Expression and purification of cytokine receptor homology domain of human granulocyte-colony stimulating factor receptor in Escherichia coli.
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.
Tanaka
R
R
Laboratory of Applied and Molecular Microbiology, Faculty of Agriculture, Kagoshima University, Japan.
Tokunaga
H
H
Hara
S
S
Arakawa
T
T
Tokunaga
M
M
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
D018121:Receptors, Cytokine; D016188:Receptors, Granulocyte Colony-Stimulating Factor; D011993:Recombinant Fusion Proteins