Active human tissue plasminogen activator secreted from insect cells using a baculovirus vector.
A baculovirus expression vector was constructed with the tissue plasminogen activator (TPA) cDNA under the control of the viral polyhedrin promoter. After infection of insect cells with the recombinant baculovirus, active TPA was secreted into the medium in which these cells were grown. TPA was isolated from the conditioned media using metal chelate affinity chromatography followed by immunoaffinity purification using mouse monoclonal anti-human TPA coupled to Sepharose. Sodium dodecyl sulfate-gel electrophoresis under reducing conditions and sequence analysis of recombinant human TPA have revealed a two-chain form of the enzyme. The N-terminal amino acid was identified to be serine, indicating that it was processed at its N-terminus by the insect cell culture in a manner similar to that observed for mammalian cells. The relative specific activity of recombinant TPA from insect cells is comparable to that of Bowes melanoma TPA standard. Its activity is stimulated in the presence of fibrinogen fragments, but by a factor about 2.3-fold lower than the Bowes melanoma TPA. The apparent molecular weight of recombinant TPA from insect cells was about 60K by fibrin agar activity gels, suggesting less complex glycosylation than recombinant TPA from mammalian cells.
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001665:Binding Sites; D002478:Cells, Cultured; D004591:Electrophoresis, Polyacrylamide Gel; D005822:Genetic Vectors; D006801:Humans; D007304:Insect Viruses / Q000235:genetics*; D007313:Insecta; D010957:Plasmids; D011994:Recombinant Proteins / Q000032:analysis / Q000235:genetics; D010959:Tissue Plasminogen Activator / Q000032:analysis / Q000235:genetics / Q000378:metabolism*
D000596:Amino Acids; D011994:Recombinant Proteins; D010959:Tissue Plasminogen Activator