Characterization of a recombinant chimeric plasminogen activator with enhanced fibrin binding.
A recombinant chimeric plasminogen activator (GHRP-scu-PA-32K), consisting of the tetrapeptide Gly-His-Arg-Pro fused to the N-terminus of the low-molecular single-chain urokinase-type plasminogen activator (Leu144-Leu411), was produced by expression in CHO cells. The stable expression cell line was selected for large-scale expression. The product was purified by antibody-Sepharose affinity chromatography with a recovery of 67%. The apparent molecular weight of purified GHRP-scu-PA-32K was 33 kDa according to SDS-PAGE. Its specific activity was 150000 IU/mg protein according to fibrin plate determination. The conversion of single-chain to two-chain molecules mediated by plasmin was comparable for GHRP-scu-PA-32K (K(m)=4.9 microM, k(2)=0.35 s(-1)) and scu-PA-32K. The activation of plasminogen by GHRP-scu-PA-32K (K(m)=1.02 microM, k(2)=0.0028 s(-1)) was also similar to that of scu-PA-32K. The fibrin binding of GHRP-scu-PA-32K was 2.5 times higher than that of scu-PA-32K at a fibrin concentration of 3.2 mg/ml. In contrast to scu-PA-32K in vitro 125I-fibrin-labeled plasma clot lysis, GHRP-scu-PA had a higher thrombolytic potency, whereas it depleted less fibrinogen in plasma. These results show that GHRP-scu-PA-32K as expected is a potential thrombolytic agent.
Jiao
J
J
Peking University, College of Life Sciences, National Laboratory of Protein Engineering, 100871, Beijing, PR China.
Yu
M
M
Ru
B
B
D000818:Animals; D016466:CHO Cells; D002846:Chromatography, Affinity; D006224:Cricetinae; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation / Q000502:physiology; D005337:Fibrin / Q000378:metabolism*; D005341:Fibrinolysin / Q000378:metabolism; D005342:Fibrinolysis / Q000187:drug effects / Q000502:physiology; D005343:Fibrinolytic Agents / Q000378:metabolism / Q000494:pharmacology; D006801:Humans; D007163:Immunosorbent Techniques; D008970:Molecular Weight; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D010958:Plasminogen / Q000378:metabolism; D010960:Plasminogen Activators / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D011485:Protein Binding / Q000502:physiology; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism* / Q000494:pharmacology; D014568:Urokinase-Type Plasminogen Activator / Q000235:genetics / Q000378:metabolism*
D005343:Fibrinolytic Agents; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; C426012:glycyl-histidyl-arginyl-prolyl-single-chain urokinase-type plasminogen activator (144-411); D005337:Fibrin; D010958:Plasminogen; D010960:Plasminogen Activators; D005341:Fibrinolysin; D014568:Urokinase-Type Plasminogen Activator