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Heterologous Expres...
Abstract
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- 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.
Subject headings
- NATURVETENSKAP -- Kemi (hsv//swe)
- NATURAL SCIENCES -- Chemical Sciences (hsv//eng)
Keyword
- human zinc transporter 1
- cation diffusion facilitator
- C-terminal domain
- small-angle X-ray scattering
- membrane proteins
Publication and Content Type
- ref (subject category)
- art (subject category)
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