Expression, purification, and characterization of the active immunoglobulin-like domain of human granulocyte-colony-stimulating factor receptor in Escherichia coli.
We succeeded in the expression, purification, and refolding of the immunoglobulin-like (Ig) domain of human granulocyte-colony-stimulating factor (G-CSF) receptor with amino-terminal His-tag in Escherichia coli. The refolded Ig domain bound to a G-CSF affinity column and could be eluted with free G-CSF as a receptor-ligand complex, demonstrating that the Ig domain has the information necessary for binding its ligand, G-CSF. The eluted His-Ig/G-CSF complex could be separated from excess G-CSF by Ni-NTA column chromatography. The yield of this active recombinant His-Ig protein is about 0.72 mg per liter of culture. Its small size and the ease of production make this receptor fragment a useful reagent for the structural analysis of its complex with G-CSF.
Laboratory of Applied and Molecular Microbiology, Faculty of Agriculture, Kagoshima University, 1-21-24 Korimoto, Kagoshima 890-0065, Japan.
M
M
Ishibashi
H
H
Tokunaga
T
T
Arakawa
M
M
Tokunaga
D019943:Amino Acid Substitution; D001667:Binding, Competitive; D002846:Chromatography, Affinity; D004926:Escherichia coli / Q000235:genetics*; D016179:Granulocyte Colony-Stimulating Factor / Q000378:metabolism; D006639:Histidine / Q000378:metabolism; D006801:Humans; D007136:Immunoglobulins / Q000737:chemistry*; D013058:Mass Spectrometry; D008970:Molecular Weight; D020673:Protein Renaturation; D017434:Protein Structure, Tertiary; D016188:Receptors, Granulocyte Colony-Stimulating Factor / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D020539:Sequence Analysis, Protein; D012995:Solubility
D007136:Immunoglobulins; D016188:Receptors, Granulocyte Colony-Stimulating Factor; D011993:Recombinant Fusion Proteins; D016179:Granulocyte Colony-Stimulating Factor; D006639:Histidine