Department of Structural Biology, Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow, Russia.
Department of Biological Sciences and Bioengineering, Indian Institute of Technology, Kanpur, 208016, India.
Protein Expr Purif. 2020 Jul;171:105617. doi: 10.1016/j.pep.2020.105617. Epub 2020 Mar 5.
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 N-, N/C-, H/N/C- 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 H/N 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.
趋化因子受体形成 G 蛋白偶联受体 (GPCR) 的主要亚家族，它们参与与我们的免疫反应和调节有关的许多细胞和生理过程。更好地了解配体结合、激活、信号转导和趋化因子受体的调节对于设计潜在的治疗干预措施以治疗由于异常趋化因子信号引起的人类疾病非常重要。研究趋化因子受体结构细节的一个关键限制是大量纯化、同质和完全功能的趋化因子配体的可用性，并且商业上可获得的产品对于深入的结构研究来说负担不起。此外，均匀同位素标记的趋化因子的生产，例如适合基于 NMR 的结构研究的趋化因子，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在毫克水平上表达和纯化人趋化因子 CCL7 及其 N-、N/C-、H/N/C-同位素标记衍生物。纯化的 CCL7 不仅在使用圆二色性和 H/NMR 分析时保持良好折叠的三维结构，而且还诱导异三聚体 G 蛋白和β-arrestin 在细胞系统中与选定的趋化因子受体偶联。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 的 N 端修饰会损害其功能。我们在这里提出的策略可能适用于其他趋化因子，因此为大量生产用于功能和结构研究的趋化因子提供了一种潜在的通用且具有成本效益的方法。
