Abstract
The mature C-terminal signaling domain of the Drosophila Decapentaplegic proprotein (DPP) can be efficiently refolded from chaotrope-solubilized inclusion bodies with the aid of a membrane protein-solubilizing detergent, high concentrations (0.75-2 M) of NaCl, and low temperatures (5-15 degreesC). The disulfide-linked homodimeric product contains N-terminal heparin-binding sites that were utilized as intrinsic affinity tags to obtain a highly enriched preparation in one chromatographic step. A subsequent C4 reverse phase high pressure liquid chromatography step provides high purity, salt-free protein that is amenable to biophysical and structural studies at a yield of approximately 3 mg/liter of bacterial culture. The dimeric protein is correctly folded as determined by electrophoretic, spectroscopic, chemical, and proteolytic analyses. Refolded DPP is also bioactive as shown by induction of chondrogenesis in embryonic chick limb bud cells and by high affinity binding to Noggin, an antagonist of bone morphogenetic protein signaling. In contrast to bone morphogenetic proteins extracted from demineralized bone or overexpressed in cell culture, the refolded Escherichia coli-expressed protein is not glycosylated at a conserved N-linked site and is therefore homogeneous. The C-terminal domain dimer is more hydrophobic and thus less soluble than its unfolded or partially folded forms, necessitating highly solubilizing conditions for recovery after folding in vitro. Hence solubilization of the mature ligand may be one of the principal roles of the large (250-400 amino acids) N-terminal prodomains of transforming growth factor-beta superfamily members, shown to act as intramolecular chaperones in vivo.
Highlights
The mature C-terminal signaling domain of the Drosophila Decapentaplegic proprotein (DPP) can be efficiently refolded from chaotrope-solubilized inclusion bodies with the aid of a membrane protein-solubilizing detergent, high concentrations (0.75–2 M) of NaCl, and low temperatures (5–15 °C)
We show that the mature C-terminal signaling domain of Drosophila DPP can be efficiently refolded in vitro from chaotrope-solubilized inclusion bodies and purified to near-homogeneity by heparin affinity chromatography followed by reverse phase HPLC
Our studies show that, in addition to the extracellular domains of heterodimeric receptor complexes, DPP binds with high affinity to heparin, a glycosaminoglycan related to heparan sulfate of extracellular matrix and cell-surface proteoglycans, and with high specificity to Noggin, a protein antagonist of BMP activity in vertebrates
Summary
Expression Vector Construct—The mature C-terminal domain of DPP (DPPC) was overexpressed in Escherichia coli with the bacteriophage T7 RNA polymerase/10 promoter system [20]. Residual unfolded monomer, folded dimeric product, and higher order multimers were separated by heparin affinity chromatography on a 5-ml Heparin HiTrap column by fast protein liquid chromatography (Amersham Pharmacia Biotech) at ambient temperature through combined step and gradient elution with NaCl. The column was equilibrated with 6 M urea, 50 mM Tris-HCl, 2 mM EDTA, pH 8.0, 0.1 M NaCl at a flow rate of 1.0 ml/min, and the Refolded Drosophila DPP concentrated, buffer-exchanged folding reaction was pumped onto the column with a 50-ml Superloop. Concentrate proteins, and remove salt, detergent, or chaotrope, trichloroacetic acid was added to a final concentration of 15–20%, and the sample was incubated on ice for 20 min. Because the disordered N-terminal arm is unresolved in the electron density map and lacking from the crystal structure of BMP-7, the structure of this polypeptide could not be incorporated into the model of the DPP homodimer
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