Site-directed
Mutagenesis of the Cysteines of Human IL-18 and Its Effect on IL-18 Activity
PEI Dong-Sheng, FU Yi, SUN
Ya-Feng, ZHAO Hui-Ren*
( Research Center for Biochemistry and Molecular Biology, Xuzhou Medical
College, Xuzhou 221002, China )
Abstract 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-γ production in
human peripheral blood mononuclear cells.
Key words interleukin 18; cysteine; site-directed
mutagenesis; mutant
*Corresponding author: Tel,
86-516-5748423, e-mail: [email protected]