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

Research Project

Project/Area Number 14572032
Research Category

Grant-in-Aid for Scientific Research (C)

Allocation TypeSingle-year Grants
Section一般
Research Field Physical pharmacy
Research InstitutionKYUSHU UNIVERSITY

Principal Investigator

UEDA Tadashi  Kyushu University, Graduate School of Pharmaceutical Sciences, Professor, 大学院・薬学研究院, 教授 (90184928)

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)
Project Period (FY) 2002 – 2003
Project Status Completed (Fiscal Year 2003)
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)
Keywordsnuclear magnetic resonance / interleukin 13 / relaxation analysis / allergy / stable-isotope-labeling / 核磁気共鳴法 / チオレドキシン / NMR
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 … 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

Report

(3 results)
  • 2003 Annual Research Report   Final Research Report Summary
  • 2002 Annual Research Report
  • Research Products

    (9 results)

All 2004 Other

All Journal Article (4 results) Publications (5 results)

  • [Journal Article] Identification of the region in Escherichia coli DnaA protein required for specific recognition of the DnaA box.2004

    • Author(s)
      Yoshida et al.
    • Journal Title

      Cell.Mol.Life Sci. 60(9)

      Pages: 1998-2008

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Journal Article] A small chimerically bifunctional monomeric protein : Tapes japonica lysozyme.2004

    • Author(s)
      Takeshita et al.
    • Journal Title

      Cell.Mol.Life Sci. 60(9)

      Pages: 1944-1951

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Journal Article] Identification of the region in Escherichia coli DnaA protein required for specific recognition of the DnaA box.2004

    • Author(s)
      Yoshida et al.
    • Journal Title

      Cell.Mol.Life.Sci. 60

      Pages: 1998-2008

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Journal Article] A small chimerically bifunctional monomeric protein : Tapes Japonica lysozyme.2004

    • Author(s)
      Takeshita et al.
    • Journal Title

      Cell.Mol.Life Sci. 60

      Pages: 1944-1951

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] Yoshida et al.: "Identification of the region in Escherichia Coli DnaA protein required for specific recognition of the DnaA box."Cell.Mol.Life Sci.. 60(9). 1998-2008 (2003)

    • Related Report
      2003 Annual Research Report
  • [Publications] Obita et al.: "Solution structure and activity of mouse lysozyme"Cell.Mol.Life Sci.. 60(1). 176-183 (2003)

    • Related Report
      2003 Annual Research Report
  • [Publications] Ohkuri et al.: "A metal binding in the polypeptide chain improves the folding efficiency of a denatured and reduced protein"Biopolymers. 64・2. 106-114 (2002)

    • Related Report
      2002 Annual Research Report
  • [Publications] Obita et al.: "Determination of the secondary structure in solution of the Escherichia coli DnaA DNA-binding domain"Biochem.Biophys.Res.Commun.. 299・1. 42-48 (2002)

    • Related Report
      2002 Annual Research Report
  • [Publications] Ohmura et al.: "Fluctuations in free or substrate-complexed lysozyme and a mutant of it detected on x-ray crystallography and comparison with those detected on NMR"J.Biochem.. 131・5. 701-704 (2002)

    • Related Report
      2002 Annual Research Report

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Published: 2002-04-01   Modified: 2016-04-21  

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