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

NMR分析白细胞介素13中SNP引起严重过敏反应的机制

基本信息

批准号：
14572032
负责人：
UEDA Tadashi
金额：
$ 2.24万
依托单位：
KYUSHU UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2002
资助国家：
日本
起止时间：
2002 至 2003
项目状态：
已结题

项目摘要

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

我们构建了表达（His）_6-D-D-D-D-K融合人白细胞介素13（Fusion IL-13）和（His）_6-D-D-D-D-K融合人突变型白细胞介素13（Fusion mutant IL-13）的表达载体。在含NH_4Cl的M_9基本培养基中，这些班氏杆菌<15>都能生长。孵育后，我们得到融合IL-13和融合突变体IL-13作为包涵体。将单个沉淀物溶解在含有6 M盐酸胍的pH 8的Tris-HCl缓冲液中。将每种溶液吸附到Ni-NTA柱上，并用含有6 M盐酸胍的pH 4.5的含有50 mM磷酸钠缓冲液的缓冲液洗脱。然后，将收集的部分的pH调节至8，并将溶液在40度下用50 mM甲醇还原30分钟。将每种溶液稀释到含有3 M尿素、30%甘油、5 mM胱胺和5 mM半胱氨酸的pH 8.5的50 mM Tris-HCl缓冲液中，并在4度下搅拌3天。在通过离心除去任何沉淀的蛋白质后， ...更多信息 NT上样于Ni-NTA柱。用含有10%甘油的pH6.1的20 mM磷酸缓冲液洗涤后，用含有250 mM咪唑的相同缓冲液洗脱可溶性蛋白质。在用含有10%甘油的pH 6.1的20 mM磷酸钠缓冲液透析后，通过在20 ℃孵育14小时，用肠激酶处理融合IL-13和融合突变体IL-13中的（His）_6-D-D-D-D-K。将各反应混合物上样到CM-Toyopolymer 650 M阳离子交换柱上。野生型IL-13和突变型IL-13用含有1 M NaCl的pH 6.1的20 mM磷酸钠缓冲液洗脱。我们通过分析它们的^1H-^<15>N HSQC光谱证实，两种IL-13都具有正确的构象。然后，我们测量了T1，T2和^1H-^<15>N NOE（有或无饱和）。我们通过使用上述参数（T1、T2和NOE）的无模型分析评估了野生型IL-13和突变型IL-13中单个残基处的序参数和雷克斯值。比较突变型IL-13与野生型IL-13的序参量和氨基酸残基处的雷克斯值，发现二者在D-螺旋IL-13处存在差异。突变分析表明IL-13的D-螺旋与IL-13受体α 2相互作用。因此，这表明突变体IL-13与白细胞介素13受体-α的亲和力比野生型IL-13的亲和力弱，这可能取决于IL-13中D-螺旋的内部运动的差异。少