Expression of the soluble extracellular domain of human thrombopoietin receptor using a maltose-binding protein-affinity fusion system.
The thrombopoietin (TPO) receptor (Mpl) belongs to the family of ligand-dependent cytokine receptors and plays a functional role in regulating platelet production. The signaling capacity largely depends on the binding of TPO to the extracellular domains of the TPO receptor (Mpl-EC). Because the expression level of Mpl in human tissue is very low, studies on the functional and spatial characteristics of its ligand-binding sites have been limited. In the present study, we report the expression and purification of Mpl-EC as a fusion with the maltose-binding protein (MBP), designated MBP-Mpl-EC. MBP-Mpl-EC was expressed in the cytoplasm of Escherichia coli as a soluble fusion protein. Specific binding of TPO to purified MBP-Mpl-EC was demonstrated by a dot-blot assay and surface plasmon resonance. We conclude that bacterial expression of MBP-Mpl-EC yields large amounts of protein with correct folding and that it can be used for further structure and function analyses.
Zhang
Qing
Q
Biotechnology Research Center, Key Laboratory of Gene Engineering of the Education Ministry, Zhongshan University, Guangzhou 510275, People's Republic of China.
Pan
Rui-Min
RM
Ge
Yi-Chen
YC
Xu
Peilin
P
D002352:Carrier Proteins / Q000235:genetics*; D004926:Escherichia coli / Q000378:metabolism; D006801:Humans; D015151:Immunoblotting; D008024:Ligands; D058619:Maltose-Binding Proteins; D009363:Neoplasm Proteins / Q000096:biosynthesis* / Q000235:genetics; D017434:Protein Structure, Tertiary; D011518:Proto-Oncogene Proteins / Q000096:biosynthesis* / Q000235:genetics; D018121:Receptors, Cytokine / Q000096:biosynthesis* / Q000235:genetics; D053628:Receptors, Thrombopoietin; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000378:metabolism; D012995:Solubility; D020349:Surface Plasmon Resonance; D013926:Thrombopoietin / Q000737:chemistry / Q000378:metabolism*
D002352:Carrier Proteins; D008024:Ligands; D058619:Maltose-Binding Proteins; D009363:Neoplasm Proteins; D011518:Proto-Oncogene Proteins; D018121:Receptors, Cytokine; D053628:Receptors, Thrombopoietin; D011993:Recombinant Fusion Proteins; C082218:MPL protein, human; D013926:Thrombopoietin