Purification and in vitro folding of recombinant human thrombopoietin receptor expressed in Escherichia coli.
Thrombopoietin receptor (TPOR) is a member of the cytokine receptor superfamily expressed primarily on hematopoietic cells. TPOR plays an important role in regulating platelet production. Due to its low expression level in human tissue, studies on the biochemical and biophysical properties of TPOR have been limited. In the present study, an extracellular domain of recombinant human TPOR (rh TPOR-EN) was expressed in Escherichia coli as inclusion bodies. Purification was achieved by metal chelated chromatography under denaturing condition and was refolded by gel filtration chromatography. Far UV 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 available for biochemical and biophysical characterizations.
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Department of Biology, Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong SAR, China.
Huxtable
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Xu
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Hsieh
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D001665:Binding Sites; D002846:Chromatography, Affinity; D002850:Chromatography, Gel / Q000379:methods; D002942:Circular Dichroism; D003001:Cloning, Molecular / Q000379:methods; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D002479:Inclusion Bodies / Q000378:metabolism; D008024:Ligands; D009363:Neoplasm Proteins; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D011518:Proto-Oncogene Proteins / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification; D018121:Receptors, Cytokine; D053628:Receptors, Thrombopoietin; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D020349:Surface Plasmon Resonance
D008024:Ligands; D009363:Neoplasm Proteins; D011518:Proto-Oncogene Proteins; D018121:Receptors, Cytokine; D053628:Receptors, Thrombopoietin; D011994:Recombinant Proteins; C082218:MPL protein, human