DHFR coamplification of t-PA in DHFR+ bovine endothelial cells: in vitro characterization of the purified serine protease.
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.
Connors
R W
RW
Department of Molecular Genetics, Smith Kline and French Laboratories, King of Prussia, PA 19406-0939.
Sweet
R W
RW
Noveral
J P
JP
Pfarr
D S
DS
Trill
J J
JJ
Shebuski
R J
RJ
Berkowitz
B A
BA
Williams
D
D
Franklin
S
S
Reff
M E
ME
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
D013762:Tetrahydrofolate Dehydrogenase; D010959:Tissue Plasminogen Activator