质膜转运蛋白xCT属于SLC7家族,具有介导谷氨酸和胱氨酸跨细胞质膜交换的生理作用,对氧化还原控制至关重要。xCT蛋白与辅助蛋白CD98形成异二聚体。多年来,由于在几乎所有人类癌症中都有过表达,xCT成为一个热门的药理学靶点,它们的进展依赖于胱氨酸的可用性。尽管如此,xCT生物学的几个未知方面仍然存在,需要一个合适的单蛋白实验模型,有待解决。为了这个目标,已利用重组宿主大肠杆菌过表达xCT的人同工型。在这个广泛使用和低成本的系统中,生长和蛋白质生产的优化是通过作用于细菌菌株的代谢需求来实现的。然后,His标记的蛋白质已通过Ni2螯合色谱纯化,并在蛋白脂质体中重建以进行转运活性测定。表达的蛋白质处于折叠/活性状态,从而允许功能和动力学表征。有趣的是,重组蛋白的特征符合从完整细胞中提取的天然蛋白的特征,进一步证实大肠杆菌作为表达人蛋白的宿主的适合性。这项研究为阐明xCT的其他分子方面开辟了前景,以及研究与内源性和外源性化合物的相互作用,与人类健康相关。
The plasma membrane transporter xCT belongs to the SLC7 family and has the physiological role of mediating the exchange of glutamate and cystine across the cell plasma membrane, being crucial for redox control. The xCT protein forms a heterodimer with the ancillary protein CD98. Over the years, xCT became a hot pharmacological target due to the documented over-expression in virtually all human cancers, which rely on cystine availability for their progression. Notwithstanding, several unknown aspects of xCT biology still exist that require a suitable single protein experimental model, to be addressed. To this aim, the recombinant host Escherichia coli has been exploited to over-express the human isoform of xCT. In this widely used and low-cost system, the optimization for growth and protein production has been achieved by acting on the metabolic needs of the bacterial strains. Then, the His-tagged protein has been purified by Ni2+-chelating chromatography and reconstituted in proteoliposomes for transport activity assays. The expressed protein was in a folded/active state allowing functional and kinetic characterization. Interestingly, the features of the recombinant protein meet those of the native one extracted from intact cells, further confirming the suitability of E. coli as a host for the expression of human proteins. This study opens perspectives for elucidating other molecular aspects of xCT, as well as for studying the interaction with endogenous and exogenous compounds, relevant to human health.