Analysis of conformational changes of a G protein-coupled receptor that are propagated to a novel intracellular factor.

我们利用大肠杆菌细胞成功构建了一种调节趋化因子受体CCR2b的新型细胞内因子FROUNT的蛋白表达系统，并开发了一种蛋白纯化方案。利用该系统和方案，我们获得了大量纯化的全长front蛋白，足以进行结构分析。我们用溶液核磁共振和表面等离子体共振证实了纯化的FROUNT蛋白具有ccr2b结合活性。我们还利用昆虫细胞成功构建了CCR2b全长蛋白表达系统，并制定了蛋白纯化方案。

We succeeded in constructing a protein expression system for a novel intracellular factor FROUNT that regulates a chemokine receptor CCR2b using Escherichia coli cells and developing a protocol for the protein purification. Utilizing this system and protocol, we obtained large amounts of purified full-length FROUNT proteins that are enough for structural analyses. We elucidated that the purified FROUNT proteins possess CCR2b-binding activity with solution NMR and surface plasmon resonance. We also succeeded in constructing a protein expression system of full-length CCR2b using insect cells and developing a protocol for the protein purification.