Hull York Medical School, University of York, York, YO10 5DD, UK.
Department of Chemistry, University of York, York, YO10 5DD, UK.
Sci Rep. 2024 Oct 15;14(1):24188. doi: 10.1038/s41598-024-75327-y.
Chemokines are proteins important for a range of biological processes from cell-directed migration (chemotaxis) to cell activation and differentiation. Chemokine C-C ligand 5 (CCL5) is an important pro-inflammatory chemokine attracting immune cells towards inflammatory sites through interaction with its receptors CCR1/3/5. Recombinant production of large quantities of CCL5 in Escherichia coli is challenging due to formation of inclusion bodies which necessitates refolding, often leading to low recovery of biologically active protein. To combat this, we have developed a method for CCL5 production that utilises the purification of SUMO tagged CCL5 from E. coli SHuffle cells avoiding the need to reform disulfide bonds through inclusion body purification and yields high quantities of CCL5 (~ 25 mg/L). We demonstrated that the CCL5 produced was fully functional by assessing well-established cellular changes triggered by CCL5 binding to CCR5, including receptor phosphorylation and internalisation, intracellular signalling leading to calcium flux, as well as cell migration. Overall, we demonstrate that the use of solubility tags, SHuffle cells and low pH dialysis constitutes an approach that increases purification yields of active CCL5 with low endotoxin contamination for biological studies.
趋化因子是一类在细胞定向迁移（趋化作用）到细胞激活和分化等多种生物学过程中发挥重要作用的蛋白质。趋化因子 C-C 配体 5（CCL5）是一种重要的促炎趋化因子，通过与其受体 CCR1/3/5 的相互作用，将免疫细胞吸引到炎症部位。由于形成包涵体，大肠杆菌中大量重组生产 CCL5 具有挑战性，这需要进行重折叠，通常导致具有生物活性的蛋白质回收率低。为了解决这个问题，我们开发了一种利用 SUMO 标记的 CCL5 从大肠杆菌 SHuffle 细胞中进行纯化的方法，避免了通过包涵体纯化来重新形成二硫键的需要，并产生了大量的 CCL5（~25mg/L）。我们通过评估 CCL5 与 CCR5 结合引发的成熟细胞的变化，包括受体磷酸化和内化、细胞内信号转导导致钙流以及细胞迁移，证明了所产生的 CCL5 是完全有功能的。总的来说，我们证明了使用可溶性标签、SHuffle 细胞和低 pH 透析的方法可以提高活性 CCL5 的纯化产量，同时降低内毒素污染，适用于生物学研究。
