Cotrim Camila A, Jarrott Russell J, Whitten Andrew E, Choudhury Hassanul G, Drew David, Martin Jennifer L
Griffith Institute for Drug Discovery, Griffith University, Nathan, QLD, Australia.
Australian Nuclear Science and Technology Organisation, Lucas Heights, NSW, Australia.
Front Chem. 2021 Apr 30;9:667803. doi: 10.3389/fchem.2021.667803. eCollection 2021.
Human zinc transporter 1 (hZnT1) belongs to the cation diffusion facilitator (CDF) family. It plays a major role in transporting zinc (Zn) from the cytoplasm across the plasma membrane and into the extracellular space thereby protecting cells from Zn toxicity. Through homology with other CDF family members, ZnT1 is predicted to contain a transmembrane region and a soluble C-terminal domain though little is known about its biochemistry. Here, we demonstrate that human ZnT1 and a variant can be produced by heterologous expression in cells and purified in the presence of detergent and cholesteryl hemisuccinate. We show that the purified hZnT1 variant has Zn/H antiporter activity. Furthermore, we expressed, purified and characterized the soluble C-terminal domain of hZnT1 (hZnT1-CTD) in a bacterial expression system. We found that the hZnT1-CTD melting temperature increases at acidic pH, thus, we used an acetate buffer at pH 4.5 for purifications and concentration of the protein up to 12 mg/mL. Small-angle X-ray scattering analysis of hZnT1-CTD is consistent with the formation of a dimer in solution with a V-shaped core.
人类锌转运蛋白1（hZnT1）属于阳离子扩散促进剂（CDF）家族。它在将锌（Zn）从细胞质运输穿过质膜进入细胞外空间的过程中发挥着主要作用，从而保护细胞免受锌毒性的影响。通过与其他CDF家族成员的同源性分析，预计ZnT1含有一个跨膜区域和一个可溶性C末端结构域，但其生物化学性质鲜为人知。在此，我们证明人类ZnT1及其一个变体可以通过在细胞中的异源表达产生，并在去污剂和胆固醇半琥珀酸酯存在的情况下进行纯化。我们表明纯化的hZnT1变体具有锌/氢反向转运活性。此外，我们在细菌表达系统中表达、纯化并表征了hZnT1的可溶性C末端结构域（hZnT1-CTD）。我们发现hZnT1-CTD的解链温度在酸性pH下升高，因此，我们使用pH 4.5的乙酸盐缓冲液进行蛋白质的纯化和浓缩，浓度可达12 mg/mL。hZnT1-CTD的小角X射线散射分析结果与溶液中形成具有V形核心的二聚体一致。
