Analysis on mechanism of severe allergic reaction caused by SNP in interleukin 13 using NMR

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We consbucted tcansibm is that express (His)_6-D-D-D-D-K fused human intedeukin 13 (Fusion IL-13) and (His)_6-D-D-D-D-K fused human mutant interieukin 13 whemArgl 10 is mutated to Gln (Fusion mutant IL-13). These bansfonnants were arlturod in M9 minimal media containing ^<15>NH_4Cl. After incubation, we got Fusion IL-13 and Fusion mutant IL-13 as inclusion bodies. Individual precipitates were dissolved in Tris-HCl buffer at pH 8 containing 6M guanidine hydrochloride. Each solution was adsorbed to Ni-NTA column and eluted with a buffer containing 50mM sodium phosphate buffer at pH 4.5 containing 6M guanidine hydrochloride. Then, the pH of the collected factions were adjusted to 8 and the solutions were reduced by 50 mM m hnol for 30min at 40 degrees. Each solution was diluted into 50 mM Tris-HCl buffer at pH 8.5 containing 3M urea, 30% glycerol, 5 mM cystamine and 5 mM cysteine and stirred for 3days at 4 degrees. After any precipitated proteins were removed by centrifugation, each sugar … More nt was applied to Ni-NTA column. After washing by 20 mM phosphate buflr at pH6.1 containing 10% glycerol, the soluble proteins were eluted with the same buffer containing 250 mM imidazole. After dialysis against 20 mM sodium phosphate buffer at pH 6.1 containing 10% glycerol, (His)_6-D-D-D-D-K in Fusion IL-13 and Fusion mutant IL-13 was processed with enterokinase by incubation for 14 hours at 20 degrees. Each reaction mixtwe was applied to cation exchange column of CM-Toyopearl 650 M. The wild-Type IL-13 and mutant IL-13 were eluted with 20 mM sodium phosphate buffer at pH 6.1 containing 1 M NaCl. We confirmed by analysis of their ^1H-^<15>N HSQC spectra that both IL-13 had correct conformations. Then, we measured T1, T2 and ^1H-^<15>N NOE with or without saturation. We evaluated order parameters and Rex values at individual residues in the wild-type IL-13 and the mutant IL-13 by means of model free analysis using above parameters (T1, T2 and NOE). Compared order parameters and Rex values at residues in the mutant IL-13 with those in the wild-type IL-13, there was difference at D-helix IL-13 between them. It was reported by mutation analysis that D-helix in IL-13 interacted with interleukin 13 receptor alpha 2. Therefore, it was suggested that the subtle weaker affinity of the mutant IL-13 than that of the wild-type IL-13 with with interleukin 13 receptor- alpha may depend on the difference in internal motions at D-helix in IL-13. Less