Expression and purification of human FROUNT, a common cytosolic regulator of CCR2 and CCR5
Expression and purification of human FROUNT, a common cytosolic regulator of CCR2 and CCR5
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DOI：
10.1016/j.pep.2010.12.012
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发表时间：
2011-05-01
影响因子：
1.6
通讯作者：
Terasawa, Hiroaki
中科院分区：
文献类型：
--
作者：
Esaki, Kaori;Terashima, Yuya;Terasawa, Hiroaki
趋化因子受体在免疫细胞对趋化因子梯度(趋化性)的反应中，在免疫细胞向炎症部位的募集中起着关键作用。趋化因子的信号转导机制，尤其是细胞内信号级联反应的启动机制还不是很清楚。我们以前发现了一种细胞质蛋白FROUNT，它与CCR2和CCR5的C末端区域结合，介导趋化因子信号转导。虽然详细的生化研究和药物筛选需要大量的纯化蛋白，但目前还没有生产重组FROUNT的方法报道。在本研究中，我们开发了一种生产重组人FROUNT的方法。利用冷休克表达系统，成功地将人FROUNT作为融合到折叠伴侣触发因子的可溶性蛋白在大肠杆菌中表达。SPR测试表明，纯化的FROUNT蛋白具有CCR2结合能力，不需要任何额外的成分。凝胶过滤分析表明，FROUNT以均低聚状态存在。这种高产量的方法对人类FROUNT生产是具有成本效益的。它应该是进一步的生化和结构研究的有力工具，以阐明GPCR调控和趋化因子信号转导，也将有助于药物的开发。(C)2010 Elsevier Inc.保留所有权利。
Chemokine receptors play pivotal roles for immune cell recruitment to inflammation sites, in response to chemokine gradients (chemotaxis). The mechanisms of chemokine signaling, especially the initiation of the intracellular signaling cascade, are not well understood. We previously identified a cytoplasmic protein FROUNT, which binds to the C-terminal regions of CCR2 and CCR5 to mediate chemokine signaling. Although large amounts of purified protein are required for detailed biochemical studies and drug screening, no method to produce recombinant FROUNT has been reported. In this study, we developed a method for the production of recombinant human FROUNT. Human FROUNT was successfully expressed in Escherichia coli, as a soluble protein fused to the folding chaperone Trigger Factor, with a cold shock expression system. The purified FROUNT protein displayed CCR2 binding ability without any additional components, as demonstrated by SPR measurements. A gel filtration analysis suggested that FROUNT exists in a homo-oligomeric state. This high-yield method is cost-effective for human FROUNT production. It should be a powerful tool for further biochemical and structural studies to elucidate GPCR regulation and chemokine signaling, and also will contribute to drug development. (c) 2010 Elsevier Inc. All rights reserved.