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.

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Citations

Apr 5, 1995·Biotechnology and Bioengineering·M M GuarnaJ M Piret
Nov 1, 1994·Transgenic Research·W N DrohanH Lubon
Mar 31, 1999·Trends in Biotechnology·M FusseneggerP P Mueller
Oct 13, 1992·Annals of the New York Academy of Sciences·W H VelanderJ L Johnson
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Jan 1, 1991·Experimental Cell Research·D T BergB W Grinnell
Jun 9, 1995·The Journal of Biological Chemistry·L V MedvedK C Ingham

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