Construction and characterization of a recombinant chimeric plasminogen activator consisting of a fibrin peptide and a low molecular mass single-chain urokinase.
A recombinant chimeric plasminogen activator (f beta/scuPA-32k), with a fibrin beta-chain peptide (comprising Gly15 through Arg 42) linked to the N-terminal of a low molecular mass (32 kDa) single-chain urokinase (scuPA-32k, comprising Leu144 through Leu 411) via a 50 amino acid linker sequence, was produced by expression the corresponding chimeric cDNA in Escherichia coli cells. After refolding in vitro, the chimeric protein was purified to homogeneity by zinc chelate-Sepharose chromatography, Sephacryl S200 chromatography and benzamidine-Sepharose chromatography in sequence. The apparent molecular mass was 36 kDa shown by SDS-PAGE analysis. The special activity was 87,000 IU/mg detected by fibrin plate determination. F beta/scuPA-32k could directly activate plasminogen following Michaelis-Menten kinetics with K(m) = 0.52 microM and k(2) = 0.0024 s(-1). Mediated by plasmin, the single-chain molecule could be converted to the active two-chain molecule. The chimeric protein had 3.3 times higher fibrin affinity than scuPA-32k in the fibrin concentration of 3.2 mg/mL, while the chimeric protein inhibited the fibrin clotting and platelet aggregation. F beta/scuPA-32k showed a higher thrombolytic potency in vitro plasma clot lysis than scuPA-32k and depleted less fibrinogen in plasma. These results showed that the chimeric protein had not only higher fibrinolytic activity but also anti-thrombus activity. Further evaluation of the thrombolytic potential in appropriate animal models is required.
Jiao
J
J
National Laboratory of Protein Engineering, College of Life Sciences, Peking University, Beijing 100871, China.
Yu
M
M
Ru
B
B
D001665:Binding Sites; D001777:Blood Coagulation / Q000187:drug effects; D004926:Escherichia coli; D005338:Fibrin Fibrinogen Degradation Products / Q000235:genetics*; D005822:Genetic Vectors; D006801:Humans; D007700:Kinetics; D008970:Molecular Weight; D010446:Peptide Fragments / Q000235:genetics*; D010960:Plasminogen Activators / Q000096:biosynthesis / Q000235:genetics* / Q000494:pharmacology; D010974:Platelet Aggregation / Q000187:drug effects; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000494:pharmacology; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000494:pharmacology; D014568:Urokinase-Type Plasminogen Activator / Q000235:genetics*
D005338:Fibrin Fibrinogen Degradation Products; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; C033786:fibrinogen Bbeta (15-42); D010960:Plasminogen Activators; D014568:Urokinase-Type Plasminogen Activator