title: Characterization of a recombinant fusion protein of the finger domain of tissue-type plasminogen activator with a truncated single chain urokinase-type plasminogen activator.
abstract: Human recombinant single chain urokinase-type plasminogen activator (recombinant scu-PA) and a hybrid between human tissue-type plasminogen activator (t-PA) and scu-PA, obtained by ligation of cDNA fragments encoding the NH2-terminal region (amino acids 1-67) of t-PA and the COOH-terminal region (amino acids 136-411) of scu-PA, were expressed in a mammalian cell system. The proteins were purified from conditioned culture media containing 2% fetal calf serum by chromatography on zinc chelate-Sepharose, immunoadsorption chromatography on an insolubilized murine monoclonal antibody directed against urokinase, benzamidine-Sepharose chromatography, and Ultrogel AcA 44 gel filtration. Between 180 and 230 micrograms of the purified proteins were obtained per liter of conditioned medium, with a yield of approximately 18% and a purification factor of 720-1900. On sodium dodecyl sulfate gel electrophoresis under reducing conditions, the proteins migrated as single bands with approximate Mr 50,000 for recombinant scu-PA and Mr 43,000 for the t-PA/scu-PA hybrid. Following conversion to urokinase with plasmin, the proteins had a specific amidolytic activity comparable to that of natural scu-PA. Both proteins activated plasminogen directly with Km = 0.53 and 1.4 microM and k2 = 0.0034 and 0.0027 s-1, respectively. Both proteins did not bind specifically to fibrin and had a comparable degree of fibrin selectivity as measured in a system composed of a whole human 125I-fibrin-labeled plasma clot suspended in human plasma. It is concluded that this chimeric protein, consisting of the NH2-terminal "finger-like" domain of t-PA and the COOH-terminal region of scu-PA, has very similar enzymatic properties as compared to scu-PA, but has not acquired the fibrin affinity of t-PA.
mesh_terms: D000230:Adenocarcinoma / Q000032:analysis; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D000911:Antibodies, Monoclonal; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D006224:Cricetinae; D004247:DNA / Q000032:analysis; D005337:Fibrin / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008175:Lung Neoplasms / Q000032:analysis; D008970:Molecular Weight; D011993:Recombinant Fusion Proteins / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics*; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D010959:Tissue Plasminogen Activator / Q000235:genetics* / Q000378:metabolism; D014568:Urokinase-Type Plasminogen Activator / Q000235:genetics* / Q000378:metabolism
chemical_list: D000596:Amino Acids; D000911:Antibodies, Monoclonal; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D005337:Fibrin; D004247:DNA; D010959:Tissue Plasminogen Activator; D014568:Urokinase-Type Plasminogen Activator