2006 Fiscal Year Final Research Report Summary
Structural and Functional Analysis of the MD-2 protein
| Project/Area Number |
17590439
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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 |
Immunology
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| Research Institution | Saga University |
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Principal Investigator |
KIMOTO Masao Saga University, Medicine, Professor, 医学部, 教授 (40153225)
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| 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)
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| Project Period (FY) |
2005 – 2006
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| Keywords | MD-2 / LPS / pET32 vector / fusion protein / E coli expression / NF κ B reporter gene / epitope / pCAGG-S1 vector |
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| 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.
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