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BioMed Research International
Volume 2013 (2013), Article ID 297305, 12 pages
http://dx.doi.org/10.1155/2013/297305
Research Article

In Vitro Characterization of a Multifunctional Staphylokinase Variant with Reduced Reocclusion, Produced from Salt Inducible E. coli GJ1158

1Centre for Bioseparation Technology, VIT University, Vellore 632014, Tamilnadu, India
2Department of Biotechnology, Acharya Nagarjuna University, Nagarjuna Nagar, Guntur 522510, Andhra Pradesh, India
3Interdisciplinary Graduate Program in Human Toxicology, University of Iowa, Iowa City, IA 52242, USA
4Department of Occupational and Environmental Health, The University of Iowa, College of Public Health, 100 Oakdale Campus No. 121 IREH, Iowa City, IA 52242-5000, USA

Received 22 April 2013; Revised 19 June 2013; Accepted 3 July 2013

Academic Editor: Eileen Hao Yu

Copyright © 2013 K. K. Pulicherla et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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 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.