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Mutants of Staphylokinase Carrying Amino and Carboxy-Terminal Extensions for Polyethylene Glycol Conjugation

a technology of polyethylene glycol and amino and carboxyterminal extensions, which is applied in the field of cysteine variants of staphylokinase, can solve the problems of hammering its use, affecting the allergic response of patients, and relatively short plasma half-life, and achieves the effect of higher temperature stability

Inactive Publication Date: 2010-09-02
SINGH SATISH +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Benefits of technology

[0031]In still another embodiment of the present invention, the PEGylated cysteine variant has increased proteolytic stability as compared to their original unmodified counterparts. In yet another embodiment, the Staphylokinase variant has proteolytic stability greater than the proteolytic stability of SEQ ID NO:1.

Problems solved by technology

However, being a bacterial product, clinical administration of SAK creates an allergic reaction and due to its short plasma half-life (3 min) relatively large therapeutic dose may be required.
Another limiting factor of Staphylokinase, that can hamper its use in thrombolytic therapy, is its relatively short plasma half-life (3-4 min) due to that repeated dose of this drug might be required to get effective recanalization during thrombolytic therapy and that in turn might exert higher allergic response in the patients.
Derivatization of cysteine substituted SAK mutants with PEG within the core region resulted in substantial loss of its plasminogen activation ability.
Therefore, ideal site for the PEG conjugation within the core region has not been found and the approach to conjugate PEG with SAK has not been successful as these SAK derivatives display significantly lower plasminogen activation ability than the native form of SAK.
The engineering of SAK for further improvement has been limited due to its smaller size and difficulty in targeting specific regions of protein without compromising functional properties of SAK as most of the regions of either involved in the interaction with the partner plasmin(ogen) or substrate plasminogen [Parry, M. A., Fernandez-Catalan, C., Bergner, A., Huber, R., Hopfner, K. P., Scholott, B., Guhrs, K. H. Bode, W (1998).

Method used

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  • Mutants of Staphylokinase Carrying Amino and Carboxy-Terminal Extensions for Polyethylene Glycol Conjugation
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  • Mutants of Staphylokinase Carrying Amino and Carboxy-Terminal Extensions for Polyethylene Glycol Conjugation

Examples

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example 1

[0073]Construction of SAK mutants carrying extended amino and carboxy terminal regions: SAK is the smallest known protein that has an efficient plasminogen activator activity. Major portion of SAK polypeptide is important for either forming a bimolecular complex with plasminogen or interaction with a substrate molecule. Therefore, addition or a deletion within the core region of SAK often results [Schlott, B., Gurhs, H. K., Hartmann, M., Rocker, A., Collen, D. (1997) Staphylokinase requires NH2-terminal proteolysis for plasminogen activation. J. Biol. Chem. 273; 22346-22350; Rajamohan, G. and Dikshit, K. L.(2000) Role of the N-terminal region of Staphylokinase (SAK) evidence for the participation of the N-terminal region of SAK in the enzyme-substrate complex formation. FEBS Lett. 474; 151-158] in a non-functional form of protein. Therefore, to generate derivatives of SAK, extension of one or more amino acid residues carrying one or multiple cysteine residues at terminal positions, ...

example 2

[0075]Intracellular production of SAK derivatives and their recovery from recombinant E. coli: E. coli cells transformed with recombinant plasmid carrying mutant SAK gene were streaked on a Luria Bertani (LB) agar plate containing 50 μg / ml Kanamycin and kept in an incubator set at 37° C. for overnight. Individual colonies appearing on the plate were used to raise seed culture in a 10 ml liquid LB medium supplemented with 50 μg / ml Kanamycin and grown at 37° C., 200 rpm on a gyratory shaker for 8-10 h. This primary seed culture (1% v / v) was used to inoculate 1 liter of LB medium containing 50 μg / ml Kanamycin and allowed to grow at 37° C. at 200 rpm till its optical density (OD600 nm) reaches to 0.4-0.5. The culture was then induced for SAK production by adding 0.1 mM IPTG and further grown at 37° C. for another 6-8 hours. Cells were then harvested by spinning them down by centrifugation at 6000.times.g in a GS-3 rotor (Sorvall) for 30 min at 4° C. The supernatants were discarded and t...

example 3

[0076]Determination of sub-unit association properties of SAK derivatives: In order to check how the extension of amino terminal regions of SAK and placement of cysteine residues within this region has affected the oligomeric state of protein, purified preparation of each SAK derivative was run on a native 10% polyacrylamide gel without adding any SDS and mercaptoethanol. Gel was stained with coomassie blue to check change in the oligomeric state of the protein. Checking the molecular mass of each SAK derivative following standard method of size exclusion chromatography on a G75 Sephadex column further substantiated change in the oligomeric state of protein.

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Abstract

The present invention relates to the development of new derivatives of a bacterial plasminogen activator, Staphylokinase (SAK), having one or more amino acid residues with single or multiple cysteines at the amino and / or carboxy terminal ends and their conjugation with PEG (Polyethylene Glycol), resulting in new Staphylokinase derivatives that display altered oligomeric states, enhanced thermal and protease stability and extended plasma half-life. Also included is the cloning and expression in a suitable bacterial host; purification of Staphylokinase derivatives to homogeneity and their chemical modification by integrating a PEG molecule to create new biologically active Staphylokinases having higher protein stability and improved in vivo plasma half life, that may enhance the clinical potential of Staphylokinase in thrombolytic therapy for the treatment of cardiovascular diseases.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS[0001]This application claims the benefit of India Application No. 2757 / DEU2008, filed Dec. 5, 2008, incorporated herein by reference in its entirety.REFERENCE TO SEQUENCE LISTING, TABLE OR COMPUTER PROGRAM[0002]A Sequence Listing is being submitted electronically via EFS in the form of a text file, created Dec. 4, 2009 and named “SAK8001seqlist.txt” (9000 bytes), the contents of which are incorporated herein by reference in their entirety.TECHNICAL FIELD[0003]The present invention relates to cysteine variants of Staphylokinase (SAK) wherein its amino and carboxy terminal ends are extended by adding extra amino acids having at least one cysteine residue where Polyethylene Glycol (PEG) can be conjugated.[0004]This invention relates to the field of cardiovascular therapeutics, particularly to the thrombolytic drugs that are utilized for the treatment of various cardiovascular diseases.[0005]These fibrinolytic agents, e.g., Tissue plasminogen acti...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): A61K38/48A61P9/10A61P9/04C12N9/48C12N9/96C12N1/21
CPCA61K38/00C12N9/48A61K47/48215A61K47/60A61K38/4886C12N9/52C12Y304/24029A61P9/04A61P9/10
Inventor SINGH, SATISHDIKSHIT, KANAK LATA
Owner SINGH SATISH
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