Sep 1, 1989

Amplification of multicistronic plasmids in the human 293 cell line and secretion of correctly processed recombinant human protein C

Gene
J D WallsB W Grinnell

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.

Mentioned in this Paper

Antibiotic Resistance, Microbial
Hygromycin A
Transfection
HEK293 Cells
Dhfr
Gene Amplification
Post-Translational Protein Processing
Anticoagulant Activity
Murine
Proteins, Recombinant DNA

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