Expression of Recombinant TGF-β2(442) Precursor and Detection in BSC-40 Cells

Abstract

Analysis of cDNA clones encoding transforming growth factor (TGF)-β2 predicts two different precursor proteins derived by alternative mRNA splicing; a 414 amino acid precursor [TGF-β2 (414)] and a 442 amino acid precursor [TGF-β2(442)]. The two proteins differ by a 28 amino acid insertion within the pro-region of TGF-β2 (442). In order to characterize the TGF-β2 related proteins encoded by the TGF-β2(442) cDNA and determine whether it could, in fact, direct the synthesis of active growth factor, we have expressed this gene in Chinese hamster ovary (CHO) cells and, after amplification with methotrexate, obtained stable clones secreting TGF-β2 (442). The TGF-β2 secreted by these cells was latent as acidification was necessary to detect optimal biological activity. In addition to mature TGF-β2, high molecular weight pro-region containing proteins were also secreted as analyzed by immunoblotting using site-specific anti-peptide antibodies. These proteins migrated differently than those secreted by CHO cells transfected with cDNA encoding TGF-β2(414), indicating that structural differences exist between the two complexes. An anti-peptide antiserum was produced in rabbits against the 28 amino acid insert region of TGF-β2(442). This sera was then used to detect the presence of TGF-β2(442) in serum-free media conditioned by BSC-40 cells. Since the TGF-β2(442) precursor is produced and secreted by a non-recombinant cell line, this suggests that it may play a physiological role in regulating the activity of TGF-β2.

Key Words:

• TGF-β2
• latency associated complex