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

我们推测，表达(His)_6-D-K融合人白介素13(Fusion IL-13)和(His)_6-D-K融合人白介素13(Interieukin 13)，而Arg1 10突变为Gln(融合突变体IL-13)。它们是在含有NH_4CL的M9最低限度的培养基中产生的。孵育后得到融合IL-13和融合突变体IL-13作为包涵体。个别沉淀物在含有6M盐酸胍的pH为8的Tris-HCl缓冲液中溶解。每种溶液都被吸附到Ni-NTA柱上，用含有50 mM磷酸二氢钠缓冲液的缓冲液洗脱，缓冲液中含有6M盐酸胍，pH 4.5。然后，将收集的各组分的pH调节到8，并将溶液在40℃下用50 mM的乙醇还原30min。每种溶液在含有3M尿素、30%甘油、5 mM半胱胺和5 mM半胱氨酸的pH 8.5的50 mM Tris-HCl缓冲液中稀释，在4度下搅拌3d。在通过离心法去除任何沉淀蛋白质后，每个糖…在Ni-NTA柱上加入更多的NT。用含10%甘油的20 mM磷脂缓冲液洗脱，再用含250 mM咪唑的缓冲液洗脱。在含有10%甘油的20 mM磷酸二氢钠缓冲液中透析后，将融合IL-13和融合突变体IL-13中的(His)_6-D-K与肠激酶在20℃孵育14h。每个反应混合物用于CM-Toyota opearl 650M阳离子交换柱，野生型IL-13和突变体IL-13用20 mM磷酸二氢钠缓冲液洗脱，pH 6.1，含1M氯化钠。我们通过分析他们的^1H-^&lt；15&gt；N HSQC谱证实了这两种IL-13都具有正确的构象。然后，我们测量了饱和前后的t1、t2和^1H-^&t；我们利用上述参数(T1、T2和NOE)通过无模型分析的方法估计了野生型IL-13和突变型IL-13的顺序参数和单个残基的Rex值。比较突变型IL-13和野生型IL-13的序列参数和残基的Rex值，发现它们在D-螺旋IL-13上存在差异。突变分析表明，IL-13中的D-螺旋与IL-13受体α2相互作用。因此，突变型IL-13与IL-13受体-α的亲和力略低于野生型IL-13，这可能与IL-13中D-螺旋的内部运动不同有关。较少

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