The TGF-B family now comprises 5 distinct isoforms. Of these, only TGF-betas I and 2 have been purified as proteins; TGF-Betas 3 and 4 have been cloned from a chicken embryo chondrocyte cDNA library and TGF-Beta-5 has been cloned from a Xenopus oocyte cDNA library. Although we have been able to use both the cDNA probes to identify certain cells which express high mRNA levels of these new TGF-Betas and anti-peptide antibodies raised to sequences of the putative novel TGF-Betas to detect expression of the proteins, TGF-Betas 3-5 have not yet been purified from natural sources or produced in any significant quantities by recombinant techniques. Because the proteins have been unavailable, it has not been possible to test their activities in various biological systems. For these reasons, it is important that natural sources of these peptides be identified and that purified, chemically characterized protein be available for testing. The purpose of this project is to purify to homogeneity proteins exhibiting TGF-Beta-like activity from a wide variety of cell and tissue extracts including mammalian cells engineered to express the various TGF-Beta isoforms under control of strong promoters. These proteins will be positively identified in terms of their amino acid sequence, or by immunoreactivity, when that is possible. TGF-Betas 3, 4, or 5 as well as any novel TGF-Beta isoforms so purified will then be assayed in a broad spectrum of both in vitro and in vivo biological assays to characterize fully their biological activity. One related aspect of this project involves an attempt to purify to homogeneity and to determine the amino acid sequence of the TGF-Beta-related mesoderminducing activity from Xenopus and to characterize any specific biological interactions of this factor with the TGF-Betas.
