[Site-directed mutagenesis of the cysteines of human IL-18 and its effect on IL-18 activity].
To study the structure-function relationships of human IL-18(hIL-18), site-directed mutagenesis was used to generate four hIL-18 cysteine mutants, C74S, C104S, C112S and C163S. The cDNAs of the four cysteine mutants were inserted into prokaryotic expression vector pJW2 and expressed as inclusion bodies in E. coli. The inclusion bodies were washed with 2 mol/L urea, dissolved in 8 mol/L urea, and purified by chromatography on Sephadex G-100 column. The purity of the purified mutants were greater than 90% as judged by SDS-PAGE. The activity of rhIL-18 C74S, C104S, C112S and C163S accounted for 5%, 81%, 58% and 11% of wild type, respectively. These results suggest that Cys74 and Cys163 play important roles in inducing IFN-gamma production in human peripheral blood mononuclear cells.
Pei
Dong-Sheng
DS
Research Center for Biochemistry and Molecular Biology, Xuzhou Medical College, Xuzhou 221002, China.
Fu
Yi
Y
Sun
Ya-Feng
YF
Zhao
Hui-Ren
HR
D019943:Amino Acid Substitution; D003545:Cysteine / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D066298:In Vitro Techniques; D007371:Interferon-gamma / Q000378:metabolism; D020382:Interleukin-18 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D007963:Leukocytes, Mononuclear / Q000187:drug effects / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D013329:Structure-Activity Relationship
D020382:Interleukin-18; D007371:Interferon-gamma; D003545:Cysteine