title: Engineering streptokinase for generation of active site-labeled plasminogen analogs.
abstract: 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.
authors.affiliation: Department of Pathology, Vanderbilt University School of Medicine, Nashville, TN 37232, USA.
authors.forename: Malabika
authors.initials: M
authors.lastname: Laha
authors.forename: Peter
authors.initials: P
authors.lastname: Panizzi
authors.forename: Matthias
authors.initials: M
authors.lastname: Nahrendorf
authors.forename: Paul E
authors.initials: PE
authors.lastname: Bock
mesh_terms: D055162:Biocatalysis; D020134:Catalytic Domain; D010450:Endopeptidases / Q000378:metabolism; D004792:Enzyme Precursors / Q000378:metabolism; D005456:Fluorescent Dyes / Q000737:chemistry; D006639:Histidine / Q000235:genetics / Q000378:metabolism; D007098:Imino Acids / Q000737:chemistry; D007700:Kinetics; D009842:Oligopeptides / Q000235:genetics / Q000378:metabolism; D010958:Plasminogen / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D015202:Protein Engineering; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013300:Streptokinase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*
chemical_list: D004792:Enzyme Precursors; D005456:Fluorescent Dyes; C471213:His-His-His-His-His-His; D007098:Imino Acids; D009842:Oligopeptides; D011993:Recombinant Fusion Proteins; D006639:Histidine; D010958:Plasminogen; D010450:Endopeptidases; D013300:Streptokinase; C099488:TEV protease; C008109:iminodiacetic acid