Chemokine receptors form a major sub-family of G protein-coupled receptors (GPCRs) and they are involved in a number of cellular and physiological processes related to our immune response and regulation. A better structural understanding of ligand-binding, activation, signaling and regulation of chemokine receptors is very important to design potentially therapeutic interventions for human disorders arising from aberrant chemokine signaling. One of the key limitations in probing the structural details of chemokine receptors is the availability of large amounts of purified, homogenous and fully functional chemokine ligands, and the commercially available products, are not affordable for in-depth structural studies. Moreover, production of uniformly isotope-labeled chemokines, for example, suitable for NMR-based structural investigation, also remains challenging. Here, we have designed a streamlined approach to express and purify the human chemokine CCL7 as well as its 15N-, 15N/13C-, 2H/15N/13C- isotope-labeled derivatives, at milligram levels using E. coli expression system. Purified CCL7 not only maintains a well-folded three-dimensional structure as analyzed using circular dichroism and 1H/15N NMR but it also induces coupling of heterotrimeric G-proteins and β-arrestins for selected chemokine receptors in cellular system. We compared cAMP response induced by histidine tagged CCL7 and native CCL7 and found that modification of the N-terminus of CCL7 compromises its functionality. Our strategy presented here may be applicable to other chemokines and therefore, provide a potentially generic and cost-effective approach to produce chemokines in large amounts for functional and structural studies.

中文翻译：

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天然 CCL7 的纯化及其与选定趋化因子受体的功能相互作用。

趋化因子受体是 G 蛋白偶联受体 (GPCR) 的一个主要亚家族，它们参与许多与我们的免疫反应和调节相关的细胞和生理过程。更好地理解趋化因子受体的配体结合、激活、信号传导和调节对于设计由异常趋化因子信号传导引起的人类疾病的潜在治疗干预非常重要。探索趋化因子受体结构细节的关键限制之一是大量纯化、同质和功能齐全的趋化因子配体的可用性，而市售产品无法进行深入的结构研究。此外，生产均匀同位素标记的趋化因子，例如，适用于基于 NMR 的结构研究，也仍然具有挑战性。在这里，我们设计了一种简化的方法，使用大肠杆菌表达系统以毫克水平表达和纯化人类趋化因子 CCL7 及其 15N-、15N/13C-、2H/15N/13C-同位素标记的衍生物。纯化的 CCL7 不仅保持良好折叠的三维结构，如使用圆二色性和 1H/15N NMR 分析的那样，而且它还诱导细胞系统中选定趋化因子受体的异源三聚体 G 蛋白和 β-抑制蛋白偶联。我们比较了组氨酸标记的 CCL7 和天然 CCL7 诱导的 cAMP 反应，发现 CCL7 N 末端的修饰会损害其功能。我们在此介绍的策略可能适用于其他趋化因子，因此，