C - Chemistry – Metallurgy – 12 – N
Patent
C - Chemistry, Metallurgy
12
N
530/13, 195/1.23
C12N 15/00 (2006.01) C07K 14/495 (2006.01) C12N 5/00 (2006.01) C12P 21/00 (2006.01) A61K 38/00 (2006.01)
Patent
CA 2003886
ABSTRACT Recombinant transforming growth factor-.beta.1 (TGF-.beta.1) is expressed to high levels in Chinese hamster ovary (CHO) cells using dihydrofolate reductase (dhfr) gene amplification. The expression plasmid was derived from the pSV2 vectors and contained, in tandem, the simian TGF-.beta.1 and mouse dhfr cDNAs. Transcription of both cDNAs was controlled by the SV40 early promoter. Stepwise selection of transfected CHO cells in increasing concentrations of methotrexate yielded cell lines expressing amplified TGF-.beta.1 nucleic acid sequences. The expression plasmid DNA was amplified greater than 35-fold in one of the methotrexate selected transfectants. The major proteins secreted by these cells consisted of latent TGF-.beta.1 and TGF-.beta.1 precursor polypeptides as judged by immunoblots using site-specific anti-peptide antibodies derived from various regions of the TGF-.beta.1 precursor. Levels of recombinant TGF-.beta.1 protein secreted by these cells approached 30 ug/24 hour/107 cells. Expression vectors encoding TGF-.beta.1 precursor variants which direct the synthesis and secretion of TGF-.beta.1 in transfected mammalian cells are also described.
Brunner Amy M.
Gentry Larry
Purchio Anthony F.
Twardzik Daniel
Oncogen Limited Partnership
Osler Hoskin & Harcourt Llp
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