Characterization of a soluble IL-6 receptor alpha mutant C277D/H280I expressed in E.coli.
The pleiotropic cytokine interleukin-6(IL-6) triggers the formation of a high affinity receptor complex constituted by the ligand-binding subunit IL-6 receptor alpha (IL-6R alpha) and the signal transducer gp130. To construct the antagonist of IL-6, a DNA segment encoding the soluble human IL-6R alpha (shIL-6R alpha) mutant with two substitutions C277D/H280I was generated by overlap extension polymerase chain reaction. The DNA segment was then subcloned into vector pET-3b and expressed in E.coli at a high level. The refolded and purified recombinant protein, which was designated as DM650, exhibited an increased IL-6-binding capability by 8-fold. DM650 antagonized IL-6 perfectly, resulting in the growth-inhibition of human myeloma AF-10 cells. DNA fragmentation assay proved that the growth of AF-10 cells was inhibited through the induction of apoptosis suppressed by IL-6.
Duan
J
J
Department of Molecular Genetics, Institute of Basic Medical Sciences, Beijing, People's Republic of China.
Wang
J
J
Cai
X
X
Wang
L
L
Liu
H
H
Zou
M
M
D015703:Antigens, CD / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D017209:Apoptosis; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics*; D005822:Genetic Vectors / Q000378:metabolism; D006801:Humans; D015850:Interleukin-6 / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D017510:Protein Folding; D018123:Receptors, Interleukin / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D019947:Receptors, Interleukin-6; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000302:isolation & purification / Q000378:metabolism; D012995:Solubility; D013329:Structure-Activity Relationship; D014407:Tumor Cells, Cultured; D000093242:Interleukin-6 Inhibitors
D015703:Antigens, CD; D015850:Interleukin-6; D018123:Receptors, Interleukin; D019947:Receptors, Interleukin-6; D011994:Recombinant Proteins; D000093242:Interleukin-6 Inhibitors