| 研究実績の概要 |
The EML4-ALK fusion protein is a strong driver of lung cancer, and to purify this well-known oncoprotein is challenge work. However, visualizing a clear structure of the EML4-ALK under the HS-AFM requires good performance in protein purification. At present, we have much improved the purification conditions of EML4-ALK variant 5 (the smallest one in all EML4-ALK variants) and gain a good quality of purified protein EML4-ALK v5, which fused with a GFP reporter at the C-terminal. We want to use the GFP as a marker in the steps of the cell transfection and the structural identification by HS-AFM. Based on this strategy, we have also visualized the protein EML4-ALK v5 dynamic structures under the HS-AFM. Currently, the quantification of the structural characters for EML4-ALK variant v5 is undergoing. As we expected in the proposal of this project, we will also continue to purify the other variants of EML4-ALK oncoprotein in the same way and then to confirm their structure under HS-AFM.
Interestingly, we have found the EML4-ALK proteins formed phase separation by strongly interacting with other unknown proteins during the purification steps. This formed phase separation in PBS buffer was very stable and did not affect even we have changed the pH and salt concentration in the buffer. Only adding the ALK inhibitor to the protein sample can dramatically decrease the phase separation amounts and size. These results indicated that the phase separation might have biological meanings. Thus, we will also focus on the phase separation in the following steps.
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| 現在までの達成度 (区分) |
現在までの達成度 (区分)
3: やや遅れている
理由
EML4-ALK fusion oncoproteins include flexible domains, which is functionally interacted with other proteins, and this property increased the difficulties of the purification steps. Indeed, the purified EML4-ALK proteins are easy to be contaminated by some unexpected proteins. These contaminations make it confused to identify the sub-domain of EML4-ALK and cause a dirty background under HS-AFM. Therefore, we have kept a lot of effort and time into this problem. Lucky, we have finally got a high-quality purified protein v5 for HS-AFM observation.
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| 今後の研究の推進方策 |
As we have planned in the submitted proposal of this project, we will continue to purify the other variants of EML4-ALK oncoprotein in the same way and to confirm their structures under HS-AFM. We finally want to find dynamic differences among the variants of EML4-ALK. As we have the new findings of EML4-ALK phase separation, we will also try some ideas on this topic.
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