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Structural and Functional Analysis of the MD-2 protein

Research Project

Project/Area Number 17590439
Research Category

Grant-in-Aid for Scientific Research (C)

Allocation TypeSingle-year Grants
Section一般
Research Field Immunology
Research InstitutionSaga University

Principal Investigator

KIMOTO Masao  Saga University, Medicine, Professor, 医学部, 教授 (40153225)

Co-Investigator(Kenkyū-buntansha) FUKUDOME Kenji  Saga University, Medicine, Associate Professor, 医学部, 助教授 (50284625)
TSUNEYOSHI Naoko  Saga University, Medicine, Assistant Professor, 医学部, 助手 (80336114)
WATANABE Keiichi  Saga University, Agriculture, Professor, 農学部, 教授 (40191754)
MOTOSHIMA Hiroyuki  Saga University, Agriculture, Assistant Professor, 農学部, 助手 (20312275)
Project Period (FY) 2005 – 2006
Project Status Completed (Fiscal Year 2006)
Budget Amount *help
¥3,600,000 (Direct Cost: ¥3,600,000)
Fiscal Year 2006: ¥1,400,000 (Direct Cost: ¥1,400,000)
Fiscal Year 2005: ¥2,200,000 (Direct Cost: ¥2,200,000)
KeywordsMD-2 / LPS / pET32 vector / fusion protein / E coli expression / NF κ B reporter gene / epitope / pCAGG-S1 vector / pCAG-S1ベクター
Research Abstract

1. Analysis of the functional binding between MD-2 and LPS.
We made a battery of transfectant cell line that expresses mutant MD-2 proteins with alanine substitutions at positions. Using these transfectant cell lines, we confirmed that these positions were critical for binding to LPS.
2. Production and purification of the E. coli recombinant MD-2 protein.
Using the pET32 vector, we succeeded to create fusion proteins of various mutant MD-2 protein and thioredoxin. After purification of these fusion proteins using colum chromatogaraphy, we found that fusion proteins were bound with LPS of the E. coli. The binding of LPS and MD-2 was found to be sensitive to the various detergents.
3. Functional analysis of LPS binding to the MD-2 protein.
Mutant MD-2 transfectant cell lines were stimulated with LPS and subjected to NFkB reporter assay. This assay allowed us to examine the functional analysis of the LPS recognizing region of the MD-2 protein.
4. Analysis of the MD-2 structure.
With multiple trial of the crystallization of MD-2 recombinant proteins, we did not succeed to obtain crystals even by using pCAGG-S1 vector. Trials using vertebrate and insect recombinant proteins are in progress.

Report

(3 results)
  • 2006 Annual Research Report   Final Research Report Summary
  • 2005 Annual Research Report
  • Research Products

    (9 results)

All 2006 2005 Other

All Journal Article (9 results)

  • [Journal Article] Expression of CD180, a Toll-like receptor homologue, is up-regulated in children with Kawasaki disease.2006

    • Author(s)
      Imayoshi M
    • Journal Title

      J. Mol. Med. 84(2)

      Pages: 168-174

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2006 Annual Research Report 2006 Final Research Report Summary
  • [Journal Article] Preparation and characterization of truncated human lipopolysaccharide-binding protein in Escherichia coli.2006

    • Author(s)
      Kohara J.
    • Journal Title

      Protein Expr Purif. 49(2)

      Pages: 276-283

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2006 Annual Research Report 2006 Final Research Report Summary
  • [Journal Article] Induction of long-term lipopolysacchride tolerance by an agonistic monoclonal antibody to the Toll-like receptor 4/MD-2 complex.2006

    • Author(s)
      Ohta S
    • Journal Title

      Clin Vaccine Immunol. 13(10)

      Pages: 1131-1136

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2006 Annual Research Report 2006 Final Research Report Summary
  • [Journal Article] Penta-acylated lipopolisaccharide binds to murine MD-2 but does not induce the oligomelization of TLR4 required for signal transduction.2006

    • Author(s)
      Tsuneyoshi N
    • Journal Title

      Cellular Immunology 244(1)

      Pages: 57-64

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2006 Final Research Report Summary
  • [Journal Article] Expression of CD180, a Toll-like receptor homologue, is up-regulated in children with Kawasaki disease.2006

    • Author(s)
      Imayoshi M
    • Journal Title

      J.Mol.Med. 84(2)

      Pages: 168-174

    • Related Report
      2005 Annual Research Report
  • [Journal Article] Comparison of lipopolysaccharide-binding functions of CD14 and MD-2.2005

    • Author(s)
      Kohara J
    • Journal Title

      Clin Diagn Lab Immunol. 12(11)

      Pages: 1292-1297

    • Related Report
      2005 Annual Research Report
  • [Journal Article] The functional and structural properties of MD-2 required for lipopolysaccharide binding are absent in MD-1.2005

    • Author(s)
      Tsuneyoshi N
    • Journal Title

      J Immunol. 174(1)

      Pages: 340-344

    • Related Report
      2005 Annual Research Report
  • [Journal Article] Preparation and characterization of monoclonal antibodies to thrombomodulin.2005

    • Author(s)
      Tsuneyoshi N
    • Journal Title

      Hybridoma 24(4)

      Pages: 189-196

    • Related Report
      2005 Annual Research Report
  • [Journal Article] Penta-acylated lipopolisaccharide binds to murine MD-2 but does not induce the oligomelization of TLR4 required for signal transduction.

    • Author(s)
      Tsuneyoshi N
    • Journal Title

      Cellular Immunology (in press)

    • Related Report
      2006 Annual Research Report

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Published: 2005-04-01   Modified: 2016-04-21  

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