Article (Scientific journals)
Characterization of a recombinant fusion protein of the finger domain of tissue-type plasminogen activator with a truncated single chain urokinase-type plasminogen activator.
Gheysen, D.; Lijnen, H. R.; Pierard, Luc et al.
1987In Journal of Biological Chemistry, 262 (24), p. 11779-84
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Keywords :
Adenocarcinoma/analysis; Amino Acid Sequence; Amino Acids/analysis; Animals; Antibodies, Monoclonal; Chemistry, Physical; Cricetinae; DNA/analysis; Fibrin/metabolism; Humans; Kinetics; Lung Neoplasms/analysis; Molecular Weight; Physicochemical Phenomena; Recombinant Fusion Proteins/genetics/metabolism; Recombinant Proteins/genetics; Structure-Activity Relationship; Substrate Specificity; Tissue Plasminogen Activator/genetics/metabolism; Urokinase-Type Plasminogen Activator/genetics/metabolism
Abstract :
[en] 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.
Disciplines :
Cardiovascular & respiratory systems
Author, co-author :
Gheysen, D.
Lijnen, H. R.
Pierard, Luc ;  Université de Liège - ULiège > Département des sciences cliniques > Cardiologie - Pathologie spéciale et réhabilitation
de Foresta, F.
Demarsin, E.
Jacobs, P.
De Wilde, Marc ;  Université de Liège - ULiège > Relations académiques et scientifiques (Sciences)
Bollen, A.
Collen, D.
Language :
English
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.
Publication date :
1987
Journal title :
Journal of Biological Chemistry
ISSN :
0021-9258
eISSN :
1083-351X
Publisher :
American Society for Biochemistry and Molecular Biology, United States - Maryland
Volume :
262
Issue :
24
Pages :
11779-84
Peer reviewed :
Peer Reviewed verified by ORBi
Available on ORBi :
since 12 July 2014

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