Amplification of multicistronic plasmids in the human 293 cell line and secretion of correctly processed recombinant human protein C.
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.
Walls
J D
JD
Department of Molecular Biology, Lilly Research Laboratories, Indianapolis, IN 46285.
Berg
D T
DT
Yan
S B
SB
Grinnell
B W
BW
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
D000941:Antigens; D005819:Genetic Markers; D011486:Protein C; D011994:Recombinant Proteins; D012313:RNA; D004247:DNA; D013762:Tetrahydrofolate Dehydrogenase