title: Cloning and expression of a new recombinant thrombolytic and anthithrombotic agent - a staphylokinase variant.
abstract: To develop a more potent antithrombin agent with thrombolytic and antiplatelet properties, a new staphylokinase (SAK) variant was constructed. The kringle 2 domain (K2) of tissue type-plasminogen activator (t-PA) containing a fibrin-specific binding site (i), the RGD sequence (Arg-Gly-Asp) for the prevention of platelet aggregation (ii) and the antithrombotic agent - hirulog (iii) was assembled to the C-terminal part of recombinant staphylokinase (r-SAK). cDNA for the hybrid protein SAK-RGD-K2-Hirul was cloned into Pichia pastoris pPIC9K yeast expression vector. The introduction of K2 t-PA, the RGD sequence and hirulog into the C-terminus of r-SAK did not alter the staphylokinase activity. We observed a higher clot lysis potency of SAK-RGD-K2-Hirul as evidenced by a faster and more profound lysis of (125)I-labeled human fibrin clots. The potency of thrombin inhibition by the hirulog C-terminal part of the recombinant fusion protein was almost identical to that of r-Hir alone. These results suggest that the SAK-RGD-K2-Hirul construct can be a more potent and faster-acting thrombolytic agent with better antithrombin and antiplatelet properties compared to r-SAK and SAK-RGD-K2-Hir.
authors.affiliation: Department of Medical Biochemistry, Medical University of Lodz, Łódź, Poland.
authors.forename: Michał
authors.initials: M
authors.lastname: Kowalski
authors.forename: George
authors.initials: G
authors.lastname: Brown
authors.forename: Magdalena
authors.initials: M
authors.lastname: Bieniasz
authors.forename: Katarzyna
authors.initials: K
authors.lastname: Oszajca
authors.forename: Ewa
authors.initials: E
authors.lastname: Chabielska
authors.forename: Tadeusz
authors.initials: T
authors.lastname: Pietras
authors.forename: Zofia
authors.initials: Z
authors.lastname: Szemraj
authors.forename: Eusebio
authors.initials: E
authors.lastname: Makandjou-Ola
authors.forename: Jacek
authors.initials: J
authors.lastname: Bartkowiak
authors.forename: Janusz
authors.initials: J
authors.lastname: Szemraj
mesh_terms: D000991:Antithrombins / Q000378:metabolism*; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers; D004220:Disulfides / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005342:Fibrinolysis; D006629:Hirudins / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D008666:Metalloendopeptidases / Q000235:genetics* / Q000378:metabolism; D010843:Pichia / Q000235:genetics; D010958:Plasminogen / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011993:Recombinant Fusion Proteins / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D000991:Antithrombins; D017931:DNA Primers; D004220:Disulfides; D006629:Hirudins; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; C521927:SAK-RGD-K2-Hir; D010958:Plasminogen; D008666:Metalloendopeptidases; C571711:SAK-RGD-K2-Hirul; C022653:auR protein, Staphylococcus aureus