Cloning and expression of simian transforming grow

专利名称：Cloning and expression of simian

transforming growth factor &bgr; 1

发明人：Anthony F. Purchio,Larry Gentry,Daniel

Twardzik,Amy M. Brunner

申请号：US07/958522申请日：19921008公开号：US05844085A公开日：19981201

摘要：Recombinant transforming growth factor-&bgr;1 (TGF-&bgr;1) is expressed tohigh levels in Chinese hamster ovary (CHO) cells using dihydrofolate reductase (dhfr)gene amplification. The expression plasmid was derived from the pSV2 vectors andcontained, in tandem, the simian TGF-&bgr;1 and mouse dhfr cDNAs. Transcription ofboth cDNAs was controlled by the SV40 early promoter. Stepwise selection oftransfected CHO cells in increasing concentrations of methotrexate yielded cell linesexpressing amplified TGF-&bgr;1 nucleic acid sequences. The expression plasmid DNAwas amplified greater than 35-fold in one of the methotrexate selected transfectants.The major proteins secreted by these cells consisted of latent TGF-&bgr;1 and TGF-&bgr;1 precursor polypeptides as judged by immunoblots using site-specific anti-peptideantibodies derived from various regions of the TGF-&bgr;1 precursor. Levels of

recombinant TGF-&bgr;1 protein secreted by these cells approached 30 ug/24 hour/10.sup.7 cells. Expression vectors encoding TGF- &bgr;1 precursor variants which direct thesynthesis and secretion of TGF-&bgr;1 in transfected mammalian cells are also described.

申请人：BRISTOL-MYERS SQUIBB PHARMACEUTICAL RESEARCH INSTITUTE-SEATTLE

代理机构：Pennie & Edmonds

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