Expression of a novel chimeric protein containing the A chain of tissue-type plasminogen activator and the B chain of pro-urokinase in insect cells using the baculovirus system.
A hybrid cDNA tu-pa, which contains Ser1-Thr263 of tissue-type plasminogen activator (t-PA) and Ser138-Leu411 of pro-urokinase (pro-UK) was constructed and expressed in the Sf9-AcNPV system. The expression level was approximate 2.5 mg/L. Tu-PA was purified via one-step affinity column conjugated with monoclonal antibody against the B chain of pro-UK, which showed a single band of approximate 60 kDa in SDS-PAGE. The specific activity of the chimeric protein on fibrin plate was 200,000 IU/mg protein. Tu-PA had a higher selectivity for fibrin than UK and pro-UK. Its activity can be promoted by CNBr degraded fibrin fragments as t-PA.
D000818:Animals; D018929:Cell Culture Techniques / Q000379:methods; D002460:Cell Line; D003001:Cloning, Molecular; D017077:Culture Media, Conditioned; D004792:Enzyme Precursors / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D005337:Fibrin / Q000378:metabolism; D015870:Gene Expression; D005822:Genetic Vectors; D017924:Nucleopolyhedroviruses; D010958:Plasminogen / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D018411:Spodoptera / Q000166:cytology; D010959:Tissue Plasminogen Activator / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D014568:Urokinase-Type Plasminogen Activator / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification