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Novel Expression of Chimeric Plasminogen Activators in Insect Cells

Bio/Technology volume 7, pages 286–292 (1989)Cite this article

Abstract

The isolated protease domain of single chain urokinase–type plasminogen activator (scu–PA) preferentially activates fibrin–associated plasminogen in plasma, yet scu–PA binds poorly to forming fibrin clots. We are investigating the effect of combining this protease domain with protein domains that are reported to be involved in the binding of other plasma proteins to forming fibrin clots; the chimeric proteins consist of the protease domain and either the plasmin kringle 1 domain or the tissue plasminogen activator kringle 2 domain. Rapid construction of expression vectors for these chimeras was possible because much of the coding region required was synthesized either by polymerase chain reaction using RNA as the initial template or by the ligation of multiple long synthetic oligonucleotides. We also used an artificial signal peptide and baculovirus expression vectors to direct the secretion of these chimeras from insect cells. The signal peptide was designed by selecting those amino acid residues that occur more frequently than expected (based on amino acid composition) at positions −13 to +2 relative to the signal peptidase cleavage site in eukaryotic proteins. The chimeric proteins expressed by insect cells were enzymatically active, but their binding to forming fibrin clots was similar to that of the poorly binding scu–PA. In an in vitro clot lysis system, these chimeras displayed a specificity for fibrin–associated plasminogen greater than the specificity of two chain urokinase–type plasminogen activator and similar to the specificity of tissue plasminogen activator and scu–PA.

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  1. James J. Devlin: Corresponding author.

Authors and Affiliations

  1. Molecular Biology Department, Cetus Corporation, Emeryville, CA, 94608

    James J. Devlin, Patricia E. Devlin, Robin Clark, Edward C. O'Rourke & David F. Mark

  2. Chemistry Department, Cetus Corporation, Emeryville, CA, 94608

    Corey Levenson

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Devlin, J., Devlin, P., Clark, R. et al. Novel Expression of Chimeric Plasminogen Activators in Insect Cells. Nat Biotechnol 7, 286–292 (1989). https://doi.org/10.1038/nbt0389-286

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