title: Type 1 transforming growth factor beta: amplified expression and secretion of mature and precursor polypeptides in Chinese hamster ovary cells.
abstract: Recombinant type 1 transforming growth factor beta (TGF-beta) was expressed to high levels in CHO cells by using dihydrofolate reductase (dhfr) gene amplification. The expression plasmid was derived from the pSV2 vectors and contained, in tandem, the simian TGF-beta and mouse dhfr cDNAs. Transcription of both cDNAs was controlled by the simian virus 40 early promoter. Stepwise selection of transfected CHO cells in increasing concentrations of methotrexate yielded cell lines that expressed amplified TGF-beta 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 and TGF-beta precursor polypeptides, as judged by immunoblots by using site-specific anti-peptide antibodies derived from various regions of the TGF-beta precursor. Levels of recombinant TGF-beta protein secreted by these cells approached 30 micrograms/24 h per 10(7) cells and required prior acidification for optimal activity; nonacidified supernatants were approximately 1% as active as acidified material. Antibodies directed toward sequences present in the mature growth factor readily identified a proteolytically processed recombinant TGF-beta which, on sodium dodecyl sulfate-polyacrylamide gels, comigrated with highly purified natural TGF-beta. In addition to mature recombinant TGF-beta, site-specific antibodies demonstrated the existence of larger TGF-beta precursor polypeptides. The availability of biologically active recombinant type 1 TGF-beta and precursor forms should provide a means to examine the structure, function, and potential in vivo therapeutic use of this growth factor.
mesh_terms: D000143:Acids; D000818:Animals; D001681:Biological Assay; D002460:Cell Line; D003001:Cloning, Molecular; D006224:Cricetinae; D005784:Gene Amplification; D005786:Gene Expression Regulation / Q000187:drug effects; D008727:Methotrexate / Q000494:pharmacology; D008970:Molecular Weight; D010452:Peptide Biosynthesis; D010455:Peptides / Q000378:metabolism; D011498:Protein Precursors / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics; D014162:Transfection; D015291:Transforming Growth Factors
chemical_list: D000143:Acids; D010455:Peptides; D011498:Protein Precursors; D011994:Recombinant Proteins; D015291:Transforming Growth Factors; D013762:Tetrahydrofolate Dehydrogenase; D008727:Methotrexate