title: In vitro characterization of a multifunctional staphylokinase variant with reduced reocclusion, produced from salt inducible E. coli GJ1158.
abstract: The thrombolytic therapy with clinically approved drugs often ensues with recurrent thrombosis caused by thrombin-induced platelet aggregation from the clot debris. In order to minimize these problems, a staphylokinase (SAK)-based bacterial friendly multifunctional recombinant protein SRH (staphylokinase (SAK) linked with tripeptide RGD and dodecapeptide Hirulog (SRH)) was constructed to have Hirulog as an antithrombin agent and RGD (Arg-Gly-Asp) as an antiplatelet agent in the present study. This multifunctional fusion protein SRH was expressed in osmotically inducible E. coli GJ1158 as soluble form and purified with a yield of 0.27 g/L and functionally characterized in vitro. SRH retained the fibrinolytic activity and plasminogen activation rate comparable to the parental counterpart SAK. The antithrombin activity of SRH was significantly higher than SAK. The platelet rich clot lysis assay indicated that SRH had enhanced platelet binding activity and T(50%) and C50 of SRH were significantly lower than that of SAK. Furthermore, SRH inhibited the ADP-induced platelet aggregation in dose-dependent manner while SAK had no significant effect on platelet aggregation. Thus, the current study suggests that the SAK variant produced from osmotically inducible GJ1158 is more potent thrombolytic agent with antithrombin and antiplatelet aggregation activities for reduction of reocclusion in thrombolytic therapy.
authors.affiliation: Centre for Bioseparation Technology, VIT University, Vellore 632014, Tamilnadu, India. kkpulicherla@gmail.com
authors.forename: K K
authors.initials: KK
authors.lastname: Pulicherla
authors.forename: Anmol
authors.initials: A
authors.lastname: Kumar
authors.forename: G S
authors.initials: GS
authors.lastname: Gadupudi
authors.forename: Seetha Ram
authors.initials: SR
authors.lastname: Kotra
authors.forename: K R S Sambasiva
authors.initials: KR
authors.lastname: Rao
mesh_terms: D002478:Cells, Cultured; D004926:Escherichia coli / Q000502:physiology*; D024861:Genetic Enhancement / Q000379:methods; D014644:Genetic Variation; D006801:Humans; D008666:Metalloendopeptidases / Q000235:genetics* / Q000378:metabolism / Q000494:pharmacology*; D010974:Platelet Aggregation / Q000187:drug effects*; D010975:Platelet Aggregation Inhibitors / Q000378:metabolism / Q000494:pharmacology; D015202:Protein Engineering / Q000379:methods*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000494:pharmacology; D012492:Salts / Q000378:metabolism
chemical_list: D010975:Platelet Aggregation Inhibitors; D011993:Recombinant Fusion Proteins; D012492:Salts; D008666:Metalloendopeptidases; C022653:auR protein, Staphylococcus aureus