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Heterologous Expression and Biochemical Characterization of the Human Zinc Transporter 1 (ZnT1) and Its Soluble C-Terminal Domain.
- Source :
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Frontiers in chemistry [Front Chem] 2021 Apr 30; Vol. 9, pp. 667803. Date of Electronic Publication: 2021 Apr 30 (Print Publication: 2021). - Publication Year :
- 2021
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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 (Zn <superscript>2+</superscript> ) from the cytoplasm across the plasma membrane and into the extracellular space thereby protecting cells from Zn <superscript>2+</superscript> 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 Zn <superscript>2+</superscript> /H <superscript>+</superscript> 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.<br />Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.<br /> (Copyright © 2021 Cotrim, Jarrott, Whitten, Choudhury, Drew and Martin.)
Details
- Language :
- English
- ISSN :
- 2296-2646
- Volume :
- 9
- Database :
- MEDLINE
- Journal :
- Frontiers in chemistry
- Publication Type :
- Academic Journal
- Accession number :
- 33996761
- Full Text :
- https://doi.org/10.3389/fchem.2021.667803