Laboratory of Biochemistry, Molecular Biotechnology, and Molecular Biology, Department DiBEST (Biologia, Ecologia e Scienze della Terra), University of Calabria, Via Bucci 4C, 6C, 87036 Arcavacata di Rende, Italy.
National Research Council (CNR), Institute of Biomembranes, Bioenergetics and Molecular Biotechnologies (IBIOM), Via Amendola 122/O, 70126 Bari, Italy.
Int J Mol Sci. 2023 Dec 30;25(1):536. doi: 10.3390/ijms25010536.
The human SLC7A10 transporter, also known as ASC-1, catalyzes the transport of some neutral amino acids. It is expressed in astrocytes, neurons, and adipose tissues, playing roles in learning, memory processes, and lipid metabolism, thus being involved in neurological and metabolic pathologies. Structure/function studies on this transporter are still in their infancy. In this study, we present a methodology for producing the recombinant human transporter in Its transport function was assayed in proteoliposomes following the uptake of radiolabeled L-serine. After the testing of several growth conditions, the hASC-1 transporter was successfully expressed in BL21(DE3) codon plus RIL in the presence of 0.5% glucose and induced with 0.05 mM IPTG. After solubilization with CE and cholesteryl hemisuccinate and purification by Ni-chelating chromatography, hASC-1 was reconstituted in proteoliposomes. In this experimental system it was able to catalyze an Na-independent homologous antiport of L-serine. A Km for L-serine transport of 0.24 mM was measured. The experimental model developed in this work represents a reproducible system for the transport assay of hASC-1 in the absence of interferences. This tool will be useful to unveil unknown transport properties of hASC-1 and for testing ligands with possible application in human pharmacology.
人类 SLC7A10 转运蛋白，也称为 ASC-1，催化一些中性氨基酸的转运。它在星形胶质细胞、神经元和脂肪组织中表达，在学习、记忆过程和脂质代谢中发挥作用，因此参与神经和代谢病理学。对该转运蛋白的结构/功能研究仍处于起步阶段。在这项研究中，我们提出了一种在大肠杆菌中生产重组人转运蛋白的方法。在摄取放射性标记的 L-丝氨酸后，在脂质体中测定其转运功能。在测试了几种生长条件后，成功地在 BL21(DE3)codon plus RIL 中表达了 hASC-1 转运蛋白，在 0.5%葡萄糖存在下，并以 0.05mM IPTG 诱导。用 CE 和胆固醇半琥珀酸溶解后，通过镍螯合层析纯化，hASC-1 被重建为脂质体。在这个实验系统中，它能够催化 L-丝氨酸的非依赖 Na 的同源反向转运。测量到 L-丝氨酸转运的 Km 为 0.24mM。本工作中开发的实验模型代表了一种在无干扰情况下 hASC-1 转运测定的可重现系统。该工具将有助于揭示 hASC-1 的未知转运特性，并测试可能在人类药理学中应用的配体。
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