title: Expression and purification of cytokine receptor homology domain of human granulocyte-colony stimulating factor receptor in Escherichia coli.
abstract: In an attempt to generate a stable non-glycosylated cytokine receptor homology (CRH) domain (Tyr97-Ala309) of human granulocyte-colony stimulating factor (G-CSF) receptor, two free cysteines in the CRH domain were converted to serine by site-directed mutagenesis. Taking advantage of the tight regulation for the expression of T7 RNA polymerase, the mutated CRH domain was successfully expressed in Escherichia coli (E. coli) with a pelB signal sequence at its NH2-terminus and with a His tag at its COOH-terminus. The processed and secreted CRH domain after solubilization and in vitro refolding retained G-CSF binding activity, and its yield (approximately 40 micrograms/30 ml culture) was more than 100-fold higher than that of the mouse CRH domain expressed by the MalE fusion system in E. coli.
authors.affiliation: Laboratory of Applied and Molecular Microbiology, Faculty of Agriculture, Kagoshima University, Japan.
authors.forename: R
authors.initials: R
authors.lastname: Tanaka
authors.forename: H
authors.initials: H
authors.lastname: Tokunaga
authors.forename: S
authors.initials: S
authors.lastname: Hara
authors.forename: T
authors.initials: T
authors.lastname: Arakawa
authors.forename: M
authors.initials: M
authors.lastname: Tokunaga
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D004926:Escherichia coli / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D018121:Receptors, Cytokine / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D016188:Receptors, Granulocyte Colony-Stimulating Factor / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification
chemical_list: D018121:Receptors, Cytokine; D016188:Receptors, Granulocyte Colony-Stimulating Factor; D011993:Recombinant Fusion Proteins