CHARACTERIZATION OF RECOMBINANT SOLUBLE HUMAN TRANSFORMING GROWTH-FACTOR-BETA RECEPTOR-TYPE-II (RHTGF-BETA-SRII)

Recombinant human transforming growth factor soluble receptor Type II (rhTGF-beta sRII) corresponding to the 159 amino acid extracellular do main of hTGF-beta RII has been expressed in insect cells using the bac ulovirus expression system or in a mouse myeloma cell line. N-terminal sequence analysis of the purified protein revealed the removal of the 23 amino acid signal peptide. In SDS-PAGE, the rhTGF-beta sRII resolv es into multiple bands due to N-linked glycosylation. Recombinant hTGF -beta sRII is a TGF-beta antagonist and will inhibit the biological ac tivities of TGF-beta 1, TGF-beta 3, and TGF-beta 5 on TGF-beta-respons ive cell lines, such as murine HT-2 or human TF-1 with an ED(50) of ap proximately 0.3 mu g/mL. However, hTGF-beta RII does not inhibit TGF-b eta 2 bioactivities in these cell lines, suggesting that hTGF-beta RII has low affinity for TGF-beta 2. Polyclonal antibodies to hTGF-beta s RII have been produced in goats and purified on Protein-G affinity col umns. This antibody can inhibit TGF-beta 1,2,3,5-dependent bioactiviti es on human cell lines such as TF-1. Additionally, this antibody has s pecies cross-reactivity and will also inhibit TGF-beta-dependent bioac tivities on murine cells. These results, taken together, suggest that hTGF-beta RII is required for signal-transduction of all TGF-beta isof orms, including TGF-beta 2 rhTGF-beta sRII has been used as a capture reagent in conjunction with a TGF-beta 1-specific polyclonal antibody as the detection antibody in a highly sensitive enzyme-linked immunoso rbent assay (ELISA) for the specific measurement of TGF-beta 1 in comp lex biological fluids with a minimum detectable dose of approximately 10 pg/mL. Latent TGF-beta 1 and other TGF-beta isoforms do not cross-r eact in this assay. rhTGF-beta sRII can also sandwich effectively with anti-TGF-beta 3 and TGF-beta 5 antibodies to detect TGF-beta 3 and TG F-beta 5, respectively. Consistent with the bioassay results, rhTGF-be ta sRII is not suitable for use either as a capture or as a detection reagent in TGF-beta 2-specific ELISAs because the sensitivity is much lower than for the other isoforms of TGF-beta. (C) 1995 Academic Press Limited.