title: Amplification of multicistronic plasmids in the human 293 cell line and secretion of correctly processed recombinant human protein C.
abstract: We have constructed multicistronic vectors containing the cDNAs for murine dihydrofolate reductase (DHFR), hygromycin phosphotransferase (HyPR), and human protein C (HPC), an antithrombotic factor. Using a sequential selection protocol with hygromycin (Hy) and methotrexate (MTX), we demonstrate the selective amplification of the murine dhfr cDNA in the adenovirus-transformed human kidney cell line 293, and the coamplification of the cDNA for HPC. Such recombinant 293 cell lines secreted HPC at levels as high as 25 micrograms/10(6) cells/day. In addition, we found that the complex vitamin K-dependent posttranslational modification of gamma-carboxylation of glutamate was not limiting at these high secretion levels, although the proteolytic processing of the protein was slightly reduced. Further, the HPC secreted from the gene-amplified cell lines had full anticoagulant activity when compared to plasma-derived HPC.
authors: Department of Molecular Biology, Lilly Research Laboratories, Indianapolis, IN 46285.
authors: J D
authors: JD
authors: Walls
authors: D T
authors: DT
authors: Berg
authors: S B
authors: SB
authors: Yan
authors: B W
authors: BW
authors: Grinnell
mesh_terms: D000818:Animals; D000941:Antigens / Q000032:analysis; D002460:Cell Line; D003001:Cloning, Molecular; D004247:DNA / Q000302:isolation & purification; D004352:Drug Resistance, Microbial / Q000235:genetics; D005784:Gene Amplification; D005796:Genes; D005819:Genetic Markers; D006801:Humans; D010957:Plasmids; D011486:Protein C / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D012313:RNA / Q000302:isolation & purification; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D013762:Tetrahydrofolate Dehydrogenase / Q000096:biosynthesis / Q000235:genetics; D014162:Transfection
chemical_list: D000941:Antigens; D005819:Genetic Markers; D011486:Protein C; D011994:Recombinant Proteins; D012313:RNA; D004247:DNA; D013762:Tetrahydrofolate Dehydrogenase