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Applied and Environmental Microbiology, February 1999, p. 506-513, Vol. 65, No. 2
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Staphylokinase as a Plasminogen Activator Component in Recombinant Fusion Proteins

S. J. Szarka,1 E. G. Sihota,1 H. R. Habibi,2 and S.-L. Wong1,*

Department of Biological Sciences, Division of Cellular, Molecular, and Microbial Biology,1 and Division of Zoology,2 University of Calgary, Calgary, Alberta, Canada T2N 1N4

Received 4 May 1998/Accepted 28 October 1998

The plasminogen activator staphylokinase (SAK) is a promising thrombolytic agent for treatment of myocardial infarction. It can specifically stimulate the thrombolysis of both erythrocyte-rich and platelet-rich clots. However, SAK lacks fibrin-binding and thrombin inhibitor activities, two functions which would supplement and potentially improve its thrombolytic potency. Creating a recombinant fusion protein is one approach for combining protein domains with complementary functions. To evaluate SAK for use in a translational fusion protein, both N- and C-terminal fusions to SAK were constructed by using hirudin as a fusion partner. Recombinant fusion proteins were secreted from Bacillus subtilis and purified from culture supernatants. The rate of plasminogen activation by SAK was not altered by the presence of an additional N- or C-terminal protein sequence. However, cleavage at N-terminal lysines within SAK rendered the N-terminal fusion unstable in the presence of plasmin. The results of site-directed mutagenesis of lysine 10 and lysine 11 in SAK suggested that a plasmin-resistant variant cannot be created without interfering with the plasmin processing necessary for activation of SAK. Although putative plasmin cleavage sites are located at the C-terminal end of SAK at lysine 135 and lysine 136, these sites were resistant to plasmin cleavage in vitro. Therefore, C-terminal fusions represent stable configurations for developing improved thrombolytic agents based on SAK as the plasminogen activator component.

* Corresponding author. Mailing address: Department of Biological Sciences, Division of Cellular, Molecular and Microbial Biology, University of Calgary, 2500 University Drive, N.W., Calgary, Alberta, Canada T2N 1N4. Phone: (403) 220-5721. Fax: (403) 289-9311. E-mail: slwong{at}ucalgary.ca.

Applied and Environmental Microbiology, February 1999, p. 506-513, Vol. 65, No. 2
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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