Bell, W. R.; Knaterund, G.; Robertson, T. L. and Terrin, M. L. (1988) J. Am. Coll. Cardiol. 11,1). One of the most common side effects of streptokinase therapy are allergic reactions, which have been noted in up to 15% of treated patients (McGrath, K. G.; Zeffren, B.; Alexander, J.; Kaplan, K. and Patterson, R. (1985) J. Allergy Clin. Immunol. 76, 453; Sorber, W. A. and Herbst, V. (1988) Cutis 42, 57; Davies, K. A.; Mathieson, P.; Winearis, C. G.; Rees, A. J.; and Walport, M. J. (1990) Clin.Exp.Immunol.80, 83; Schweitzer, D. H.; Van der Wall, E. E.; Bosker, H. A.; Scheffer, E. and Macfarlane, J. D. (1991) Cardiology 78, 68; Bruserund, O. L.; Sollid, L. and Foyn-Jorgensen, P. (1986) J. Clin. Lab. Immun. 20, 69-74). The streptokinase also induces a strong cellular immune response (Bruserund, O. (1990) APMIS 98, 1077-1084; Bruserund, O.; Elsayed, S. and Pawelec, G. (1992) Mol. Immunol. 29,1097-1104; Youkeles, L. H.; Solirnan, M. Y. and Rosenstreich, D. L. (1991) J. Allergy Clin. Immunol. 88, 166-171; Randall, K.; Gelfond, D. H.; Stoffel, S.; Scharf, S.; Higuchi, R.; Horn, G. T.; Mullis, K. B. and Erlich, H. A. (1988) Science 239, 487-491).
The widespread use of streptokinase in humans makes its antigenicity an important clinical problem.
Despite the rich clinical information about the immunogenicity of streptokinase, little is known about the structural basis for its antigenicity. There is no X ray crystallographic data on the structure of streptokinase and it is not known whether certain regions of the molecule are more immunogenic than others, nor have there been studies of the molecular mechanisms responsible for antibody-mediated neutralization of streptokinase activity.
Previous reports have shown different antigenic regions in streptokinase mapped with murine anti-streptokinase monoclonal antibodies, soluble recombinant streptokinase fragment and anti streptokinase antibodies from human sera from patients treated with streptokinase (Reed, G. L.; Kussie, P. and Parhami-Seren, B. (1993) J. Immunol. 150, 4407-4415; Parhami-Seren, B.; Lynch, M.; White, H. D. and Reed, G. L. (1995) Mol. Immunol. 32, 717-724; Parhami-Seren, B.; Keel, T. and Reed, G. L. (1996) Hybridoma 15, 169-176; Gonzalezgronow, M.; Enghild, J. J.; Pizzo, S. V. (1993) Biochimica et Biophysica Acta 1180, 283-288; U.S. Pat. No. 5,240,845).
The object of the present invention is to achieve streptokinase mutants from modifications of skc-2 gene previously described (European Patent No. EP 0 489 201 B1; Estrada et al (1992) Biotechnology 10, 1138-1142) and coding for streptokinase SKC-2 (Heberkinase®, Heber Biotec SA, Havana, Cuba), such that the obtained mutants conserve their capacity for plasminogen activator complex formation and having reduced antigenicity that could constitute a preferred alternatives to native streptokinase for thrombolytic therapy. Heberkinase® contains a recombinant SKC-2 obtained after the expression of the skc-2 gene in
E. coli
(European Patent No. EP 0 489 201 B1; Estrada et al (1992) Biotechnology 10, 1138-1142).
The present invention relates to the mapping of antigenic regions located on SKC-2 using cellulose-bound peptide scans and human total sera from patients treated with Heberkinase®.
The present invention also relates to the immunological features of a synthetic 42 amino acids peptide resembling amino acids 373-414 from the SKC-2 C-terminal region using a panel of sera collected from patients before and after Heberkinase® therapy and tested in anti-SKC-2(373-414) peptide ELISA and SKC-2 (373-414) direct binding assay.
The present invention relates to a method for the cloning and expression of SKC-2 mutants corresponding to the fragments 40-1245 and 1-1119 from the skc-2 gene, which codes for SKC-2, previously described in the European Patent No. EP 0 489 201 B1, which products are proteins presenting:
a deletion of the first 13 amino acids residues at the N-terminal region, called SKC-2-N 13, which sequence corresponds to the Seq. Ident. No. 1.
a deletion of the first 13 amino acids residues at the N-terminal region with Asp-Ile-Val-Asp-Gly-Gly-6xHis tail fused at the C-terminus of the protein, called SKC-2-N13-Asp-Ile-Val-Asp-Gly-Gly-6xHis which sequence corresponds to the Seq. Ident. No. 2.
A deletion of the last 42 amino acids residues at the C-terminal region from position 373 to 414, called SKC-2-C42, which sequence corresponds to the Seq. Ident. No. 3.
A deletion of the last 42 amino acids residues at the C-terminal region from position 373 to 414 with Asp-Ile-Val-Asp-Gly-Gly-6xHis tail fused at the C-terminus of the protein, called SKC-2-C42-Asp-Ile-Val-Asp-Gly-Gly-6xHis, which sequence corresponds to the Seq. Ident. No. 4.
The present invention also relates to these mutant proteins, which molecular weight is 46.000 dalton for SKC-2-N13, 47.000 dalton for SKC-2-N13-Asp-Ile-Val-Asp-Gly-Gly-6xHis, 42,000 dalton for SKC-2-C42 and 43.000 dalton for SKC-2-C42-Asp-Ile-Val-Asp-Gly-Gly-6xHis, which amino acids sequences corresponds to the Seq. Ident. No. 1-4. The fragments of nucleotide sequence from skc-2 gene were obtained from pEKG3 plasmid (european patent No. .EP 0 489 201 B1), by genetic amplification using the polymerase chain reaction (PCR) with 6 synthetic oligonucleotides denominated sk1, sk2, sk3, sk4, sk5 and sk6, having sequences identified with the Seq. Ident. No. 5-10.
The present invention also relates to recombinant DNA including the nucleotide fragments 40-1245 and 1-1119 from skc-2 gene, such as vectors pEMI-1 (FIG.
2
), pSKH-11 (FIG.
3
), pIJ-4 (
FIG. 4
) and pMC-8 (
FIG. 5
) for the expression of these fragments in bacteria. For expression in
E. coli
these fragments were cloned under the tryptoph

Affiliated with

Also associated with

Rating

Streptokinase mutants does not yet have a rating. At this time, there are no reviews or comments for this patent.

If you have personal experience with Streptokinase mutants, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Streptokinase mutants will most certainly appreciate the feedback.

Rate now

Comments { 0 }

Profile ID: LFUS-PAI-O-2551183