Asp126,Asp130
and Asp134 are Necessary for Human IL-18 to Elicit IFN-γ Production
from PBMC
FU Yi, PEI Dong-Sheng, ZHAO Hui-Ren*
( Research Center for Biochemistry and Molecular Biology, Xuzhou Medical
College, Xuzhou 221002, China )
Abstract To identify
the amino acid residues which are critical to interleukin 18 (IL-18) function,
three highly-conserved amino acids (Asp126,Asp130 and Asp134)
were mutated to Asn, Lys and Lys. The wild type and mutant recombinant human
interleukin-18 (rhIL-18) were expressed in E.coli,renatured by
stepwise dilution and purified by Sephadex G-75 chromatography. The purity of
the recombinant proteins was over 95% and Western blot showed that the mutant
rhIL-18 had the same immunogenicity as that of wild type rhIL-18. The
activities of wild type and mutant rhIL-18s were defined as the ability to
induce interferon-gamma(IFN-γ) production from human peripheral blood
mononuclear cells(PBMC). The results showed that the three mutants induced
significantly less amount of IFN-γ from PBMC(32%, 8% and 10% of wild type for
hIL-18D126N, hIL-18D130K and hIL-18D134K,
respectively) indicating that the three highly conserved amino acids are
necessary for human IL-18 function.
Key words human interleukin 18； protein structure and
function；
site-directed mutagenesis； interferon-γ
*Corresponding author： Tel, 86-516-5748423； e-mail, [email protected]