High-level expression of secreted proteins from cells adapted to serum-free suspension culture.
We have developed a host cell/vector system based on the use of adenovirus-transformed cells and a promoter, designated GBMT, capable of being activated by the Ela tumor antigen produced in these cells. GBMT-based vectors were constructed with hygromycin phosphotransferase and murine dihydrofolate reductase as selective markers. We demonstrate their utility in two adenovirus-transformed cell lines, human kidney 293 and Syrian hamster AV12-664. Further, we describe methods and conditions for the direct adaptation of isolated recombinant clones to serum-free suspension growth conditions. For exemplary purposes, we describe the generation of stable recombinant 293 cell lines with single-copy integrated vectors secreting the highly complex clotting factor human protein C at levels as high as 20 mg/l in serum-free suspension culture. In addition, using the AV12-664 cell line with GBMT and direct dominant selection of the dhfr gene, we have isolated clones secreting a tissue plasminogen activator derivative at levels of about 40 mg/l under serum-free suspension conditions. The distinct advantages of this vector/host cell system are 1) the direct selection of stable clones expressing relatively high levels of recombinant protein, eliminating the need for the tedious stepwise gene amplification process and 2) the direct adaptation to serum-free suspension culture.
Berg
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Lilly Research Laboratories, Lilly Corporate Center, Indianapolis, IN 46285-0424.
McClure
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Grinnell
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D000222:Adaptation, Physiological; D000256:Adenoviridae / Q000235:genetics; D000818:Animals; D001483:Base Sequence; D002461:Cell Line, Transformed; D002472:Cell Transformation, Viral; D006224:Cricetinae; D016895:Culture Media, Serum-Free; D005822:Genetic Vectors; D006801:Humans; D008647:Mesocricetus; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D011486:Protein C / Q000096:biosynthesis; D011994:Recombinant Proteins / Q000096:biosynthesis*; D013535:Suspensions; D010959:Tissue Plasminogen Activator / Q000096:biosynthesis; D014162:Transfection
D016895:Culture Media, Serum-Free; D011486:Protein C; D011994:Recombinant Proteins; D013535:Suspensions; D010959:Tissue Plasminogen Activator