High-level expression of TGF-beta 2 and the TGF-beta 2(414) precursor in Chinese hamster ovary cells.
Chinese hamster ovary (CHO) clones secreting high levels of transforming growth factor-beta 2 (TGF-beta 2) were obtained after transfection with a cDNA clone coding for the 414-amino acid TGF-beta 2 precursor and subsequent amplification with methotrexate. The TGF-beta 2 was secreted in a latent form since acidification was necessary for detection of maximal levels of bioactivity. Amino- and carboxy-terminal sequencing of purified recombinant TGF-beta 2 indicated that correct processing of mature TGF-beta 2 had occurred. In addition to mature TGF-beta 2, the recombinant CHO clones secreted larger proteins having molecular weights of 85, 105, and 130 kD, which consisted of both mature and pro-region sequences when analyzed by immunoblotting using site-specific anti-peptide antibodies. Analysis of serum- and cell-free media from recombinant CHO cells metabolically labeled with [3H]glucosamine and [32P]orthophosphate indicated that pro-TGF-beta 2 was glycosylated and phosphorylated. Two-dimensional electrophoretic analysis of acid hydrolysates showed that the 32P was incorporated into mannose-6-phosphate.
Oncogen, Seattle, Washington 98121.
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Madisen
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Lioubin
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Marquardt
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Purchio
D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D006224:Cricetinae; D005786:Gene Expression Regulation; D006031:Glycosylation; D006863:Hydrogen-Ion Concentration; D008360:Mannosephosphates / Q000032:analysis; D008727:Methotrexate / Q000494:pharmacology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010766:Phosphorylation; D011498:Protein Precursors / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D014162:Transfection; D016212:Transforming Growth Factor beta / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification
D008360:Mannosephosphates; D011498:Protein Precursors; D016212:Transforming Growth Factor beta; C027693:mannose-6-phosphate; D008727:Methotrexate