Purification and in vitro folding of recombinant human thrombopoietin receptor expressed in Escherichia coli

Thrombopoietin receptor (TPOR) is a member of the cytokine receptor superfa mily expressed primarily on hematopoietic cells. TPOR plays an important ro le in regulating platelet production. Due to its low expression level in hu man tissue, studies on the biochemical and biophysical properties of TPOR h ave been limited. In the present study, an extracellular domain of recombin ant human TPOR (rh TPOR-EN) was expressed in Escherichia coli as inclusion bodies. Purification was achieved by metal chelated chromatography under de naturing condition and was refolded by gel filtration chromatography. Far U V circular dichroism spectroscopy and surface plasmon resonance experiments were performed to demonstrate that the protein was in a refolded state and could bind with its ligand. Thus, a production and purification scheme was developed by which sufficient quantities of rh TPOR-EN can be made availab le for biochemical and biophysical characterizations. (C) 2001 Academic Pre ss.