Engineering streptokinase for generation of active site-labeled plasminogen analogs.

Laha M, Panizzi P, Nahrendorf M, Bock PE
Anal Biochem. 2011 415 (2): 105-15

PMID: 21570944 · PMCID: PMC3114107 · DOI:10.1016/j.ab.2011.04.025

We previously demonstrated that streptokinase (SK) can be used to generate active site-labeled fluorescent analogs of plasminogen (Pg) by virtue of its nonproteolytic activation of the zymogen. The method is versatile and allows stoichiometric and active site-specific incorporation of any one of many molecular probes. The limitation of the labeling approach is that it is both time-consuming and low yield. Here we demonstrate an improved method for the preparation of labeled Pg analogs by the use of an engineered SK mutant fusion protein with both COOH- and NH(2)-terminal His(6) tags. The NH(2)-terminal tag is followed by a tobacco etch virus proteinase cleavage site to ensure that the SK Ile(1) residue, essential for conformational activation of Pg, is preserved. The SK COOH-terminal Lys(414) residue and residues Arg253-Leu260 in the SK β-domain were deleted to prevent cleavage by plasmin (Pm) and to disable Pg substrate binding to the SK·Pg(∗)/Pm catalytic complexes, respectively. Near elimination of Pm generation with the SKΔ(R253-L260)ΔK414-His(6) mutant increased the yield of labeled Pg 2.6-fold and reduced the time required more than 2-fold. The versatility of the labeling method was extended to the application of Pg labeled with a near-infrared probe to quantitate Pg receptors on immune cells by flow cytometry.

Copyright © 2011 Elsevier Inc. All rights reserved.

MeSH Terms (14)

Biocatalysis Catalytic Domain Endopeptidases Enzyme Precursors Fluorescent Dyes Histidine Imino Acids Kinetics Oligopeptides Plasminogen Protein Binding Protein Engineering Recombinant Fusion Proteins Streptokinase

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