| 研究課題/領域番号 |
20K16246
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| 研究機関 | 大阪公立大学 |
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研究代表者 |
アワスティ シャルダ 大阪公立大学, 大学院獣医学研究科, 客員研究員 (30751888)
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| 研究期間 (年度) |
2020-04-01 – 2024-03-31
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| キーワード | Cholix toxin |
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| 研究実績の概要 |
The objectives for the fiscal year 2022-23 were to conduct expression and purification of histidine-tagged recombinant candidate receptor protein (cRP) for use in toxin overlay or binding assays. Further, to analyze in vitro binding between cRP and recombinant cholix toxin (rChxA) and perform structural studies using X-ray crystallography upon finding binding between cRP and rChxA.
Histidine-tagged cRP-gene was cloned in pFASTBac1, recombinant bacmids were purified and Spodoptera frugiperda (Sf9) cells were transformed to generate recombinant baculovirus particles. The Sf9 cells were infected with baculovirus to express the histidine-tagged cRP. The expression of histidine-tagged cRP was confirmed by Western blotting using anti-cRP or anti-His-tag antibodies. When analyzed for the solubility, the histidine-tagged cRP was insoluble and could only be detected in pellet fraction. Thus, histidine-tagged cRP could not be purified using affinity chromatography.
As the histidine-tagged cRP could not be purified, commercially available cRP were purchased. Whole cRP was not available and therefore extra-cellular domain of cRP was purchased. Binding and overlay assays were carried out between rChxA and extra-cellular domain of cRP. No binding could be observed either in toxin overlay or binding assays. This might suggest that extra-cellular domain of cRP could not bind to rChxA.
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| 現在までの達成度 (区分) |
現在までの達成度 (区分)
4: 遅れている
理由
The progress of our research plan for the year 2022-23 was delayed due to unexpected experimental results and the impact of the corona pandemic. Some of our efforts were diverted to other experiments and works related to pandemic.
As expected for year 2022-23, we successfully completed the cloning and expression of the histidine-tagged cRP using the baculovirus system. However, due to the protein's insoluble nature, we faced challenges in purifying the recombinant cRP. We are currently conducting further experiments to purify the recombinant cRP from inclusion bodies.
Unfortunately, we were unable to carry out structural studies as no binding was observed between the cRP and rChxA. I expect to progress this work in the current fiscal year.
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| 今後の研究の推進方策 |
The histidine-tagged cRP will be purified from insoluble fraction by denaturation using urea. The purified histidine-tagged cRP will be used for toxin overlay assay with rChxA. The binding assay will be also carried out. Finally, if the binding between the purified histidine-tagged cRP and rChxA will be successful, they will be used for X-ray crystallography.
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| 次年度使用額が生じた理由 |
Some of the experimental objectives could not be completed due to unexpected results and corona pandemic. Those experiments will be carried out along with other objectives in the current fiscal year. The funds will be required to accomplish the incomplete objectives of the research.
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