BIOCHEMICAL AND BIOPHYSICAL CHARACTERIZATION OF REFOLDED DROSOPHILA DPP, A HOMOLOG OF BONE MORPHOGENETIC PROTEIN-2 AND PROTEIN-4

The mature C-terminal signaling domain of the Drosophila Decapentapleg ic proprotein (DPP) can be efficiently refolded from chaotrope-solubil ized inclusion bodies with the aid of a membrane protein solubilizing detergent, high concentrations (0.75-2 M) of NaCl, and low temperature s (5-15 degrees C), The disulfide-linked homodimeric product contains N-terminal heparin-binding sites that were utilized as intrinsic affin ity tags to obtain a highly enriched preparation in one chromatographi c step. A subsequent C4 reverse phase high pressure liquid chromatogra phy step provides high purity, salt-free protein that is amenable to b iophysical and structural studies at a yield of approximately 3 mg/lit er of bacterial culture. The dimeric protein is correctly folded as de termined by electrophoretic, spectroscopic, chemical, and proteolytic analyses. Refolded DPP is also bioactive as shown by induction of chon drogenesis in embryonic chick limb bud cells and by high affinity bind ing to Noggin, an antagonist of bone morphogenetic protein signaling. In contrast to bone morphogenetic proteins extracted from demineralize d bone or overexpressed in cell culture, the refolded Escherichia coli -expressed protein is not glycosylated at a conserved N-linked site an d is therefore homogeneous. The C-terminal domain dimer is more hydrop hobic and thus less soluble than its unfolded or partially folded form s, necessitating highly solubilizing conditions for recovery after fol ding in vitro. Hence solubilization of the mature ligand may be one of the principal roles of the large (250-400 amino acids) N-terminal pro domains of transforming growth factor-beta superfamily members, shown to act as intramolecular chaperones in vivo.