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J Biol Chem, Vol. 273, Issue 44, 29052-29065, October 30, 1998
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From the 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). 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- Department of Cell Biology and
¶ Department of Biophysical Chemistry Biozentrum, University of
Basel, CH-4056 Basel, Switzerland, ** Regeneron Pharmaceuticals, Inc.,
Tarrytown, New York 10591, and
Theodor-Boveri-Institut (Biozentrum),
Physiologische Chemie II, Am Hubland,
D-97074 Würzburg, Germany
superfamily members, shown to act as intramolecular chaperones in
vivo.
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