title: DHFR coamplification of t-PA in DHFR+ bovine endothelial cells: in vitro characterization of the purified serine protease.
abstract: High-level expression of human tissue-type plasminogen activator was accomplished in endothelial cells by a novel approach to dihydrofolate reductase (DHFR) coamplification in DHFR+ cells. A tripartite mammalian expression vector coding for DHFR, neomycin phosphotransferase, and the t-PA gene was introduced into bovine endothelial cells by transfection and selection for G418 resistance. Upon methotrexate selection of these transformants, we obtained endothelial cells that had amplified the plasmid-encoded DHFR and t-PA genes. As a result, cell lines were isolated that efficiently produced t-PA (greater than 4 pg/cell.day). This t-PA was purified and compared with recombinant t-PA produced in Chinese hamster ovary cells. These two t-PA samples differed in carbohydrate composition, and amounts of 530 and 527 amino acid forms but had similar in vitro activity.
authors.affiliation: Department of Molecular Genetics, Smith Kline and French Laboratories, King of Prussia, PA 19406-0939.
authors.forename: R W
authors.initials: RW
authors.lastname: Connors
authors.forename: R W
authors.initials: RW
authors.lastname: Sweet
authors.forename: J P
authors.initials: JP
authors.lastname: Noveral
authors.forename: D S
authors.initials: DS
authors.lastname: Pfarr
authors.forename: J J
authors.initials: JJ
authors.lastname: Trill
authors.forename: R J
authors.initials: RJ
authors.lastname: Shebuski
authors.forename: B A
authors.initials: BA
authors.lastname: Berkowitz
authors.forename: D
authors.initials: D
authors.lastname: Williams
authors.forename: S
authors.initials: S
authors.lastname: Franklin
authors.forename: M E
authors.initials: ME
authors.lastname: Reff
mesh_terms: D000818:Animals; D002417:Cattle; D002460:Cell Line; D005822:Genetic Vectors; D006801:Humans; D021141:Nucleic Acid Amplification Techniques; D010957:Plasmids; D013762:Tetrahydrofolate Dehydrogenase / Q000096:biosynthesis* / Q000235:genetics; D010959:Tissue Plasminogen Activator / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification
chemical_list: D013762:Tetrahydrofolate Dehydrogenase; D010959:Tissue Plasminogen Activator