Analysis on mechanism of severe allergic reaction caused by SNP in interleukin 13 using NMR
| Project/Area Number |
14572032
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Physical pharmacy
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| Research Institution | KYUSHU UNIVERSITY |
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Principal Investigator |
UEDA Tadashi Kyushu University, Graduate School of Pharmaceutical Sciences, Professor, 大学院・薬学研究院, 教授 (90184928)
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| Co-Investigator(Kenkyū-buntansha) |
IZUHARA Kenji SAGA University, Faculty of Medicine, Professor, 医学部, 教授 (00270463)
IMOTO Taiji Sojo University, Faculty of Engineering, Professor, 工学部, 教授 (90038282)
ABE Yoshito Kyushu University, Graduate School of Pharmaceutical Sciences, Associate Professor, 大学院・薬学研究院, 助教授 (60315091)
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| Project Period (FY) |
2002 – 2003
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| Project Status |
Completed (Fiscal Year 2003)
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| Budget Amount *help |
¥3,500,000 (Direct Cost: ¥3,500,000)
Fiscal Year 2003: ¥1,100,000 (Direct Cost: ¥1,100,000)
Fiscal Year 2002: ¥2,400,000 (Direct Cost: ¥2,400,000)
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| Keywords | nuclear magnetic resonance / interleukin 13 / relaxation analysis / allergy / stable-isotope-labeling / 核磁気共鳴法 / チオレドキシン / NMR |
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| Research Abstract |
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
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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
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Report
(3 results)
Research Products
(9 results)
