Characterization of latent recombinant TGF-β2 produced by chinese hamster ovary cells

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Abstract
Latent recombinant transforming growth factor‐β2 (LrTGF‐β2) complex has been purified from serum‐free media conditioned by Chinese hamster ovary cells transfected with a plasmid encoding the TGF‐β2 (414) precursor. Under neutral conditions, LrTGF‐β2 had an apparent molecular weight of 130 kDa. The complex contained both mature and pro‐region sequences. Acidification of LrTGF‐β2 resulted in the release of mature 24 kDa TGF‐β2 from the high molecular weight pro‐region‐containing complex, suggesting that TGF‐β2 was non‐covalently associated with this complex. These results were confirmed by crosslinking experiments performed on partially purified LrTGF‐β2. Protein sequence analysis of the purified TGF‐β2 pro‐region indicated that signal peptide cleavage occured between ser(20) and leu(21). The pro‐region, which previously was found to contain mannose‐6‐phosphate, bound to the mannose‐6‐phosphate receptor. Proteolytic cleavage of mature TGF‐β2 from pro‐TGF‐β2 was inhibited by monensin and chloroquin suggesting that binding to this receptor and subsequent transport to acidic vesicles may be involved in the processing of rTGF‐β2 precursor.