Heterologous Expression and Biochemical Characterization of the Human Zinc Transporter 1 (ZnT1) and Its Soluble C-Terminal Domain
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Jarrott, RJ
Whitten, AE
Choudhury, HG
Drew, D
Martin, JL
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Abstract
Human zinc transporter 1 (hZnT1) belongs to the cation diffusion facilitator (CDF) family. It plays a major role in transporting zinc (Zn2+) from the cytoplasm across the plasma membrane and into the extracellular space thereby protecting cells from Zn2+ 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 Saccharomyces cerevisiae cells and purified in the presence of detergent and cholesteryl hemisuccinate. We show that the purified hZnT1 variant has Zn2+/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.
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Frontiers in Chemistry
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9
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© 2021 Cotrim, Jarrott, Whitten, Choudhury, Drew and Martin. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
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Chemical sciences
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Cotrim, CA; Jarrott, RJ; Whitten, AE; Choudhury, HG; Drew, D; Martin, JL, Heterologous Expression and Biochemical Characterization of the Human Zinc Transporter 1 (ZnT1) and Its Soluble C-Terminal Domain, Frontiers in Chemistry, 2021, 9